Systems and methods for enzymatic oligonucleotide synthesis

Abstract

The present invention provides for methods and systems for template-free synthesis of an oligonucleotide, and methods of using the systems as disclosed herein for template-free synthesis of an oligonucleotide.

Description

SYSTEMS AND METHODS FOR ENZYMATIC OLIGONUCLEOTIDE SYNTHESISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 633,644, filed April 12, 2024; U.S. Provisional Application No. 63 / 634,877, filed April 16, 2024; and U.S. Provisional Application No. 63 / 646,600, filed May 13, 2024; all of which are incorporated by reference herein.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The Sequence Listing concurrently submitted herewith as file name CX10- 264WO4_ST26.xml, created on April 11, 2025, with a file size of 47,860 bytes, is part of the specification and is incorporated by reference herein.FIELD

[0003] Described herein are methods for synthesizing oligonucleotides using an enzymatic process. Also described are systems that can be used to perform such methods.BACKGROUND

[0004] Synthetic oligonucleotides (oligos) are significant reagents in research and medicine and are currently poised to be in greater demand. At present, the pharmaceutical industry is projected to demand hundreds to thousands of kilograms of oligonucleotides per year for active pharmaceutical ingredients (see, e.g., Andrews, Benjamin I et al. “Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing.” The Journal of Organic Chemistry, 2021, 86:49-61). A barrier to widescale and commercial adoption of synthetic oligos as products in multiple fields is the ability to efficiently synthesize RNA, DNA, and other polynucleotides. Current manufacturing methods rely on phosphoramidite chemistry as a means for synthesizing oligonucleotides, but the method is best suited for producing small quantities of DNA and RNA. Additionally, RNA synthesis using phosphoramidite synthesis chemistry is limited to producing short oligonucleotides of approximately 200 base pairs (Beaucage & Caruthers, Tetrahedron Lett., 1981, 22(20): 1859) Despite the growing demand, optimization of the technology has only been able to achieve incremental gains in oligonucleotide length, yield and efficiency.

[0005] Phosphoramidite chemistry also requires large amounts of environmentally harmful reagents, such as acetonitrile and dichloroacetic acid, to drive conversion efficiency. For example, 1 kilogram of small interfering RNA (siRNA) may require as much as 1000 kg of acetonitrile. This results in thousands of kilograms of chemical waste per kilogram of active pharmaceutical ingredient.SUMMARY OF THE INVENTION

[0006] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising:elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed. In some aspects, the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide. In some aspects, the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the method further comprises separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase. In some aspects, the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety. In some aspects, at least two different types of transferases are used in separate cycles. In some aspects, the elongating occurs in a first reaction chamber. In some aspects, the removing the 3' blocking moiety occurs in a second reaction chamber. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotidecomprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0007] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: in a first reaction chamber, elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the second reaction chamber into the first reaction chamber. In some aspects, the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide. In some aspects, the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber. In some aspects, the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety. In some aspects, at least two different types of transferases are used in separate cycles. In some aspects, the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the secondreaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the first reaction chamber. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the second reaction chamber. In some aspects, the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the second reaction chamber. In some aspects, the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the first reaction chamber. In some aspects, the flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into the first reaction chamber comprises: flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into one or more reservoirs, and flowing the elongated oligonucleotide without the 3' blocking moiety from the one or more reservoirs into the first reaction chamber. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blockingmoiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0008] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising, in a plurality of reaction chambers: in at least a first reaction chamber, elongating an oligonucleotide in solution by attaching a first nucleotide comprising a 3' blocking moiety to the oligonucleotide using a first transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution from the at least the first reaction chamber into at least a second reaction chamber, wherein the first transferase is retained in the at least the first reaction chamber; in the at least the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the at least the second reaction chamber into at least a third reaction chamber comprising a second transferase; and in the at least the third reaction chamber, elongating the elongated oligonucleotide without the 3' blocking moiety the solution by attaching a second nucleotide comprising a 3' blocking moiety to the elongated oligonucleotide without the 3' blocking moiety using the second transferase to make a further elongated oligonucleotide comprising the 3' blocking moiety of the second nucleotide. In some aspects, the first transferase and the second transferase are different types of transferases. In some aspects, the first nucleotide and the second nucleotide are preselected types of nucleotides. In some aspects, the first nucleotide and the second nucleotide are different types of nucleotides. In some aspects, the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the hydrolase is retained in the at least the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows from the second reaction chamber into the at least the third reaction chamber. In some aspects, the first reaction chamber, the second reaction chamber, and the third reaction chamber are connectedthrough a plurality of conduits, wherein: the elongated oligonucleotide comprising the 3' blocking moiety flows from the at least the first reaction chamber into at least a second reaction chamber through a first portion of the plurality of conduits; and the elongated oligonucleotide without the 3' blocking moiety flows from the at least the second reaction chamber into at least a third reaction chamber through a second portion of the plurality of conduits. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more pumps. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more valves. In some aspects, the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog. In some aspects, the 5' phosphate analog is a 5'-(a- P-thio)phosphate moiety. In some aspects, the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase removes three 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase is immobilized on a solid support. In some aspects, the hydrolase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the transferase is a polymerase from the DNA polymerase X family. In some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). In some aspects, the transferase is immobilized on a solid support. In some aspects, the transferase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, elongating produces an inorganic pyrophosphate byproduct. In some aspects, the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the pyrophosphatase is immobilized on a solid support. In some aspects, the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the elongating and the degrading occur within the same reaction chamber. In some aspects, the transferase and the inorganic pyrophosphatase are fused together. In some aspects, the transferase and the pyrophosphatase are immobilized on the same solid support. In some aspects, the transferase and the pyrophosphatase are immobilized on different solid supports. In some aspects, the transferase is immobilized on a solid support and the pyrophosphatase is in the solution. In some aspects, the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide. In some aspects, the elongating and the degrading occur within different reaction chambers. In some aspects, the method comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety and the inorganicpyrophosphate byproduct, in the solution, from a reaction chamber comprising the transferase into a reaction chamber comprising the pyrophosphatase, followed by flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reaction chamber comprising the pyrophosphatase into a reaction chamber in which the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the first reaction chamber or the second reaction chamber is a column. In some aspects, the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allow s passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of hydrolase and allows passage of the oligonucleotide. In some aspects, the first reaction chamber or the second reaction chamber is a batch reaction chamber. In some aspects, the batch reaction chamber comprises an impeller. In some aspects, the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support. In some aspects, the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support. In some aspects, the method further comprises separating unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography. In some aspects, the liquid chromatography comprises size exclusion chromatography. In some aspects, the liquid chromatography comprises reverse phase chromatography. In some aspects, the liquid chromatography comprises ion exchange chromatography. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the 3' blocking moiety is a phosphate moiety. In some aspects, the nucleotide comprising the 3' blocking moiety is a ribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a 2' modification. In some aspects, the 2' modification is 2'-F or 2'-0-Me (2’-O-methyl). In some aspects, the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate. In some aspects, the oligonucleotide comprises a 5' modification. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with thetransferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0009] Provided herein is a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase; wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber. In some aspects, the system further comprises a temperature regulator that controls a temperature of the solution in the system. In some aspects, the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber. In some aspects, the systemfurther comprises one or more conduits that connects the first reaction chamber and the second reaction chamber. In some aspects, the temperature regulator is a jacketed stir tank. In some aspects, the system further comprises an in-line temperature regulator that controls a temperature of the solution in at least one of the one or more conduits. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber. In some aspects, the system comprises one or more pumps configured to flow the solution from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber. In some aspects, the one or more pumps are configured to control a flow rate of the solution. In some aspects, the system further comprises a third reaction chamber comprising a second transferase, wherein the system is further configured to selectively flow said oligonucleotide in the solution from the second chamber to the third reaction chamber while retaining the hydrolase in the second reaction chamber, and flow the oligonucleotide in the solution from the third reaction chamber to the second reaction chamber while retaining the second transferase in the third reaction chamber. In some aspects, the transferase and the second transferase are different types of transferase. In some aspects, the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system. In some aspects, the hydrolase can remove a 3' blocking moiety from the oligonucleotide in the solution. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, transferase can react a nucleotide triphosphate (NTP) comprising a 3' blocking moiety, or an analog thereof comprising a 5' phosphate analog, with the oligonucleotide to elongate the oligonucleotide. In some aspects, the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a ribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a deoxyribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof comprises a 2' modification. In some aspects, the 2' modification of the NTP or the analog thereof is 2'-F or 2'-0Me. In some aspects, the 3' blocking moiety of the NTP or the analog thereof is a phosphate moiety. In some aspects, the hydrolase can remove a 3' blocking moiety from unreacted nucleotides comprising a 3' blocking moiety in the solution. In some aspects, the unreacted nucleotides are ribonucleotides. In some aspects, the unreacted nucleotides comprise a 2' modification. In some aspects, the 2' modification of the unreacted nucleotides is 2'-F or 2'-0Me. In some aspects, the 3' blocking moiety of the unreacted nucleotides is a phosphate moiety. In some aspects, the unreacted nucleotides comprise a 5' phosphate analog. In some aspects, the 5' phosphate analog of the unreacted nucleotides is a 5'-(a-P-thio)phosphate moiety. In some aspects, the hydrolase can remove one or more 5' phosphate moieties and / or 5'-(a-P-thio)phosphate moieties from the unreacted nucleotides in the solution. In some aspects, the hydrolase is immobilized on a solid support. In some aspects, the hydrolase is immobilized using a covalent, electrostatic, or ionic bond.In some aspects, the transferase is a polymerase from the DNA polymerase X family. In some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). In some aspects, the transferase is immobilized on a solid support. In some aspects, the transferase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the first reaction chamber further comprises a pyrophosphatase. In some aspects, the system further comprises a third reaction chamber comprising a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the pyrophosphatase is immobilized on a solid support. In some aspects, the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the transferase and phosphatase are fused together. In some aspects, the transferase and phosphatase are immobilized on the same solid support. In some aspects, the first reaction chamber or the second reaction chamber is a column. In some aspects, the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide. In some aspects, the first reaction chamber or the second reaction chamber is a batch reaction chamber. In some aspects, the batch reaction chamber comprises an impeller. In some aspects, the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support. In some aspects, the second reaction chamber comprises a rotating bed reactor comprising the hydrolase immobilized on a solid support. In some aspects, the system further comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide. In some aspects, the purification chamber comprises a column. In some aspects, the purification chamber comprises a liquid chromatography column. In some aspects, the purification chamber is a size exclusion column. In some aspects, the purification chamber is an ion exchange column. In some aspects, the purification chamber is reverse phase column. In some aspects, purification chamber is part of a tangential flow filtration system. In some aspects, the first reaction chamber or the second reaction chamber are cleanable or replaceable. In some aspects, the first reaction chamber comprises an inletand an outlet; the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet; and wherein the system is configured to flow the oligonucleotide in the solution from an outlet of one of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from the outlet of the second reaction chamber to an inlet of the reagent reservoir. In some aspects, the reagent reservoir comprises an impeller. In some aspects, the reagent reservoir is replaceable or cleanable. In some aspects, the method further comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the reagent reservoir. In some aspects, the system further comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the first reaction chamber. In some aspects, the product reservoir comprises an impeller. In some aspects, the product reservoir is replaceable or cleanable. In some aspects, the reagent reservoir comprises the oligonucleotide and a nucleotide comprising a 3' blocking moiety. In some aspects, the system further comprises the purification chamber, wherein the purification chamber comprises an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution, in order, from the outlet of the second reaction chamber to the inlet of the purification chamber, and from the outlet of the purification chamber to the inlet of the reagent reservoir. In some aspects, the system comprises the purification chamber, wherein the system is configured to flow the oligonucleotide, in the solution, from the outlet of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, from the outlet of the second reaction chamber to the inlet of the purification chamber, from the outlet of the purification chamber to the inlet of the product reservoir, and from the outlet of the product reservoir to the inlet of the reagent reservoir. In some aspects, the first reaction comprises an inlet and an outlet; the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet, and one or more diverter valves configured to control a flow pathway of the solution; wherein the system is configured to controllably flow the oligonucleotide in the solution through the flow pathway selected from a plurality of flow pathways comprising (i) a first flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the first reaction chamber without flowing through the second reaction chamber, and (ii) a second flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the second reaction chamber without flowing through the first reaction chamber. In some aspects, the system is configured to automatically select the flow pathway. In some aspects, the first flow pathway comprises flow of the solution from the outlet of the first reaction chamber to the inlet of the reagent reservoir without flowing through thesecond reaction chamber; and the second flow pathway comprises flow of the solution from the outlet of the second reaction chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber. In some aspects, the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a third flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent reservoir to the inlet of the purification chamber without flowing through the first reaction chamber or the second reaction chamber. In some aspects, the third flow pathway further comprises flow of the solution from the outlet of the purification chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber or the second reaction chamber. In some aspects, the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a fourth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the first reaction chamber, from an outlet of the first reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir. In some aspects, the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a fifth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the second reaction chamber, from an outlet of the second reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir. In some aspects, the reagent reservoir comprises an impeller. In some aspects, the transferase in the first reaction chamber is immobilized on a solid support, and the first reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the transferase; and the hydrolase in the second reaction chamber is immobilized on a solid support, and the second reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the hydrolase. In some aspects, the transferase in the first reaction chamber is immobilized on a solid support within a rotating bed reactor, and the first reaction chamber comprises an inlet and an outlet; and the hydrolase in the second reaction chamber is immobilized on a solid support within a rotating bed reactor, and the second reaction chamber comprises an inlet and an outlet. In some aspects, the system comprises one or more diverter valves configured to alternatively direct flow of the solution through (i) a first flow pathways comprising flow of the solution from the outlet of the first reaction chamber to an inlet of the purification chamber, and from an outlet of the purification chamber to the inlet of the first reaction chamber, or (ii) a second flow pathway comprising flow of the solution from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from an outlet of the second reaction chamber to the inlet of the first reaction chamber. In some aspects, the system further comprises a reaction chamber comprising a primase. In some aspects, the primase is immobilized on a solid support. In some aspects, the reaction chamber comprising the primase comprises a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide. In someaspects, the system comprises a degassing system or a sparging system. In some aspects, the system comprises a degassing system, and the degassing system comprises a vacuum pump. In some aspects, the system comprises the sparging system. In some aspects, the sparging system is an in-line sparging system or is configured to sparge liquids in a reservoir. In some aspects, the unit for sparging system is configured to sparge using an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, the system comprises a degasser configured to remove oxygen from the system. In some aspects, the purification chamber is configured to concentrate the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the purification chamber comprises a membrane with a molecular weight cutoff of about 500 kDa to about 5000 kDa.

[0010] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: flowing an oligonucleotide, in a solution, from a reagent reservoir into a first reaction chamber comprising a transferase; in the first reaction chamber, elongating the oligonucleotide by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using the transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the nucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber comprising a hydrolase, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety in the solution to make an elongated oligonucleotide without the 3' blocking moiety; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, into the reagent reservoir. In some aspects, the method further comprises mixing the oligonucleotide and the nucleotides comprising a 3'-blocking moiety in the reservoir. In some aspects, the method further comprises flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, in the solution, through a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety before flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety into the reagent reservoir. In some aspects, the method further comprises flowing the oligonucleotide without the 3' blocking moiety into a product reservoir before flowing the oligonucleotide without the 3' blocking moiety into the reagent reservoir. In some aspects, the method further comprises replacing or cleaning the reagent reservoir before flowing the elongated oligonucleotide without the 3' blocking moiety into the reagent reservoir. In some aspects, the method repeats the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the method further comprising flowing buffer without the oligonucleotide through the system between cycles. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide withthe transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0011] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: (a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and nucleotides comprising a 3' blocking moiety from a reagent reservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir; and (b) removing the 3' blocking moiety from the oligonucleotide in the solution to make an elongated oligonucleotide without the 3' blocking moiety, comprising subjecting the oligonucleotide to one or more deblocking flow pathwaycycles comprising: flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir to a second reaction chamber comprising a hydrolase, and flowing the elongated oligonucleotide from the second reaction chamber to the reagent reservoir. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0012] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: (a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and 3'-blocked nucleotides from a reagentreservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir; (b) flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir to a second reaction chamber comprising a hydrolase; and (c) removing the 3' blocking moiety from the oligonucleotide in the solution to make an elongated oligonucleotide without the 3' blocking moiety, comprising subjecting the oligonucleotide to one or more deblocking flow pathway cycles comprising: flowing the elongated oligonucleotide comprising the 3' blocking moiety from the second reaction chamber to a product reservoir, and flowing the elongated oligonucleotide from the product reservoir to the second reaction chamber. In some aspects, the elongating comprises subjecting the oligonucleotide to a plurality of elongation flow path cycles. In some aspects, the removing comprises subjecting the oligonucleotide to a plurality of deblocking flow pathway cycles. In some aspects, the method comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the separating comprises subjecting the oligonucleotide to one or more purification flow pathway cycles comprising: flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir or the product reservoir to a purification chamber configured to separate the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety from the purification chamber to the reagent reservoir or the product reservoir. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography. In some aspects, the liquid chromatography comprises size exclusion chromatography. In some aspects, the liquid chromatography comprises reverse phase chromatography. In some aspects, the liquid chromatography comprises ion exchange chromatography. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the method comprises repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In someaspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0013] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: in first reaction chamber, elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the an elongated oligonucleotide comprising the 3' blocking moiety in the solution using a hydrolase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide without the 3' blocking moiety; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, into the first reaction chamber. In some aspects, the method further comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moietyor the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the separating comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety from the first reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the separating comprises flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography. In some aspects, the liquid chromatography comprises size exclusion chromatography. In some aspects, the liquid chromatography comprises reverse phase chromatography. In some aspects, the liquid chromatography comprises ion exchange chromatography. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the method comprises repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber. In some aspects, the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves. In some aspects, the nucleotide comprising the 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog. In some aspects, the 5' phosphate analog is a 5'-(a- P-thio)phosphate moiety. In some aspects, the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In someaspects, the hydrolase removes three 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase is immobilized on a solid support. In some aspects, the hydrolase is immobilized using a covalent, electrostatic, or in some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). In some aspects, the transferase is immobilized on a solid support. In some aspects, the transferase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the elongating produces an inorganic pyrophosphate byproduct. In some aspects, the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase. In some aspects, he pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the pyrophosphatase is immobilized on a solid support. In some aspects, the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the elongating and the degrading occur within the same reaction chamber. In some aspects, the transferase and the inorganic pyrophosphatase are fused together. In some aspects, the transferase and the pyrophosphatase are immobilized on the same solid support. In some aspects, the transferase and the pyrophosphatase are immobilized on different solid supports. In some aspects, the transferase is immobilized on a solid support and the pyrophosphatase is in the solution. In some aspects, the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide . In some aspects, the elongating and the degrading occur within different reaction chambers. In some aspects, the first reaction chamber or the second reaction chamber is a column. In some aspects, the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of hydrolase and allows passage of the oligonucleotide. In some aspects, the first reaction chamber or the second reaction chamber is a batch reaction chamber. In some aspects, the batch reaction chamber comprises an impeller. In some aspects, the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support. In some aspects, the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support. In some aspects, the 3' blocking moiety is a phosphate moiety. In some aspects, the nucleotide comprising the 3' blocking moiety is a ribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a 2' modification. In some aspects, the 2' modification is 2'-F or 2'-0Me. In some aspects, the nucleotide comprising the 3' blocking moietycomprises a nucleoside 5'-(a-P-thio)phosphate. In some aspects, the oligonucleotide comprises a 5' modification. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0014] Provided herein is a method of template-free synthesis of an oligonucleotide, comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed. In some aspects, the method comprises separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase comprises flowing the solution through a column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column. In some aspects, the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide comprising the 3' blocking moiety. In some aspects, the method comprises flowing the oligonucleotide and the transferase, in the solution, from a reagent reservoir to the column that retains the transferase, and flowing the oligonucleotide comprising the 3' blocking moiety, in the solution, from the column that retains the transferase to the reagent reservoir. In some aspects, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety comprises adding a hydrolase to the solution after separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase. In some aspects, the method further comprises separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety. In some aspects, separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety from the hydrolase comprises flowing the solution through a column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety comprises a filter that prevents passage of the hydrolase and allows passage of the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the method comprises flowing the elongated oligonucleotide without the 3' blocking moiety and the hydrolase, in the solution, from a reagent reservoir to the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety to the reagent reservoir. In some aspects, the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the method comprises replacing or cleaning the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety or the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the cleaning comprises flowing a buffer through the column to elute the transferase or the hydrolase. In some aspects, the method further comprisesseparating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety. In some aspects, separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety comprises flowing the solution through a column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a reverse phase column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is an ion exchange column. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide. In some aspects, the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog. In some aspects, the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety. In some aspects, the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase removes three 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the transferase is a polymerase from the DNA polymerase X family. In some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). In someaspects, elongating produces an inorganic pyrophosphate byproduct. In some aspects, the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the transferase and the inorganic pyrophosphatase are fused together. In some aspects, the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety further separates the pyrophosphatase from the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the 3' blocking moiety is a phosphate moiety. In some aspects, the nucleotide comprising the 3' blocking moiety is a ribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a 2' modification. In some aspects, the 2' modification is 2'-F or 2'-0Me. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate. In some aspects, the oligonucleotide comprises a 5' modification. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase. In some aspects, making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor. In some aspects, the transferase comprises a single strand RNA ligase. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker. In some aspects, the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof. In some aspects, the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH. In some aspects, the oligonucleotide is elongated under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the3' blocking moiety is performed between 45 °C and 55 °C. In some aspects, the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber. In some aspects, the desalting chamber is maintained between 20 °C and 30 °C.

[0015] Provided herein is a system for template-free synthesis of an oligonucleotide comprising: a reagent reservoir; a first column configured to substantially separate a transferase from an oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety; wherein the system is configured to (i) flow a solution comprising an oligonucleotide comprising the 3' blocking moiety and a transferase from the reagent reservoir to the first column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the transferase in the first column, (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the first column to the reagent reservoir, (iv) flow the solution comprising an oligonucleotide without the 3' blocking moiety and a hydrolase to the second column, (v) substantially separate the oligonucleotide without the 3' blocking moiety from the hydrolase in the second column, and (vi) flow the solution comprising the oligonucleotide without the 3' blocking moiety from the second column to the reagent reservoir. In some aspects, the system comprises a plurality of conduits that connects the first column, the second column, and the reagent reservoir. In some aspects, the system further comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety. In some aspects, the system further comprises one or more conduits that connects the purification chamber and the reagent reservoir. In some aspects, the purification chamber comprises a column. In some aspects, the purification chamber comprises a liquid chromatography column. In some aspects, the purification chamber is a size exclusion column. In some aspects, the purification chamber is an ion exchange column. In some aspects, the purification chamber is reverse phase column. In some aspects, the purification chamber is part of a tangential flow filtration system. In some aspects, the system is further configured to (vii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the reagent reservoir to the purification chamber, (viii) separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety in the purification chamber, and (ix) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the purification chamber to the reagent reservoir. In some aspects, the system further comprises one or more diverter valves configured to controllably flow the solution from the reagent reservoir to the first column or the second column. In some aspects, the one or more diverter valves is configured to select a flow path for the solution, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the solution from the reagent reservoir to the firstcolumn and from the first column to the reagent reservoir and (ii) flow of the solution from the reagent reservoir to the second column and from the second column to the reagent reservoir. In some aspects, the system comprises the purification chamber, and the one or more diverter valves is further configured to controllably flow the solution from the reagent reservoir to the purification chamber. In some aspects, the plurality of flow paths further comprises (iii) flow of the solution from the reagent reservoir to the purification chamber and from the purification chamber to the reagent reservoir. In some aspects, the system further comprises one or more wash buffer reservoirs connected to the first column, the second column, or the purification chamber. In some aspects, the one or more diverter valves is configured to controllably flow wash buffer from the wash buffer reservoir to the first column, the second column, or the purification chamber. In some aspects, the one or more diverter valves is configured to select a wash buffer flow path for wash buffer in the one or more wash buffer reservoirs, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the wash buffer from the one or more wash buffer reservoirs to the first column and from the first column to a system waste outlet and (ii) flow of the wash buffer from the one or more wash buffer reservoirs to the second column and from the second column to the system waste outlet. In some aspects, the system comprises the purification chamber, and wherein the plurality of flow paths further comprises (iii) flow of the wash buffer from the one or more wash buffer reservoirs to the purification chamber and from the purification chamber to the system waste outlet. In some aspects, the system further comprises a temperature regulator that controls a temperature of the solution in the system. In some aspects, the temperature regulator is configured to control the temperature of the solution in the first column or the second column. In some aspects, the system comprises an in-line temperature regulator that controls a temperature of the solution in one or more conduits of the system. In some aspects, the temperature regulator is a jacketed stir tank. In some aspects, the system comprises one or more pumps configured to flow the solution from the reagent reservoir to the first column or from the reagent reservoir to the second column. In some aspects, the system comprises the purification chamber, and the one or more pumps are further configured to flow the solution from the reagent reservoir to the purification chamber. In some aspects, the one or more pumps are configured to control a flow rate of the solution. In some aspects, the first column is further configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety. In some aspects, the system further comprises a third column configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety, wherein the system is further configured to flow a solution comprising the oligonucleotide comprising the 3' blocking moiety and the pyrophosphatase from the reagent reservoir to the third column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the pyrophosphatase in the third column, and (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the third column to the reagent reservoir. In some aspects, the first column comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, thefirst column comprises a resin that binds the transferase. In some aspects, the first column is an ion exchange column, an affinity column, a size exclusion column, or reverse phase column. In some aspects, the second column comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide. In some aspects, the second column comprises a resin that binds the transferase. In some aspects, the second column is an ion exchange column, an affinity column, a size exclusion column, or reverse phase column. In some aspects, the reagent reservoir comprises an impeller. In some aspects, the reagent reservoir comprises a reagent port. In some aspects, the first column or the second column are cleanable or replaceable. In some aspects, the system further comprises a reaction chamber comprising a primase. In some aspects, the primase is immobilized on a solid support. In some aspects, the reaction chamber comprising the primase comprises a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide. In some aspects, the system comprises a degassing system or a sparging system. In some aspects, the system comprises a degassing system, and the degassing system comprises a vacuum pump. In some aspects, the system comprises the sparging system. In some aspects, the sparging system is an in-line sparging system or is configured to sparge liquids in a reservoir. In some aspects, the unit for sparging system is configured to sparge using an inert gas. In some aspects, the inert gas is argon or nitrogen. In some aspects, the system comprises a degasser configured to remove oxygen from the system. In some aspects, the purification chamber is configured to concentrate the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the purification chamber comprises a membrane with a molecular weight cutoff of about 500 kDa to about 5000 kDa.

[0016] Provided herein is a composition comprising the oligonucleotide made according to the methods described herein. In some aspects, the oligonucleotide is substantially free of depurination or depyrimidination impurities. In some aspects, the oligonucleotide is substantially free of N-3- cyanoethylthymine (CNET) impurities. In some aspects, the oligonucleotide is substantially free of N(2)-actyl-2,6-diaminopurine and / or an isobutyryl diaminopurine impurities. In some aspects, the oligonucleotide is substantially free of methylcytosine.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Various aspects of the disclosed systems and methods are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed systems and methods can be obtained by reference to the detailed description of illustrative aspects and the accompanying drawings.

[0018] FIG. l shows a schematic of one cycle of template-free synthesis of an oligonucleotide, according to some aspects.

[0019] FIG. 2A shows an exemplary rotating bed reactor that may be used in a reaction chamber described herein, according to some embodiments.

[0020] FIG. 2B shows an exemplary reaction chamber, which is a flow-through column having a fixed bed reactor, in accordance with some embodiments. See Basso et al., Industrial applications of immobilized enzymes - A review, Molecular Catalysis, vol. 479, no. 110607 (2019).

[0021] FIG. 2C shows another exemplary reaction chamber, which is a flow-through column having a fluidized bed reactor, in accordance with some embodiments.

[0022] FIG. 2D shows another exemplary reaction chamber, which is a batch reaction chamber having an impeller that can stir contents of the batch reaction chamber, according to some embodiments. This type of reaction chamber may be referred to as a stirred reactor.

[0023] FIG. 3 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container / chamber and a second container / chamber that are fluidly connected, in accordance with some embodiments. The first container / chamber comprises a transferase, and the second container / chamber comprises a hydrolase.

[0024] FIG. 4 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container / chamber and a second container / chamber that are fluidly connected, and flow is regulated by a control valve, in accordance with some embodiments. The first container / chamber comprises a transferase, and the second container / chamber comprises a hydrolase.

[0025] FIG. 5 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container / chamber and a second container / chamber that are fluidly connected, and the flow is regulated by a pump, in accordance with some embodiments. The first container / chamber comprises a transferase, and the second container / chamber comprises a hydrolase.

[0026] FIG. 6 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container / chamber and a second container / chamber that are fluidly connected to each other, in accordance with some embodiments. The flow is regulated by control valves and pumps. The first container / chamber comprises a transferase, and the second container / chamber comprises a hydrolase.

[0027] FIG. 7 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container / chamber and a second container / chamber that are fluidly connected to each other, in accordance with some embodiments. The flow is regulated by control valves and pumps. The first container / chamber comprises a transferase, and the second container / chamber comprises a hydrolase. The system comprises a reaction chamber 3, which can comprise an enzyme such as another transferase.

[0028] FIG. 8 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container and a second container that are fluidly connected to each other, in accordance with some embodiments. The flow is regulated by control valves and pumps. The firstcontainer comprises a transferase, and the second container comprises a hydrolase. The system comprises a reagent reservoir that can also act as a product reservoir.

[0029] FIG. 9 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container and a second container that are fluidly connected to each other, in accordance with some embodiments. The flow is regulated by control valves and pumps. The first container comprises a transferase, and the second container comprises a hydrolase. The system comprises a reagent reservoir that can also act as a product reservoir. One or more reagent reservoirs can be connected upstream of the primary reagent reservoir.

[0030] FIG. 10 shows an exemplary system for template-free synthesis of an oligonucleotide using chambers, such as in-line reaction chambers with enzymes being retained in the reaction chambers as the synthesized oligonucleotide can flow out of the reaction chambers, in accordance with some embodiments. The oligonucleotide is transferred through the system for extension and cycling using linear flow.

[0031] FIG. 11 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as in-line columns, in accordance with some embodiments. In this particular schematic, the enzymes are immobilized on a solid support, such as a fluidized bed or a fixed bed. The oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.

[0032] FIG. 12 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as stirred reactor, in accordance with some embodiments. The oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.

[0033] FIG. 13 shows an exemplary system for template-free synthesis of an oligonucleotide using a reaction chamber such as a stirred reactor, in accordance with some embodiments. Purification chambers such as in line columns can be used for removing enzyme. The oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.

[0034] FIG. 14 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as columns, in accordance with some embodiments. Desalting chambers can be used for separating or removing salts, reagents and other small molecule byproducts from the oligonucleotide. The oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.

[0035] FIG. 15 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as columns, in accordance with some embodiments. Desalting chambers can be used for separating or removing salts, reagents and other small molecule byproducts from theoligonucleotide. The oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.

[0036] FIG. 16 shows an impurity profile of an oligonucleotide synthesized by template-free synthesis.DETAILED DESCRIPTION

[0037] The present disclosure relates generally to methods of template-free synthesis of an oligonucleotide, as well as systems related thereto. The systems can be embodied by any of the system designs and / or combination of the system designs as described herein. More specifically, the present disclosure relates to the discovery of systems and methods that enable template-free synthesis, and especially commercial scale template-free synthesis, of oligonucleotides in an efficient and economical manner. Also provided herein are methods that use these systems described herein.

[0038] Oligonucleotides were previously known to be synthesized in a manner that requires a large, upfront investment in reagents, such organic solvents, to drive the large-scale production at the expense of producing large amounts of chemical waste is also costly to dispose. Additionally, current enzymatic and or chemical methods for oligonucleotide synthesis do not guarantee commercially viable yields. Current methods produce oligonucleotides at lower scales because the process of elongation and extension compromise product quality through degradation, or product yield is reduced because it must be recovered from contaminants and other byproducts that are generated from the synthesis process. Additionally, current synthesis techniques that aim to increase purity, increase oligonucleotide length and / or reduce incorporation error or degradation require manual intervention at the completion of each cycle, and thus are optimized to be operated under batch production conditions, where reagents are discarded due to contaminating solvents or otherwise rendered inoperable for further use.

[0039] For enzymatic synthesis of oligonucleotides, one such reagent that is limited in quantity and cost prohibitive is enzyme. The quantity of enzyme required using existing systems and methods for template independent enzymatic oligonucleotide synthesis would be cost prohibitive at a commercial scale. The inventors have discovered systems and methods that retain the enzyme in their reaction chamber, while the oligonucleotide (e.g., the substrate oligonucleotide) is flowed in solution between chambers and reservoirs. Retaining enzyme in the reaction chamber permits for re-use, decreasing the total quantity of enzyme required for synthesis of oligonucleotide of a predetermined sequence.

[0040] The oligonucleotides that can be synthesized using these systems and methods are of a predetermined sequence can be made with high target yields and accuracy in solution (e.g., under flow manufacturing conditions), requiring little user intervention between iterative extension cycles and enhancing reagent efficiency, therefore making the oligonucleotides commercially viable as a reagent or active pharmaceutical ingredient. These systems and methods also reduce reagent use and cost andremove the barriers that inhibit large scale oligonucleotide synthesis. Moreover, the inventors have surprisingly discovered that systems and methods permit large scale production of pre-determined oligonucleotides lengths that are longer compared to what is presently achievable by cunent synthesis techniques in an economic manner.

[0041] As such, the systems and methods disclosed herein offer a substantial improvement over enzyme mediated oligonucleotide synthesis and chemical methods of oligonucleotide synthesis.

[0042] Disclosed herein are methods that provide for controlled elongation of an oligonucleotide in solution is achieved using a nucleotide comprising a 3' blocking moiety and a transferase, yielding an elongated oligonucleotide comprising a 3' blocking moiety. The 3' blocking moiety prevents additional addition of nucleotides because it sterically hinders further elongation of the oligonucleotide chain by the transferase. The oligonucleotide comprising the 3' is retained in solution, then is then separated from the enzyme, and deblocked by removing a 3' blocking moiety to produce an elongated oligonucleotide. The deblocking is also conducted with the oligonucleotide in solution. The methods described herein describe a method of template-free synthesis of an oligonucleotide, where the oligonucleotide remains in solution (e.g., the liquid phase). As such, the discovery of the systems and the methods that use the systems as described herein enable efficient and economical template-free synthesis of an oligonucleotide through batch or flow production methods.

[0043] Disclosed herein are also systems and methods that provide for elongating the oligonucleotide in a first reaction chamber, separating the elongated oligonucleotide comprising the 3' blocking moiety by flowing it to a second reaction chamber, and removing the 3' blocking moiety in a second reaction chamber. Separating the elongated oligonucleotide comprising the 3' blocking moiety is achieved by retaining transferase in the first reaction chamber. After removal of the 3' blocking moiety from the elongated oligonucleotide, the elongated oligonucleotide is flowed from the second reaction chamber back to the first reaction chamber to re-initiate the cycle of elongation and 3' blocking moiety removal (e.g., an exemplary cycle as in FIG. 1) to further elongate the oligonucleotide by another nucleotide. Because the nucleic acid (e.g., oligonucleotide, elongated oligonucleotide comprising a 3' blocking moiety, elongated oligonucleotide) remains in solution, the nucleic acid can be moved through flowing the solution through any of the systems as described herein to perform the any of the methods of template-free synthesis disclosed herein. In some aspects, the systems and methods described herein comprise at least a third reaction chamber, wherein the elongated oligonucleotide can be further elongated by a second transferase to produce a further elongated oligonucleotide comprising the 3' blocking moiety of the second nucleotide.

[0044] Disclosed herein are also methods and systems that can be used for methods of template-free synthesis of an oligonucleotide by elongating an oligonucleotide in solution using a transferase in solution, then separating the elongated oligonucleotide from the transferase, and removing the 3'blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety.

[0045] Disclosed herein are also systems and methods for template-free synthesis of an oligonucleotide comprising a first reaction chamber that comprises a transferase, and a second reaction chamber that comprises a hydrolase. These reaction chambers are fluidly connected such that an oligonucleotide (such as an oligonucleotide in solution) can be flowed between the chambers. In the first chamber, an oligonucleotide can be elongated by a transferase, which attaches a 3' blocking moiety to the oligonucleotide, producing an elongated oligonucleotide comprising a 3' blocking moiety. The elongated oligonucleotide comprising a 3' blocking moiety is then flowed to a second reaction chamber, while the transferase is retained in the first chamber. The elongated oligonucleotide comprising a 3' blocking moiety enters the second chamber, which comprises a hydrolase. The hydrolase removes the 3' blocking moiety from the elongated oligonucleotide comprising a 3' blocking moiety, producing an elongated oligonucleotide. The elongated oligonucleotide can then be flowed from the second chamber back to the first chamber, where it can be further elongated by the transferase that was retained in the first chamber. This cycle can be iterated any number of times with the addition of a predetermined nucleotide comprising a 3' blocking moiety to achieve an oligonucleotide of a predetermined sequence.

[0046] Disclosed herein are also systems that further comprise additional modules that such as reservoirs. In some aspects, the reservoir is a reagent reservoir that holds additional reagents necessary for the methods described herein, such as buffers, cofactors, and predetermined nucleotides comprising a 3' blocking moiety. In some aspects, the reservoir is a product reservoir that holds or collects product (e.g., an elongated oligonucleotide) after the 3' moiety is removed from the elongated oligonucleotide comprising a 3' moiety. In some aspects, the product reservoir is fluidly connected to the first reaction chamber or the reagent reservoir. In some aspects, the reagent reservoir also acts as a product reservoir.

[0047] Disclosed herein are systems that are configured to separate the transferase from the oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety. The system is further configured with a reagent reservoir, wherein the solution comprising the oligonucleotides are flowed into said reagent reservoir to substantially separate the nucleic acid (e.g., an elongated oligonucleotide comprising a 3' blocking moiety or an elongated oligonucleotide) from the enzyme (e.g., a hydrolase or a transferase) prior to being flowed to the next reaction chamber.

[0048] Disclosed herein are also systems that further comprise additional modules that control the flow or regulate system conditions. In some aspects, the systems comprise one or more reaction pumps. In some aspects, the systems comprise one or more valves that selectively control a pathwayof the solution in the system. These optional features may be incorporated into the systems described herein to permit enzymatic synthesis.

[0049] These systems as described herein can be automated or configured to flow the substrate oligonucleotide in solution between chambers while retaining the enzymes (e.g., the hydrolase or transferase) in their respective reaction chambers such that they can be reused in the next iterative cycle of oligonucleotide elongation and removal of the 3' blocking moiety from the elongated oligonucleotide. The systems and methods described herein are also configured retain and enable the re-use of the hydrolase, transferase, and / or pyrophosphatase in the downstream cycles of template- free synthesis of the oligonucleotide. In certain aspects, the systems described herein can be automated or partially automated. In certain aspects, these systems can be embodied by the systems that are described in further detail herein. In certain aspects, these systems described herein can further comprise optional features such as one or more temperature regulators, thermometers, valves, pumps, degassers (such as vacuum pumps), spargers (e.g., an in-line sparger and / or a sparger in a reservoir), and flow regulators.Definitions

[0050] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0051] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0052] It is to be understood that where descriptions of various aspects use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.” It is to be further understood that where descriptions of various aspects use the term “optional” or “optionally” the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. It is to be understood that both the foregoing general description, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.

[0053] “Substantially separated” as used herein refers to dividing, excluding, partitioning, or removing 95% or more of a macromolecular species by percent weight. For example, an enzyme (e.g., transferase, hydrolase, pyrophosphatase, etc.) is considered substantially separated from an oligonucleotide if not more than 5% of the starting quantity is included in the solution after the enzyme has been substantially separated from the oligonucleotide (such as an elongated oligonucleotide comprising the 3' blocking moiety).

[0054] “Substantially free” as used herein refers to a composition that contains less than 5% or less of the identified component (e.g., impurity) by percent weight. For example, a composition that is substantially free of an identified impurity contains 5% or less of that impurity.

[0055] As used herein, “nucleotide triphosphate” or “NTP” as used herein refers to a nucleoside with three 5' phosphate groups (or 5' phosphate group analogs) but does not exclude other nucleotides comprising additional phosphate (or phosphate analog) moieties. NTPs may also comprise additional modifications, such as on the 3' position of the ribose sugar (e.g., a 3' phosphate group such that the NTP comprises a 3' phosphate group), on the 2'-position of the ribose sugar (OMe, F, H, MOE), locked and glycine NTPs. NTPs may also be also substituted at any of the 5' phosphate groups to create NTP analogs. For example, a non-bridging oxygen on the a-phosphate may be substituted with a sulfur to make nucleoside 5'-(a-P-thio)triphosphates. NTPs may also be modified to remove a nucleobase to generate an abasic nucleotide.

[0056] As used herein, “nucleotide analog” or “NTP analog” refers to a modified NTP that has been chemically modified and has biosimilar properties and functionality to naturally occurring NTPs. NTP analogs are structurally similar to NTPs. For example, an analog of a NTP can comprise a nucleoside 5 '-(a-P-thio)triphosphate moiety .

[0057] As used herein, “polynucleotide,” “oligonucleotide,” and “nucleic acid’ ’ are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and / or 2' deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2' substitutions (e.g., 2'-flouro, 2'-O- methyl, 2'-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphothiolated linkages. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some aspects, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobasesthat are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase.

[0058] As used herein, “template-free synthesis” refers to synthesis of an oligonucleotide or a polynucleotide without the use of template strand as a guide for synthesis of a complementary oligo or polynucleotide strand. Thus, template-free synthesis refers to an iterative process, whereby, successive nucleotides are added to a growing oligo or nucleotide chain or oligonucleotide substrate.

[0059] “ Substrate” or “reagent” in the context of an enzyme mediated reaction refers to the molecule acted on by the enzyme (e.g., a transferase or a hydrolase). For example, a transferase used in the systems and methods disclosed herein act on a substrate (e.g., an oligonucleotide). In another example, a hydrolase used in the systems and methods disclosed herein act on a substrate (e.g., an elongated oligonucleotide comprising a 3' blocking moiety or an unreacted nucleotide such as a nucleotide comprising a 3' blocking moiety). In another example, a pyrophosphatase used in the systems and methods disclosed herein act on a substrate (e.g., inorganic pyrophosphate).

[0060] ‘ ‘Product” in the context of an enzyme mediated reaction refers to the molecule resulting from the action of the enzyme. In some aspects, the product is the target molecule. For example, an exemplary product for a transferase used in the systems and methods described herein is an elongated oligonucleotide comprising a 3' blocking moiety.

[0061] “Byproduct” in the context of an enzyme mediated reaction is a secondary product that is generated in addition to the product resulting from the action of the enzyme. For example, byproducts can include, but are not limited to, nucleosides, NDPs, residual NDPs comprising a 3' blocking moiety, NMP, residual NMPs comprising a 3' blocking moiety, phosphate, and residual pyrophosphate.

[0062] “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein.

[0063] “Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0064] As used herein, where the description refers to “flowing,” for example without limitation, flowing of reaction solution from a reaction chamber to another reaction chamber, or a reaction chamber to a reservoir, it may also described as being in “fluid communication” between the recited elements (e.g., reaction chamber, reservoir, etc.) through conduits, such as tubes or channels. For example, a reaction chamber in fluid communication with a reservoir allows for flowing of reactionsolution between the reaction chamber and the reservoir such as through a conduit. In some embodiments, the flow of solution between the elements can be regulated, such as by valves or diverters, as described herein.

[0065] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well- known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0066] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the invention as a whole.

[0067] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0068] The entire disclosure of each patent, patent application, and publication (including patent publication or non-patent publication) referred in this application are hereby incorporated herein by reference for all purposes. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control.Nucleotides and Nucleotide Analogs

[0069] The present disclosure provides systems and methods that use nucleotides and / or nucleotide analogs such as modified nucleotides. A nucleotide comprises a sugar moiety, which can be further modified at different positions. For example, modified nucleotides contemplated include nucleotideswith blocking groups on the 3' positions of the sugar (e.g., nucleotides comprising a 3' blocking moiety), as well as nucleotides with modified bases or thiol derivates for the formation of more stable oligonucleotide phosphorothioate backbone bonds. The blocking group, also known to those skilled in the art as an inhibitor or reversible terminating group, may include a variety of groups that prevent the transferase from adding additional nucleotides to the oligonucleotides. This may include charged molecules, large molecules and moieties, or other blocking groups known to those skilled in the art. Appropriate removable blocking groups may include carbonitriles, phosphates, carbonates, carbamates, esters, ethers, borates, nitrates, sugars, phosphoramidates, phenylsulfenates, and sulfates. Other 3' blocking groups are also known in the art, including 3'-0-amines and methylamines. Nucleotides comprising a 3' blocking moiety

[0070] The systems and methods provided herein can be used to elongate an oligonucleotide in solution (e.g., in a liquid) by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide, for example, by using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety. The 3' blocking moiety can then be removed from the elongated oligonucleotide comprising the 3' blocking moiety, producing an elongated oligonucleotide.

[0071] In some aspects, the nucleotide comprising a 3' blocking moiety may comprise a nucleotide triphosphates (NTP). The NTP further comprises a 3' blocking moiety (e.g., a blocking moiety on the 3' position of the sugar of the nucleotide). As used herein, a NTP does not exclude the inclusion of other phosphates (e.g., phosphates introduced in the 3' blocking moiety).

[0072] In some aspects, the nucleotide comprising a 3' blocking moiety comprises a nucleoside tetraphosphate. NTPs with a phosphate group at the 3' position of the sugar (nucleoside tetraphosphates, pppNps or otherwise known herein as “NQPs”), with or without additional modifications to the nucleobase, sugar, and / or phosphate chain, are useful for the systems and methods of template-free synthesis as provided herein. An exemplary process of converting a NTP into a NTP with a phosphate group at the 3' position of the sugar (e.g., a NQP) is depicted in Scheme 1.Scheme 1

[0073] As depicted in Scheme 1, the NTP is converted by a 3'0-kinase to a nucleoside tetraphosphate (NQP or pppNp) with the fourth phosphate group at the 3' position of the sugar, thereby generating a nucleotide comprising a 3' blocking moiety. The group R at the 2' position of the sugar (“2'-R group”) may be an atom or group selected from H, OH, OCHj, OCH2CH2OCH3, F, and CO2R’ (where R’ is any alkyl or aryl), or another atom or chemical group. Additionally, the sugar may have other modifications at other positions. The nucleobase may be a uridine, thymine, cytosine, adenine, guanine or another nucleobase known to those skilled in the art. Although not depicted in Scheme 1, the nucleotide comprising a 3' blocking moiety may also have modifications of the nucleobase or of the 5' phosphate chain.

[0074] In some aspects, the nucleotide comprising a 3' blocking moiety may comprise one or more modifications.

[0075] In some aspects, the nucleotide comprising a 3' blocking moiety may comprise ribonucleosides, deoxyribonucleosides, dideoxynucleosides, or modified nucleosides.

[0076] In some aspects, the nucleotide comprising a 3' blocking moiety may comprise one or more modifications to the sugar. In some aspects, nucleotide comprising a 3' blocking moiety may comprise one or more modifications to the nucleobase.

[0077] In some aspects, the nucleotide comprising a 3' blocking moiety has at the 2'-position of the sugar moiety a H or OH.

[0078] In some aspects, the nucleobase of the nucleotide comprising a 3' blocking moiety is adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine,6, 8 -diaminopurine, 5-methylcytosine (m5C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, or 7-methylguanosine (m7G).

[0079] In some aspects, the nucleotide comprising a 3' blocking moiety has a nitrogenous base that is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2'-position of the sugar moiety an OH. In some aspects, the nucleotide comprising a 3' blocking moiety is a ribonucleotide.

[0080] In some aspects, the nucleotide comprising a 3' blocking moiety has a nitrogenous base that is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2'-position of the sugar moiety a H. In some aspects, the nucleotide comprising a 3' blocking moiety is a deoxyribonucleotide.

[0081] In some aspects, the nucleotide comprising a 3' blocking moiety comprises a modified nucleoside. In some aspects, the nucleoside has a modified sugar moiety or modified nucleobase, or combination thereof.

[0082] In some aspects, the nucleotide comprising a 3' blocking moiety comprises a modified 2'- position of the sugar moiety (e.g., a 2' modification). In some aspects, the 2' modification is halo, 2'- O-R’, or 2'-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some aspects, R’ is a Cl-C4alkyl. In some aspects, the modified 2'-position is a 2'-O-R’, wherein in R' is alkyloxy alkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some aspects, the 2'-position of the sugar moiety of the nucleoside substrate is -O-R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3. In some aspects, the modified 2'-position is 2'-O-(2-methoxyethyl), 2'-O-ally 1, 2'-O-propargyl, 2'-O-ethylamine, 2'-O- cyanoethyl, or 2-O-acetate ester. In some aspects, the 2'-position of the sugar moiety is halo. In some aspects, the 2'-position of the sugar moiety is F (i.e., 2'-F) or Br (i.e., 2'-Br). In some aspects, the 2' modification is 2'-F. In some aspects, the 2' modification is 2'-OMe.

[0083] In some aspects, the nucleotide comprising a 3' blocking moiety may comprise an a- thiophosphate or dithiophosphate or other modification to the 5' phosphate chain. In some aspects, the nucleotide comprising a 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate. A nucleotide that comprises a nucleoside 5'-(a-P-thio)phosphate is also known as an a-phosphate modified nucleotides (e.g., dNTPaS and NTPaS) and are presently used to prepare phosphorothiolate DNAs and RNAs, respectively. A phosphorothiolate bond introduces a sulfur atom in place of a non- briding oxygen in the phosphate backbone. Oligonucleotides comprising phosphorothiolate bonds are more resistant to degradation by nucleases. The present systems and methods as disclosed herein canutilize nucleotides comprising a 3' blocking moiety, and further comprising a 5 '-(a-P-thio)phosphate to elongate an oligonucleotide.

[0084] In some aspects, nucleotide comprising a 3' blocking moiety comprises a modified nucleobase. In some aspects, the modified nucleobase of the nucleoside substrate is 5-bromo-uracil,5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

[0085] In some aspects, the nucleotide comprising a 3' blocking moiety comprises a nucleobase that is, among others, 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines, 5-alkyluridines, 5-halouridines,6-azapyrimidines, 6- alkylpyrimidines, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, quesosine, 2-thiouridine, 4-thiouridine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine,5- carboxymethylaminomethyluridine, -D-galactosylqueosine, 1 -methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6- methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethy luridine, 5-methylcarbon ylmethyluridine, 5- methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D- mannosylqueosine, uridine-5-oxy acetic acid, 2-thiocytidine, Nl-methyl-adenine, N6-methyl- adenine, 8'-azido-adenine, N,N-dimethyl-adenosine, aminoallyl -adenosine, 5 '-methyl -uridine, pseudouridine, N1 -methyl -pseudouridine, 5'-hydroxy-methyl -uridine, 2'-thio-uridine, 4’-thio- uridine, hypoxanthine, xanthine, 5'-methyl- cytidine, 5'-hydroxy-methyl-cytidine, 6’-thio- guanine, or N7-methyl-guanine.

[0086] In some aspects, the nucleotide comprising a 3' blocking moiety comprises a noncanonical nucleobase, a removable tag, a cleavable linker, or a radio, a photo, and / or a chemical sensor.

[0087] In some aspects, the nucleotide comprising a 3' blocking moiety further comprises a conjugate moiety. In some aspects, the conjugate moiety is on the nucleobase of the nucleotide. In some aspects, the nucleotide comprising a 3' blocking moiety further comprises a 2' conjugate moiety. In some aspects, the 2' conjugate moiety is a ligand. The conjugate moiety (i.e., non-nucleotide moiety) includes, among others, carbohydrates (e.g. GalNAc), lipids, sterols, drug substances, hormones, polymers (e.g., polyethylene glycol, etc.), proteins, peptides, toxins (e.g. bacterial toxins, etc.), vitamins (e.g., folate, tocopherol, retinoic acid, etc.), or combinations thereof. In some embodiments, the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and / or oligonucleotide cell targeting.

[0088] In some embodiments, the conjugate moiety can be attached to the 5’ -terminal nucleotide, the 3’ -terminal nucleotide, or in a polynucleotide or oligonucleotide an internal nucleotide. In some embodiments, the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, forexample, to the 2’ -OH. In some embodiments, the conjugate moiety is attached to the 3’ -position of the sugar moiety of the nucleoside, for example 3’ -OH. In some embodiments, the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res. 2019 Feb 20; 47(3): 1082-1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker.

[0089] In some embodiments, the conjugate moiety comprises a C6-C22 alkyl, C6-22 alkenyl, or C6-C22 alkynyl. In some embodiments, the conjugate moiety comprises a Ce-alkyl, Ck-alkyl. Ck-alkyl, Cg- alkyl, Cio-alkyl, Cn-alkyl, Ci2-alkyl, Ci3-alkyl, Cu-alkyl, Ci5-alkyl, Ci6-alkyl, Cn-alkyl, Cis-alkyl, Cig-alkyl, C2o-alkyl, C2i-alkyl, or C22-alkyl. In some embodiments, the conjugate moiety comprises a Cg alkenyl, C7 alkenyl, Ck alkenyl Cg alkenyl, C10 alkenyl, Cn-alkenyl, Ci2-alkenyl, Cn-alkenyl, C14- alkenyl, Cis-alkenyl, Ci6-alkenyl, Cn-alkenyl, Cn-alkenyl, Cig-alkenyl, C2o-alkenyl, C21 -alkenyl, or C22-alkenyl. In some embodiments, the conjugate moiety comprises a Cr, alkynyl, C7 alkynyl, Cs alkynyl, Cg alkynyl, C10 alkynyl, Cn-alkynyl, Ci2-alkynyl, Cn-alkynyl, Cn-alkynyl, Cn-alkynyl, C16- alkynyl, Cn-alkynyl, Cn-alkynyl, Cig-alkynyl, C2o-alkynyl, C2i-alkynyl, or C22-alkynyl.

[0090] In some embodiments, the conjugate moiety comprises a heteroalkyl, heteroalkenyl, or heteroalkynyL In some embodiments, the heteroalkyl, heteroalkenyl or heteroalkynyl has one or more carbon atoms replaced with a heteroatom, such as O, S, or N.

[0091] In some embodiments, the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the cycloalkyl includes, by way of example and not limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -cyclohexenyl, and cycloheptyl. In some embodiments, the heterocycloalkyl includes, among others, l-(l,2,5,6-tetrahydropyridine, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.

[0092] In some embodiments, the conjugate moiety comprises an aryl or heteroaryl moiety. In some embodiments, the aryl group includes, by way of example and not limitation, phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, anthracenyl, fluorenyl, indenyl, and azulenyl. In some embodiments, a heteroaryl group includes, among others, pyridyl, furanyl, thienyl, pynolyl, oxazolyl, oxadiazolyl, imidazolyl ihiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isoihiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, and indazolyl.

[0093] In some embodiments, the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl- heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenylheterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl- groups.

[0094] In some embodiments, the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid. In some embodiments, the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid. In some embodiments, the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans-vaccenic acid, linoleic acid, alpha-linoleic acid, gammalinoleic acid, arachidonic acid, eicosapentaenoic acid, decanoic acid, docosahexaenoic acid (DHA), and docosanoic acid (DCA) conjugate moieties (see, e.g., Kubo et al., ACS Chem. Biol., 2021, 16, 150-164; see also, W02024 / 040041; incorporated herein by reference).

[0095] In some embodiments, the conjugate moiety comprises a sterol. In some embodiments, the sterol comprises cholesterol, alpha-cholesterol, cholesterol ester (e.g., cholesteryl palmitate, etc.), cholesterol sulfate, phytosterol, cholic acid, or lithocholic acid.

[0096] In some embodiments, the conjugate moiety comprises a vitamin or vitamin derivative, including, by way of example and not limitation, folate, tocopherol, retinoic acid, vitamin D, and the like (see, e.g., US Patent No. 9789197).

[0097] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.

[0098] In some embodiments, the conjugate moiety comprises a carbohydrate, particularly a carbohydrate moiety acting as a ligand for a cellular receptor for cellular targeting of the oligonucleotide. In some embodiments, the carbohydrate moiety comprises galactose or galactose derivatives. In some embodiments, the carbohydrate moiety is attached to the nucleoside via a linker. In some embodiments, carbohydrates moiety include the following:

[0099] In some embodiments, the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety. In some embodiments, the oligonucleotide acceptor and / or nucleotide donor may be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the conjugate moiety is monovalent, divalent, trivalent or tetravalent, GalNAc.

[0100] In some embodiments, the GalNAc moiety has the following structure,

[0101] where L is a linker, and W is a heteroatom, such as O or S. In some embodiments, the W is the 2’ -OH of the sugar moiety of a nucleoside. An exemplary monovalent GalNAc moiety is

[0102] wherein the monovalent GalNAc is attached via the linker to the 2’ -position of a nucleoside, such as adenine or guanine. These conjugate moieties can be present in contiguous nucleotides in a polynucleotide or oligonucleotide (see, e.g., W02024 / 040041).

[0103] In some embodiments, the conjugate moiety is a trivalent GalNAc. Tri- valent N- acety [galactosamine conjugate moieties are described in, for example, International patent publication WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620. “Trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. An exemplary trivalent GalNAc conjugate moieties are depicted below:

[0104] In some embodiments, the conjugate moiety comprises a reporter molecule. Example of reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy- fluorescein, 6-carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy -X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxy coumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), TexasRed, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy- 3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), IRDyes (e.g., IRD40, IRD 700, IRD 800). (See, e.g., “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., R.P. Haugland, 2002, Molecular Probes, Inc., Eugene, Oregon).

[0105] In some embodiments, the reporter moiety is a chemiluminescent moiety, for example acridinium esters, ruthenium derivatives (e.g., tris(2,2'-bipyridyl) ruthenium), and dioxetanes.

[0106] In some embodiments, the conjugate moiety comprises an affinity or capture tag. Exemplary affinity or capture tag includes, among others, biotin, desthiobiotin, digoxigenin, 3-amino-3- deoxydigoxigenin, and a hapten (e.g., dinitrophenol, Alexa Fluor 40, Alexa Fluor 488, dansyl, Lucifer yellow, Oregon Green 488, fluorescein).

[0107] In some embodiments, the conjugate moiety comprises a peptide. In some embodiments, the peptide comprises a cellular targeting peptide and / or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide. In some embodiments, the cell penetrating peptide is attached via a linker, including a cleavable linker. Cell penetrating peptides, include among others, TAT, penetratin, MAP, transportan / TPIO, VP22, polyarginine, MPG, Pep-1, pVEC, YTA2, YTA4, M918, and CADY. In some embodiments, the conjugate moiety comprises an RGD (Arg- Gly-Asp) peptide. Sequences of some penetrating peptides are described in Copolovici et al., 2014, 8(3): 1972-1994 and some are provided below:

[0108] Other cell penetrating peptides, including those conjugated to nucleic acids, are disclosed in, among others, patent publications WO24063570, WO24044663, US2024083949, WO24026141, W023230600, WO23219933, WO23177261, WO23178327, WO23093960, WO23086342, WO23081893, WO23069332, W023070108, WO23034515, US2023248630, US2023053924, W023003380, WO23277628, WO23277575, US2022378946, WO22171972, W022162200, WO2020144233, WO22180242, WO22132520, WO22129926, WO22125673, WO22120276, WO22101193, US2023287086, US2023357334, US2023144488, and US2023048338; incorporated by reference herein. In some embodiments, the peptide can be attached using a thiol group on the 5’ - phosphate of a polynucleotide or oligonucleotide.

[0109] In some aspects, the nucleotide comprising a 3' blocking moiety further comprises a targeting moiety.

[0110] In some aspects, the nucleotide comprising a 3' blocking moiety further comprises a reactive moiety. In some aspects, the reactive moiety is on the nucleobase of the nucleotide. In some aspects, the nucleotide comprising a 3' blocking moiety further comprises a 2' reactive moiety. In some embodiments, the reactive group is attached to the nucleoside via a linker. In some embodiments, the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine. In some embodiments, the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below:

[0111] Other reactive groups used in click chemistry, particularly for nucleic acids, is described in Fantoni et ah, Chem. Rev. 2021, 121, 7122-7154, incorporated by reference herein.Linker

[0112] In some embodiments, as described above, the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker. Various linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups. Linkers can be, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene, and heteroarylalkenylene. In some embodiments, the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol. In some embodiments, the linkers have functional groups for conjugation.

[0113] In some embodiments, the linker comprises a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, 1 to 20 carbon atoms, or 1 to 14 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain is optionally replaced by -O-, -NR1-, -NR1-C(=O)-, -C(=O)-NR', or -S-, and wherein R1is hydrogen or (Ci- Cejalkyl, wherein the hydrocarbon chain, is optionally substituted with one or more (e.g. 1, 2, 3, or 4) substituents selected from (Ci-Cejalkoxy, (Cs-Cejcycloalkyl, (Ci-Crjalkanoyl, (Ci-Ce)alkanoyloxy, (Ci-Cejalkoxycarbonyl, (Ci-Cejalkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0114] In some embodiments, the L is attached to the nucleoside and / or conjugate through -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-.

[0115] In some embodiments, the linker L has the structure below:

[0116] In some embodiments, the linker comprises a substituted or unsubstituted polyethylene glycol linker. In some embodiments, the polyethylene glycol linker has the formula:

[0117] In some embodiments, n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0118] In some embodiments, the polyethylene glycol linker has the structure below:

[0119] In some embodiments, exemplary polyethylene glycol linkers have the structure below:

[0120] In some embodiments, the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety. Example of a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g., Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2ndEd., S.S. Mark ed„ 2011, Humana Press).

[0121] In some embodiments, bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra). In some embodiments, an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When an activating group is attached to an oxygen atom of a carboxylic acid, the activating group can be a group that is derivable from a known coupling reagent. Examples of such coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethylcarbonate (EDC), (denzotriazol- 1 - yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-l-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU).Modified nucleotides comprising additional moieties

[0122] In some aspects, the modified nucleotides that may be used by the methods and systems as disclosed herein may comprise targeting moieties, such as targeting moieties that have therapeuticrelevance. In some aspects, the modified nucleotides may comprise reactive and / or sterically bulky side chains. In some aspects, the nucleotides that can be used for the systems and methods as described herein may be modified as shown herein.Initiating nucleotides acceptors

[0123] The systems and methods provided herein may start from an initiating nucleotide (e.g., an initiating nucleotide acceptor) to generate oligonucleotides that can be elongated in solution (e.g., in a liquid) by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide, for example, by using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety. Depending on the transferase, it may require an initiating oligonucleotide or an initiating nucleotide to act as an acceptor. For example, terminal deoxynucleotidyl transferases (TdTs), poly(N) polymerase, and certain DNA polymerases, such as polymerase > require initiating oligonucleotides, preferably 4- 6 nucleotides in length. Primase, however, is an example of a transferase which is a specialized polymerase that can use nucleotides to generate oligonucleotides that can be further elongated using the systems and methods provided herein.

[0124] In some aspects, the initiating nucleotide is a NTP, NDP, NMP, or a nucleoside. In some aspects, the initiating nucleotide comprises a 5' blocking moiety. In some aspects, the initiating nucleotide is a NTP, NDP, NMP, or a nucleoside that comprises a 5' blocking moiety. As a nonlimiting example, a 5' blocking moiety may be used to inhibit primase from using the nucleotide acceptor as the nucleotide donor. In some aspects, the 5' blocking moiety is a 5' OH. In some aspects, the initiating nucleotide (e.g., an initiating nucleotide acceptor) is an NTP, which can further serve as a nucleotide donor.Reaction Chambers

[0125] The systems and methods provided herein comprise reaction chambers. Reaction chambers are vessels in which an enzymatic reaction occurs. More specifically, reaction chambers are vessels or containers. Reaction chambers are made of an inert material and are suited for holding solutions, liquids, and / or reagents during enzymatic reactions or processes. In some aspects, the reaction chamber comprise buffers and / or solutions that may be degassed (e.g., using an vacuum pump) and / or sparged (e.g., an in-line sparger or a reservoir that is operably linked to a sparger or tank configured to flow an inert gas through the buffer and / or solution before or during operation of the system.Exemplary inert gases include argon and nitrogen. In some aspects, the enzymatic reaction or process uses multiple reaction chambers before the enzymatic reaction or process is complete. In some aspects, the enzymatic reaction or process is completed in one reaction chamber. Examples of a reaction or process include, but are not limited to, elongating an oligonucleotide in solution using a transferase and removing the removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide using a hydrolase.

[0126] The reaction chambers contemplated herein enable, promote, maintain, and / or retain favorable conditions for controlled enzymatic reactions or processes (e.g., elongating an oligonucleotide in solution using a transferase, removing the removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety using a hydrolase).

[0127] Reaction chambers contemplated for use include, but not limited to, reaction chambers fabricated from inert or non-reactive plastics, glass, or inert or non-reactive metals. In some aspects, the reaction chambers are coated. In some aspects, the coating comprises one or more polymers. In some aspects, the coating is hydrophilic.

[0128] In some aspects, the reaction chambers are sterile. In some aspects, the reaction chambers are reusable. In some aspects, the reaction chambers are cleanable and / or replaceable. In some aspects, the reaction chambers are single-use.

[0129] The reaction chambers can be of varying sizes and volumes. In some aspects, reaction chambers are suitable for commercial scale production of oligonucleotides. In some aspects, the reaction chambers are 1 mL to 50 mL in volume. In some aspects, the reaction chambers are 50 mL to 250 mL in volume. In some aspects, the reaction chambers are 30 mL to 150 mL in volume. In some aspects, the reaction chambers are 500 mL to 10 L in volume. In some aspects, the reaction chambers are 1 L to 10 L in volume. In some aspects, the reaction chambers are about 5 mL, about 10 mL, about 15 mL, about 50 mL, about 100 mL, about 300 mL, about 500 mL, or about 1 L in volume. In some aspects, the reaction chambers are about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L in volume. In some aspects, the reaction chambers are about 1L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L in volume.

[0130] In some aspects, the reaction chamber is insulated. In some implementations, the reaction chamber is temperature regulated. The temperature regulation can be achieved by, for example, water recirculation, a thermometer, a heating element, a jacketed reservoir (e.g., a jacketed reagent reservoir such as ajacketed stir tank), a jacketed chamber (e.g., any of the chambers as described herein), or a cooling element. In some aspects, the reaction chamber is maintained at a temperature. In some aspects, the temperature of the reaction chamber is 25 °C, about 30 °C, about 40 °C, about 50 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, or about 95 °C. In some aspects, the temperature of the reaction chamber is about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C. In some aspects, the temperature of the reaction chamber is 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C. In some aspects, the temperature of the reaction chamber is 40 °C. In some aspects, the first reaction chamber comprising the transferase is about 30°C to about 50 °C (such as about 40 °C). In some aspects, the temperature of the reaction chamber is 50 °C. In some aspects, the temperature of the second reaction chamber (e.g., containing the hydrolase) is about 40 °C to about 60 °C (e.g., about 50 °C).

[0131] In some aspects, the reaction chambers further comprise an apparatus that can be used to stir or mix the solution. In some aspects, the apparatus is an impeller. In some aspects, the apparatus comprises a magnetic stir bar and a stir plate. In some aspects, apparatus is a rod, such as a glass rod.

[0132] In some aspects, the reaction chambers are vessels that can be adapted for flow manufacturing.

[0133] In some aspects, the reaction chambers are batch reaction chambers (any suitable for batch reaction) . Non-limiting examples of the reaction chambers are beakers, drum, conical tubes, microfuge tubes, Erlenmeyer flasks, welled plates, and culture flasks. The reaction chambers can be round bottom, flat bottom, baffled bottom, or conical bottom vessels. Reaction chambers may be cylindrical, spherical, or flask shaped. The vessels typically associated with batch processing can be applied to flow manufacturing methods, for example, by using adapters such as rubber stoppers with tubing. A non-limiting example for using a batch vessel for the systems and processes described herein is adapting an Erlenmeyer flask using a rubber stopper and tubing to form an inlet and an outlet.

[0134] In some aspects, the batch reaction chamber comprises an apparatus for stirring or mixing the solution. Stirring or mixing ensures even distribution of reagents and maintenance of homogeneous conditions throughout the solution. Stirring or mixing is useful for maintaining proper reaction conditions such as by enhancing heat transfer (e.g., preventing heat build up in certain areas of thereaction chamber), promoting buffer and phase homogeneity and uniform distribution of reactants. These are all factors that influence enzyme reaction kinetics and product yield. In some aspects, the apparatus for stirring or mixing is an impeller. In some aspects, the batch reaction chamber comprises one or more impellers. Impellers are rotating components that can be used to mix the solution in a batch reaction chamber. An impeller is a mechanical agitator and can have different geometries. In some aspects, the impeller is a jet impeller, a blade impeller, a propeller, a paddle or a turbine impeller. In some aspects, the apparatus for stirring or mixing the solution is a rotator or a shaker. A rotator or shaker physically rotates or rocks the batch vessel to create movement in the solution. In some aspects, the apparatus for stirring or mixing the solution is a stir bar and a stir plate. A magnetic stir bar is placed inside of batch reaction chamber and placed on top of a magnetic stir plate, which uses a rotating magnetic field to cause the stir bar to spin or rotate at a set speed.

[0135] Other reaction chambers contemplated herein include rotating bed reactors (such a rotating bed reactor as shown in FIG. 2A). Rotating bed reactors are hollow cylinders with a basket inside that is packed with a solid phase or material, such as a packed bed. The basket is immersed in a solution (e.g., a liquid phase or a fluid phase) and spun at a rate to create a flow throughout the basket. Rotating bed reactors are effective for maintaining homogeneous reaction conditions and confer properties such as efficient heat transfer. In some aspects, the packed bed comprises immobilized enzyme on a solid support, such as any of the enzymes described herein. In some aspects, the immobilized enzyme is a transferase or a hydrolase. In some aspects, the immobilized enzyme is a pyrophosphatase. In some aspects, the immobilized enzyme is a transferase, and the transferase is fused to a pyrophosphatase. In some aspects, the packed bed comprises two or more immobilized enzymes. In some aspects, the two or more immobilized enzymes are at least one transferase and at least one pyrophosphatase.

[0136] Other reaction chambers contemplated for use in the systems and methods disclosed herein include columns (such as those shown in FIG. 2B-2D). Columns are typically manufactured from a rigid, inert material. Examples include glass columns (e.g., chromatography columns) or columns made of inert plastic. Columns useful for the systems and methods described herein include, but are not limited to, columns from 5-500 cm in length and 0.5 to 10 cm in width. Columns may be capped and operably linked such that they are fluidly connected. The columns may be packed with resin or beads to create a bed. In some aspects, the resin or beads are coupled to enzyme. In some aspects, the enzyme is a transferase. In some aspects, the enzyme is hydrolase. In some aspects, the enzyme is a pyrophosphatase. In some aspects, the enzyme is a transferase fusion, such as a transferase fused to a pyrophosphatase. In some aspects, the enzyme is a transferase-pyrophosphatase fusion. Solutions can migrate through the bed packed in the column via gravity flow or be flowed through using an apparatus that controls flow rate, such as a peristaltic pump.

[0137] In some aspects, the column comprises a bed that can be a fixed bed (e.g., a packed bed) as shown in FIG. 2B.

[0138] In some aspects, the bed can be fluidized as shown in FIG. 2C. A fluidized bed is when particulates or solids are suspended in solution such that the bed has fluid-like properties.

[0139] In some aspects, the column does not comprise a bed or solid support. In some aspects, the column is used as a vessel to hold the solution. In some aspects, the column comprises free enzyme, such as free transferase (e.g., transferase in solution) or free hydrolase (e.g., hydrolase in solution). In some aspects, the enzyme is retained in the column by a porous medium such as a membrane or a filter. In some aspects, the pores of the membrane or filter comprise pores of a determined diameter. In some aspects, the pores of the membrane or filter have a specific molecular weight cut off (MWCO) value. MWCO permits molecules below a specific size to pass through but retains molecules larger than the cut off size. In some aspects, the column comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the column comprises a membrane that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the column comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.

[0140] In some aspects, the systems and methods disclosed herein comprise a reaction chamber comprising a transferase, and another reaction chamber comprising a hydrolase. In some aspects, the reaction chambers are batch reaction chambers that optionally include an impeller. In some aspects, one or more of the reaction chambers comprise a rotating bed reactor. In some aspects, the rotating bed reactor is packed with a solid support. In some aspects, the transferase is immobilized on a solid support. In some aspects, the hydrolase is immobilized on a solid support. In some aspects, the reaction chambers are columns, such as fixed bed reactors (see FIG. 2B) or fluidized bed reactors (see FIG. 2C). In some aspects, the reaction chambers are stirred reactors and comprise at least one impeller (see, for example, FIG. 2D). The reaction chambers can be cleanable or replaceable. In some aspects, the reaction chambers comprises an inlet and an outlet.

[0141] In some aspects, the reaction chambers are fluidly connected by one or more conduits. A conduit, such as a conduit that fluidly connects two reaction chambers, may be used to operably link two reaction chambers. In some implementations, the one or more conduits operably links two or more reaction chambers. Non-limiting examples of conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone. In some aspects, the conduit may be rigid, semi-rigid, or flexible. In some implementations, a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings. In some aspects, a conduit may be monolithic (e.g., a single, integrated piece). In some aspects, the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the secondreaction chamber. In some aspects, the conduit is capable of bi-directional flow. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.

[0142] In some aspects, the systems and methods described herein further comprise at least one or more reaction chambers in addition to the first reaction chamber comprising a transferase and the second reaction chamber comprising a hydrolase. Addition reaction chambers (e.g., one or more reaction chambers) comprise enzymes useful for the systems and methods disclosed herein. Additional enzymes include primases, additional transferases, single stranded RNA ligases, or pyrophosphatases. In some aspects, the one or more additional reaction chambers may comprise one or more enzymes. In some aspects, the one or more additional reaction chambers comprise a transferase and a pyrophosphatase.

[0143] In some aspects, the systems and methods described herein further comprise at least a third reaction chamber, at least a fourth reaction chamber, at least a fifth reaction chamber, a least a sixth reaction chamber, at least a seventh reaction chamber, comprising another enzyme, such as a transferase, a single stranded RNA ligase, a primase, or a pyrophosphatase.

[0144] In some aspects, the one or more reaction chamber comprises a second transferase. It is contemplated that some transferase variants will exhibit greater efficiency when conjugating certain nucleotides comprising a 3' blocking moiety. As such, multiple transferases may be used to conjugate different nucleotides comprising a 3' blocking moiety, depending on the pre-selected nucleotide. The one or more reaction chambers can be fluidly connected to the other reaction chambers, and the flow path is selected such that the reaction chamber comprising the second transferase is utilized for elongating the oligonucleotide when appropriate.

[0145] In some aspects, the one or more reaction chambers comprises a pyrophosphatase. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. Inorganic phosphate is generated as a byproduct when the transferase conjugates the nucleotide to the oligonucleotide. Inorganic phosphate can inhibit enzymes such as hydrolases used for removing the 3' blocking moiety. Thus, treating the solution comprising the oligonucleotide to break down inorganic phosphate can be used to maintain efficient reaction kinetics. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the inorganic pyrophosphatase is fused to the transferase. The one or more reaction chambers comprising the pyrophosphatase can be fluidly connected to the other reaction chambers, and the flow path is selected such that the reaction chamber comprising the pyrophosphatase is utilized after the 3' blocking moiety is removed from the elongated oligonucleotide comprising a 3' blocking moiety.

[0146] In some aspects, the one or more reaction chamber comprises a primase. Primase is a polymerase that is capable of synthesizing oligonucleotides from single nucleotides or elongating short oligonucleotides. Primase is also more efficient than some hydrolases in elongating oligonucleotides shorter than 10 nucleotides in length. Thus, adding primase in a reaction chamber is useful for synthesizing and / or extending oligonucleotides productive for transferase extension. In some aspects, the one or more reaction chambers comprising the primase can be fluidly connected to the other reaction chambers, and the flow path is selected such that a reaction chamber (e.g., one of the one or more reaction chambers) comprising the primase is utilized to produce an oligonucleotide that can be elongated by the transferase in the first chamber.

[0147] In some aspects, the one or more reaction chamber comprises a single stranded RNA ligase (ssRNA ligase). ssRNA ligase is an enzyme that can ligate a nucleotide donor to an oligonucleotide acceptor, or ligate an oligonucleotide donor to an oligonucleotide acceptor. Elongated nucleotides produced by the systems and methods described herein may be ligated together to produce a longer oligonucleotide. Thus in some aspects, the one or more reaction chambers comprising the ssRNA ligase can be fluidly connected to the other reaction chambers in the system, and the flow path is selected such that a reaction chamber (e.g., one of the one or more reaction chambers) comprising the ssRNA ligase is utilized after an oligonucleotide is elongated by the systems and methods as disclosed herein. In some aspects, one or more oligonucleotides are introduced into the reaction chamber comprising the ssRNA ligase. In some aspects, a nucleotide donor (e.g., a single nucleotide donor or an oligonucleotide donor) is introduced into the reaction chamber comprising the ssRNA ligase along with an oligonucleotide acceptor.

[0148] In some aspects, any of the reaction chambers described herein are fluidly connected by one or more conduits. In some aspects, the first reaction chamber and the second reaction chamber are fluidly connected by one or more conduits. In some aspects, the third reaction chamber is fluidly connected to the first reaction chamber and the second reaction chamber by one or more conduits. A conduit, such as a conduit that fluidly connects two or more reaction chambers, may be used to operably link two or more reaction chambers. In some implementations, the one or more conduits operably links two or more reaction chambers. Non-limiting examples of conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone. In some aspects, the conduit may be rigid, semi-rigid, or flexible. In some implementations, a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings. In some aspects, a conduit may be monolithic (e.g., a single, integrated piece). In some aspects, the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber. In some aspects, the conduit is capable of bidirectional flow. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and asecond set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.Enzymes

[0149] The enzymes for the process and methods described herein can use wild-type enzymes, engineered enzymes, and combinations of wild- type enzymes and engineered enzymes. Various combinations of such enzymes can be applied to the methods and processes as appropriate where such enzymes are used. In some aspects, any of the enzymes described herein may be modified (e.g., comprise amino acid mutations relative to the wild-type protein) to enhance activity, enhance binding, alter activity (such as, but not limited to activity under different conditions and in the presence of various co-factors) to catalyze a reaction using modified nucleotides, increase product yield, increase protein expression, increase thermoactivity, increase thermostability, increase stability, increase substrate specificity and / or affinity, increase substrate range, increase specific activity, increase resistance to substrate and / or end-product inhibition, increase chemical stability, improve solvent stability, increase solubility, and increase inhibitor resistance or tolerance.

[0150] In some aspects, it is further contemplated that any of the systems or methods using the primase, transferase, single stranded RNA ligase, hydrolase and / or pyrophosphatase can be carried out by retaining the transferase, hydrolase and / or pyrophosphatase in a chamber (e.g., a reaction chamber). Retaining the enzyme can be accomplished through binding or immobilizing the primase, transferase, single stranded RNA ligase, hydrolase and / or pyrophosphatase on a substrate, such as a solid support, a filter (e.g., a porous filter), a porous substrate, a membrane (e.g., a porous membrane), or particles (e.g., beads or resin). Retaining the primase, transferase, single stranded RNA ligase, hydrolase and / or pyrophosphatase permits recycling and reuse of the primase, transferase, single stranded RNA ligase, hydrolase and / or the pyrophosphatase in each iterative cycle until a predetermined nucleotide sequence is achieved. Retaining the enzyme confers additional cost savings because the enzyme is not used once then discarded. An example of a cycle (e.g., one round of elongating and deblocking the oligonucleotide) is depicted in FIG. 1.

[0151] In some aspects, the polypeptide can be entrapped in matrixes or membranes. In some aspects, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, and collagen. In some aspects, the solid matrices, includes, among others, activated carbon, porous ceramic, and diatomaceous earth. In some aspects, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0152] In some aspects, the enzymes described herein (such as the enzymes described below) can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, immobilization on a support medium, orfusion to another protein. In some aspects, the enzyme / polypeptide comprises an affinity tag. In some aspects, the affinity tag is located at the N-terminus or the C-terminus of the enzyme. In some aspects, the enzyme is fused to a polylysine, for example, for conjugation to a support medium via the amino group of the polylysine. In some aspects, the polylysine is from 2-10 lysine units in length.

[0153] An affinity tag can be used as an adapter to conjugate the enzyme to a solid support (e.g., a resin) that comprises the binding moiety that binds and captures the affinity tag. In some aspects, the affinity tag is a polypeptide tag. Non-limiting examples of a polypeptide tag include a histidine tag (such as a glycine -histidine, penta-histidine or a hexa-histidine tag), a GST tag, a streptavidin tag, a SUMO tag, a MBP tag, a GFP tag, or an epitope tag (such as c-Myc, Flag tag (or iterations thereof), V5, or hemagglutinin (HA)). In some embodiments, the fusion is selected or designed to preserve the activity of the enzyme.

[0154] In some aspects, the affinity tag is recognized by a resin and is used to capture, to purify, to immobilize, and / or to enrich the enzyme by using the tag to immobilize the enzyme to the resin or solid support.

[0155] In some aspects, the enzyme / polypeptide is immobilized on the surface of a support material. In some aspects, the polypeptide is adsorbed on the support material. In some aspects, the enzyme is immobilized on the support material by a covalent, electrostatic, or ionic bond. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE- cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.

[0156] In some aspects, solid supports useful for immobilizing the enzyme / polypeptide in the present disclosure, include beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate with octadecyl functional groups. Exemplary solid supports useful for immobilizing the enzyme / polypeptide include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120).

[0157] Methods of enzyme immobilization are known in the art. The engineered polypeptides can be bound non-covalently or covalently. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are known in the art (see e.g., Yi et al., Proc. Biochem., 2007, 42(5): 895-898; Martin et aL, Appl. Microbiol. BiotechnoL, 2007, 76(4): 843-85; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 2010, 63:39-44; Truppo et al., Org.Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques, 2nd Ed., Academic Press, Cambridge, MA (2008); Mateo et ah, BiotechnoL Prog., 2002,18(3):629-34; and “Bioconjugation Protocols: Strategies and Methods,” In Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY (2004); the disclosures of each which are incorporated by reference herein).Transferase

[0158] A transferase is an enzyme that is capable of elongating an oligonucleotide by covalently ligating a nucleotide or a nucleotide analog (such as a nucleotide comprising a 3' blocking moiety) to the oligonucleotide. In some aspects, the transferase can react a nucleotide triphosphate (NTP) comprising a 3' blocking moiety, or an analog thereof comprising a 5' phosphate analog, with the oligonucleotide to elongate the oligonucleotide. In some aspects, the 5' phosphate analog is a 5'-(a-P- thio)phosphate moiety. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a ribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a deoxyribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof comprises a 2' modification. In some aspects, the 2' modification of the NTP or the analog thereof is 2'-F or 2'-OMe. In some aspects, 3' blocking moiety of the NTP or the analog thereof is a phosphate moiety.

[0159] In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). TdT is a member of the Pol X family of polymerases. Members of the diverse Pol X family are known to share certain residues, which are conserved across family members. TdT also has a high level of conservation across species for residues thought to be involved in binding divalent metal ions, ternary complex formation, and binding dNTP and DNA ligands (Dominguez et al. “DNA polymerase mu (Pol p), homologous to TdT, could act as a DNA mutator in eukaryotic cells” EMBO, 19(7), 1731- 1742 (2000).) The TdTs that are contemplate for use in the systems and methods described herein may be engineered (e.g., mutated or comprise amino acid mutations) to have improved thermostability, activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased affinity for NTP-3'-O-RBG and other natural or modified NTP substrates, increased affinity for oligo acceptor substrates, increased activity or specific activity on NTP-3'-O-RBG and other natural or modified NTP substrates, and / or increased activity or specific activity on various oligo acceptor substrates as compared to a wild-type TdT or other TdTs or template-independent polymerases known to those of skill in the art. These engineered TdTs are capable of template-independent synthesis of oligonucleotides and polynucleotides. TdTs useful in the systems and methods described herein are disclosed, for example, in PCT / US2023 / 076667, filed October 12, 2023, which is incorporated by reference herein in its entirety.

[0160] Other template-independent polymerases (e.g., template-independent transferases) (including, but not limited to polyA polymerases, polyU polymerases and terminal urildylytransferases) are also known in the art and may be used to practice the invention. Similarly, other polymerases are known tobe capable of template-independent synthesis (including but not limited to reverse transcriptases, lambda, polymerase mu, and members of the X family of DNA polymerases) many of which participate in DNA repair processes be used in the systems and methods described herein.

[0161] In some aspects, the transferase comprises a single stranded RNA ligase (ssRNA ligase).Hydrolase

[0162] A hydrolase is an enzyme that is catalyzes bond cleavage with water. In some aspects, the hydrolase catalyzes the removal of the 3' blocking moiety from an oligonucleotide comprising a 3' blocking moiety. Hydrolases within the scope of the invention include, but are not limited to, phosphatases (e.g., alkaline phosphatase) and include, but are not limited to, enzymes acting on ester bonds. Hydrolases contemplated for the methods and systems described herein include phosphatases, such as alkaline phosphatase. Alkaline phosphatase is a dimeric metalloenzyme that hydrolyzes monophosphate esters and generates inorganic phosphate as a byproduct. Alkaline phosphatases are widely distributed enzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of phosphate monoesters, with an optimal activity at alkaline pH. In mammals, alkaline phosphatases are present in the intestine (i.e., intestinal alkaline phosphatase) and placenta (i.e., placental alkaline phosphatase). Phosphatases are widely used in molecular biological applications, for example for the removal of 5' -phosphate from polynucleotides or oligonucleotides for subsequent labeling with labeled ATP, reducing or preventing ligation of polynucleotides or oligonucleotides, and reducing susceptibility of polynucleotides or oligonucleotides to certain nucleases, e.g., 1 exonuclease. In some aspects, the hydrolase is a recombinant phosphatase. In some aspects, the recombinant phosphatase is used for cleaving phosphate monoesters or analogs thereof. In some aspects, the hydrolase is alkaline phosphatase. In some aspects, the hydrolase is a recombinant alkaline phosphatase. In some aspects, the hydrolase is a recombinant alkaline phosphatase, or a portion thereof. Alkaline phosphatases and variants thereof are disclosed, for example, in U.S. provisional application titled “Recombinant Phosphatases” filed April 16, 2024, and in PCT / US2023 / 076667, filed October 12, 2023, both of which are incorporated by reference herein in their entirety.

[0163] In some aspects, the hydrolase cleaves a phosphate monoester or analog thereof. In some aspects, the substrate phosphate monoester or analog thereof comprises a nucleotide triphosphate (NTP), a nucleotide diphosphate (NDP), nucleotide monophosphate (NMP), 3’-P-NTP, 3’-P-NDP, 3’- P-NMP, Np, NTP-D -S, NDP-D -S, NMP-D -S, 3’-P-NTP-D-S, 3’-P-NDP-f -S, 3’-P-NMP-D -S, NpS, or any combination thereof, phosphatase and a polynucleotide or oligonucleotide having a 5'- phosphate (5'-P) and / or a 3'-phosphate (3’-P). In some embodiments, the substrate phosphate monoester or analog thereof comprises a polynucleotide or oligonucleotide with a 5'-phosphate, a 3'- phosphate, a 5'-phosphorothioate (5'-S), a 3'-phosphorothioate (3'-S), or any suitable combinationthereof. In some aspects, the hydrolase can remove a 3' blocking moiety from the oligonucleotide in the solution.Primase

[0164] In some aspects, the systems and methods disclosed herein comprises the use of a primase, which conjugates a nucleotide acceptor having a 3’ -OH group and a nucleotide donor to extend the nucleotide acceptor. In some aspects, the nucleotide acceptor is an initiating nucleotide acceptor as disclosed herein. Primase (e.g., a primase-polymerase) refers to an enzyme in the class of RNA polymerases that is involved in the replication of DNA by catalyzing the synthesis of short RNA molecules from ribonucleoside triphosphates. In some aspects, a primase is a polymerase that can generate oligonucleotides de novo from an initiating nucleotide. In some aspects, the primase reacts a nucleotide acceptor having a 3 ’-OH group with a nucleotide donor. In some aspects, the nucleotide acceptor comprises a polynucleotide acceptor, an oligonucleotide acceptor, or an initiating nucleotide acceptor. In some aspects, the oligonucleotide acceptor is at least 2, 3, 4, 5, 6, 8, 9, 10 nucleotides in length. In some aspects, the polynucleotide or oligonucleotide acceptor is DNA, RNA, or a mixture of DNA and RNA. In some aspects, the 3 ’-terminal nucleotide of the oligonucleotide acceptor is a ribonucleotide. In some aspects, the 3 ’-terminal nucleotide of the oligonucleotide acceptor is a deoxyribonucleotide. In some aspects, the initiating nucleotide acceptor comprises NTP, NDP, NMP, or a nucleoside. In some aspects, the nucleotide donor comprises a blocking group or the terminating nucleotide donor comprises a 3’ -blocking group to form a 3’ -blocked extended polynucleotide or extended oligonucleotide. In some aspects, the primase extends (e.g., elongates) the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor, some aspects, the primase is a recombinant primase. In some aspects, the recombinant primase comprises a polypeptide fragment of a primase polypeptide, wherein the polypeptide fragment comprises a primase domain. In some aspects, the recombinant primase has template-independent terminal nucleotidyl transferase activity. In some aspects, the recombinant primase has nucleotidyl transferase activity at least for a nucleotide donor dATP, ddATP, ddCTP, ddGTP, 3’ -O-methyl ATP, and / or 2’ -F-ATP.

[0165] In some aspects, a primase may be used to elongate a nucleotide (e.g., a monomeric nucleotide acceptor) or an oligonucleotide. In some aspects, the oligonucleotide is a 2-mer, a 3-mer, a 4-mer, a 5-mer, a 6-mer, a 7-mer, a 8-mer, a 9-mer, or a 10-mer. In some aspects, the systems and methods disclosed herein comprise a reaction chamber comprising a primase. In some aspects, the primase is a recombinant primase.

[0166] The primase may be a recombinant primase, which may be cloned, produced, and isolated by known methods. The primase need not need a full-length primase, as active polypeptide fragments are suitable for the methods and systems described herein. The primase should include an active primes domain, and moreover should exhibit template-independent terminal nucleotidyl transferase activity.Primases and variants thereof are disclosed, for example, in U.S. provisional application titled “Recombinant Primases And Methods Of Use” filed April 16, 2024, which is incorporated by reference herein in its entirety.

[0167] In some aspects the primase is retained in a reaction chamber. In some aspects, the primase is immobilized on a solid support. In some aspects, the primase is retained by a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.Single stranded RNA ligase (ssRNA ligase)

[0168] Single stranded RNA ligases (ssRNA ligase) can ligate short oligonucleotides to form a longer oligonucleotide in an ATP dependent manner by joining the 5'-PC>4 of a nucleotide donor to the 3'-OH of a nucleotide acceptor. In some aspects, the nucleotide donor is a single nucleotide donor. In some aspects, the nucleotide donor comprises a nucleoside monophosphate. In some aspects, the nucleoside monophosphate is further modified by a 3'-blocking moiety. In some aspects, the nucleotide donor is a pNp. In some aspects, the single nucleotide donor comprises a 5 '-phosphate and a 3'-phosphate. In some aspects, the nucleotide donor is an oligonucleotide donor. In some aspects, the oligonucleotide donor comprises a 5'-phosphate and a 3'-phosphate. In some aspects, the nucleotide acceptor is an oligonucleotide. In some aspects, the oligonucleotide comprises a 5'-OH. In some aspects, the oligonucleotide comprises a 5' blocking moiety.

[0169] In some aspects, the systems and methods disclosed herein comprise a reaction chamber comprising a ssRNA ligase, such as those disclosed in U.S. provisional application titled “Methods of RNA ligase mediated oligonucleotide synthesis,” filed April 16, 2024, which is incorporated by reference herein in its entirety.

[0170] In some aspects the ssRNA ligase is retained in a reaction chamber. In some aspects, the ssRNA ligase is immobilized on a solid support. In some aspects, the ssRNA ligase is retained by a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.Pyrophosphatase

[0171] In some aspects, the present disclosure further provides auxiliary enzymes for template-free synthesis of an oligonucleotide. One such auxiliary enzyme is pyrophosphatase such as inorganic pyrophosphatase, which catalyzes the conversion of conversion of inorganic pyrophosphate (PPi) to two orthophosphate ions (Pi). In some aspects, the systems and methods disclosed herein provide for the use of IPP enzymes (e.g., recombinant IPP enzymes) with higher specific activity, reduced off- target hydrolysis and other side reactions, increased thermostability, and increased solubility compared to type I IPP enzymes.

[0172] Pyrophosphate is often released from reactions that utilize nucleoside triphosphates (NTP). Many cellular enzymes are capable of catalyzing pyrophosphate conversion; however, the activity of most of these enzymes is not specific to a pyrophosphate substrate. For example, alkaline phosphatase will fully degrade pyrophosphate but will also hydrolyze other phosphates including the 5'-phosphates of nucleotides. Inorganic pyrophosphatases (IPP enzymes or IPPases) are known to have both high activity in the conversion of pyrophosphate to orthophosphate and relatively low activity on other phosphorylated substrates including, but not limited to, NTPs and sugar phosphates. Inorganic pyrophosphate is also a known inhibitor of hydrolases such as alkaline phosphatase, and accumulation can block alkaline phosphatase activity, which interferes with the hydrolase-mediated removal of the 3' blocking moiety from the NTP. In some aspects, the systems and methods disclosed herein comprise a reaction chamber comprising an inorganic pyrophosphatase. There are three types of inorganic pyrophosphatases (Type I, Type II, Type III). Type I IPPs have been used commercially in coupled reactions to facilitate enzymatic reactions involving DNA / RNA polymerase and other reactions that produce pyrophosphate. Type I IPP enzymes generally drive the hydrolysis of pyrophosphate to release heat, the H(+)-PPases couple the energy of inorganic pyrophosphate hydrolysis to proton movement across biological membranes. However, these enzymes are not readily conducive to in vitro and industrial processes. Type II IPP enzymes have generally been characterized from bacterial and archaeal sources (see for example, Zyryanov et al. “Rates of Elementary Catalytic Steps for Different Metal Forms of the Family II Pyrophosphatase from Streptococcus gordonii.” Biochemistry 43(4): 1065-1074 (2004), Gajadeera et al. “Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.” J Struct Biol. 189(2):81- 86 (2015), Parfenyev et al. “Quaternary Structure and Metal Ion Requirement of Family II Pyrophosphatases from Bacillus subtilis, Streptococcus gordonii, and Streptococcus mutans*” J Biol Chem. ;276(27):24511-24518 (2001)). It has been suggested that Type II IPP enzymes may prefer manganese and / or cobalt in their active sites (Zyryanov et al. “Mechanism by which metal cofactors control substrate specificity in pyrophosphatase.” Biochemical Journal. 367(Pt 3):901-906 (2002)), may have higher specific activity than type I IPP enzymes, and may be more selective toward pyrophosphate and have lower activity toward other substrates, such as NTPs, compared to type I IPP enzymes (Baykov et al. “Inorganic pyrophosphatases of Family II — two decades after their discovery.” FEBS Letters 591 (20) :3225-3234 (2017)). Thus in some aspects, the systems and methods described herein include the use of Type II IPPs or Type I and Type II IPP variants thereof (such as those disclosed in U.S. provisional application titled “Uses of Type II Inorganic Pyrophosphatases, filed April 16, 2024, incorporated by reference herein in its entirety).

[0173] In some aspects, any of the enzymes described herein can be provided as a fusion protein. In some aspects, the inorganic pyrophosphatase is provided as a fusion to a transferase. In some aspects, the enzyme is fused to a variety of polypeptide sequences, such as, by way of example and notlimitation, polypeptide tags that can be used for detection and / or purification. In some aspects, the fusion polypeptide of the enzyme comprises a glycine-histidine or histidine-tag (His-tag). In some aspects, the fusion polypeptide of the enzyme comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some aspects, the fusion polypeptide of the enzyme comprises a GST, SUMO, Strep, MBP, or GFP tag. In some aspects, the fusion is to the amino (N-) terminus of engineered enzyme polypeptide. In some aspects, the fusion is to the carboxy (C-) terminus of the enzyme polypeptide.

[0174] In some aspects, the fusion polypeptide comprises a transferase and the pyrophosphatase. In some aspects, the pyrophosphatase is fused to the C terminus of the transferase. In some aspects, the transferase and the pyrophosphatase are connected by a polypeptide linker.Purification Chambers

[0175] In some aspects, the systems and methods described herein further comprise one or more purification chambers. In some aspects, the systems and methods described herein may comprise two or more different types of purification chambers, such as two or more of the types of purification chambers as described herein. Purification chambers can be used to purify the oligonucleotide from reaction byproducts such as inorganic phosphate, or remove unreacted nucleotides comprising a 3' blocking moiety from the solution. In some aspects, the purification chambers isolate and / or substantially separate the oligonucleotide from other reagents and reaction byproducts. In some aspects, a purification chamber can be used to substantially separate and / or isolate at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the total mole or percent amount of oligonucleotide from other reagents (e.g., enzymes) and reaction byproducts after the reservoirs. Purification chambers can be vessels or chambers, and are composed of an inert material. In some aspects, the purification chambers are columns. In some aspects, purification chamber comprises a column. In some aspects, the purification chamber comprises a liquid chromatography column. Liquid chromatography comprises a mobile phase and a stationary phase, and certain molecules are retained in the stationary phase based on their inherent properties while others prefer to travel with the mobile phase. Based on these properties, molecules will exit the chromatography column at different times or in different fractions. In some aspects, the purification chamber comprises a size exclusion chromatography. Size exclusion chromatography separates molecules by parameters such as molecular weight and hydrodynamic radius. In some aspects, the purification chamber comprises an ion exchange column. Ion exchange separates molecules by inherent charge parameters when the target molecule (e.g., the oligonucleotide) is in different buffers that modify whether the molecule prefers to interact more or less with the stationary phase. In some aspects, the purification chamber is a reverse phase column. Reverse phase chromatography relies on polar / nonpolar characteristics of the molecule for separation of the target molecule from the mixture. In some aspects, the purificationchamber is part of a tangential flow filtration system. A tangential flow filtration system involves using a filter to remove or separate contaminating particles from samples.

[0176] The purification chamber may be configured to remove salts, reaction byproducts and / or unreacted reagents, for example as a desalting chamber. Desalting chambers are useful for separating the oligonucleotide from buffer components such as salts, small molecules (such as small molecule byproducts), impurities and / or metals. Desalting chambers are also useful for buffer exchanging the oligonucleotide, and for concentrating the oligonucleotide into a smaller volume. Desalting chambers may be used outside of the flow system, for example by collecting the product and desalting it prior to re-introducing the oligonucleotide for further reactions or collecting the product at the end of oligonucleotide synthesis. Desalting chambers that are not coupled to the systems as described herein include spin cartridge filters. The sample is applied to the spin cartridge filter, and centrifugal force is applied to the unit to draw the permeate through the filter. The filter comprises pores of a specified molecular weight cut off, such that any of the components of the sample that are larger than the size of the cut off are significantly more retained in the retentate, and components of the sample that are smaller than the size of the cut off are flowed through with the permeate. Desalting chambers may also be used as a part of the flow system, such as a tangential flow system or a cross flow filtration system. Filtration techniques, such as ultrafiltration, may be used, and the cartridges or conditions (for example, the flow rate to maintain appropriate transmembrane pressure, or the membrane surface required for appropriate permeate flux) are compound (e.g., product) dependent. The oligonucleotide (such as an oligonucleotide of about 9 or about 10 nucleotides in length or more) is retained with the retentate, but the permeate is flowed to the waste or discarded. In some aspects, the oligonucleotide is desalted using a desalting chamber as needed. In some aspects, the oligonucleotide is desalted at defined intervals and / or at defined cycle numbers, is In some aspects, the desalting chamber is a vessel, e.g., a column that is packed with resin for desalting and buffer exchange, such as G25 resin. In some aspects, the desalting chamber is a cartridge with a membrane of a designated molecular weight cut off. In some aspects, desalting chamber is a tangential flow device or a ultrafiltration and / or diafiltration device (e.g., cassette), such as those used in ultrafiltration / diafiltration (UF / DF). Use of ultrafiltration / diafiltration methods (e.g., use of ultrafiltration, diafiltration cassettes) also concentrates the oligonucleotides while substantially separating it from salts, reaction byproducts and / or unreacted reagents. In some aspects, the cassette comprises pores with a molecular weight cut off of 1 kilodaltons. In some aspects, the cassette comprises pores with a molecular weight cut off 3 kilodaltons. In some aspects, the desalting chamber comprises a membrane. In some aspects, the membrane is a polyethersulfone (PES) membrane. In some aspects, the temperature of the desalting chamber is 25 °C. In some aspects, the desalting chamber is cleanable. In some aspects, the desalting chamber is cleaned in place. In some aspects, the desalting chamber is reusable. In some aspects, the desalting chamber is replaceable. In some aspects, the desalting chamber is disposable.Enzyme capture chambers

[0177] In some aspects, the systems and methods described herein further comprise one or more enzyme capture chambers. Enzyme capture chambers can be used to further separate trace levels of enzymes that were not retained from the reaction reservoirs. Trace amounts of the enzymes disclosed herein may not be retained by the reaction chambers. For example, the transferase or the hydrolase may be immobilized to a solid support, but the enzyme or its active subunits may decouple during the cycle run. Thus, the enzymes described herein may be further modified with an affinity tag or another purification tag to capture any free enzyme in solution. A chamber, such as an in-line column, may be placed at the outlet of the reaction chamber (e.g., downstream of the reaction chamber) to bind any enzyme that is flowed in solution with the oligonucleotide. In some aspects, the chamber comprises a solid support that captures the enzyme and allows passage of the oligonucleotide. The resin may be, for example, an ion exchange resin or an affinity resin that can capture the enzyme. For example, the enzyme may include a His-tag and the resin may be nickel, cobalt, or IMAC resin. In some aspects, the enzyme capture chambers to further retain, separate, or exclude the enzyme from the solution comprising the oligonucleotide. In some aspects, the enzyme capture chambers further remove, separate and / or isolate the enzyme such that at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% of the enzyme is retained or removed from the solution comprising the oligonucleotide.Reservoirs

[0178] In some aspects, the systems and methods described herein further comprise one or more reagent reservoirs. Reagent reservoirs are used to hold solutions, buffers, or mixtures that provide essential reagents for template-free oligonucleotide synthesis. Reagents include co-factors, buffers, and nucleotides comprising 3' blocking moiety. In some aspects, the reagent reservoir comprises an impeller, such as an impeller as described herein. In some aspects, the reagent reservoir is replaceable or cleanable. The reagent reservoir may comprise an outlet. In some aspects, the reagent reservoir comprises an inlet and an outlet. In some aspects the reagent reservoir is fluidly connected to any of the reaction chambers or product reservoirs as described herein. In some aspects, the reagent reservoir is insulated. In some aspects, the reagent reservoir is temperature regulated. The temperature regulation can be achieved by, for example, a jacketed reservoir (e.g., a jacketed reagent reservoir such as a jacketed stir tank), or a cooling element. In some aspects, the reagent reservoir is maintained at a temperature. In some aspects, the temperature of the reagent reservoir is about 2 °C to 8 °C. In some aspects, the temperature of the reagent reservoir is about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, or about 8 °C. In some aspects, the temperature of the reagent reservoir is 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C. In some aspects, the temperature of the reagent reservoir is 2 °C. In some aspects, the temperature of the reagent reservoir is 3 °C. In some aspects, the temperature of the reagent reservoir is 4 °C. In some aspects, the temperature of the reagent reservoiris 5 °C. In some aspects, the temperature of the reagent reservoir is 6 °C. In some aspects, the temperature of the reagent reservoir is 7 °C. In some aspects, the temperature of the reagent reservoir is 8 °C.

[0179] In some aspects, the systems and methods described herein further comprise one or more product reservoirs. In some aspects, the product reservoir holds the product and any byproducts that are produced from the enzymatic reaction. In some aspects, the product reservoir comprises an inlet. In some aspects, the product reservoir comprising an inlet and an outlet. In some aspects, the system comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the reagent reservoir. In some aspects, the product reservoir comprises an impeller. In some aspects, the product reservoir is replaceable or cleanable. In some aspects, the product reservoir is insulated. In some aspects, the product reservoir is temperature regulated. The temperature regulation can be achieved by, for example, jacketed reservoir (e.g., a jacketed product reservoir such as a jacketed stir tank), or a cooling element. In some aspects, the product reservoir is maintained at a temperature. In some aspects, the temperature of the product reservoir is about 2 °C to 8 °C. In some aspects, the temperature of the product reservoir is about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, or about 8 °C. In some aspects, the temperature of the product reservoir is 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C. In some aspects, the temperature of the product reservoir is 2 °C. In some aspects, the temperature of the product reservoir is 3 °C. In some aspects, the temperature of the product reservoir is 4 °C. In some aspects, the temperature of the product reservoir is 5 °C. In some aspects, the temperature of the product reservoir is 6 °C. In some aspects, the temperature of the product reservoir is 7 °C. In some aspects, the temperature of the product reservoir is 8 °C.Systems for Template-Free Synthesis of an Oligonucleotide

[0180] The present disclosure describes systems for template-free synthesis of an oligonucleotide. Also described herein are systems for performing the methods described herein. In some aspects, the system comprises a first reaction chamber and a second reaction chamber. In some aspects, the system includes one or more reaction chambers. In some aspects, the system comprises two or more reaction chambers. In some aspects, the system comprises three or more reaction chambers. In some aspects, the system comprises four or more reaction chambers. In some aspects, the system further comprises any of the reagent reservoirs, product reservoirs, and purification chambers as described herein. In some aspects, the systems described herein includes mechanisms for limiting oxidation, such as by limiting or removing oxygen from the system. Mechanisms for preventing oxidation include the use of inert gasses (such as argon or nitrogen) for inerting, purging, or sparging. In some aspects, the systems described herein may be operated in an inert atmosphere or under inert atmospheric conditions.

[0181] In some aspects, provided herein is system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase: wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber. In some aspects, the first reaction chamber comprises a transferase. In some aspects, the transferase is a TdT. In some aspects, the transferase is a member of the Pol X family. In some aspects, the first reaction chamber further comprises a pyrophosphatase. In some aspects, the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the first reaction chamber comprises a transferase-pyrophosphatase fusion. In some aspects, the second reaction chamber comprises a hydrolase. In some aspects, the hydrolase is a phosphatase. In some aspects, the phosphatase is an alkaline phosphatase.

[0182] In some aspects, the systems described herein may be operated in parallel. For example, multiple and identical reaction chambers may be operated in parallel (e.g., simultaneously operated). In some aspects, the systems described herein may be operated serially.

[0183] In some aspects, the first reaction chamber or the second reaction chamber is a column. In some aspects, the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the fluidized bed further comprises a pyrophosphatase, such as an inorganic pyrophosphatase. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allow s passage of the oligonucleotide. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and the pyrophosphatase and allows passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide.

[0184] In some aspects, the first reaction chamber or the second reaction chamber is a batch reaction chamber. In some aspects, the batch reaction chamber comprises an impeller. In some aspects, the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support. In some aspects, wherein the second reaction chamber comprises a rotating bed reactor comprising the hydrolase immobilized on a solid support.

[0185] The system can further comprise a temperature regulator. The use of varied temperatures may be beneficial, for example, to optimize enzyme activity or promote substrate / product stability. Temperature can also impact liquid viscosity, product separation, and product polishing. Theseparameters impact the efficiency and yield of each cycle of determined nucleotide addition to reach an oligonucleotide of a pre-defined sequence. Temperature regulators can be column wall thermostats, a still air thermostat, forced air thermostats, passive pre-heaters, active pre-heaters, post-column liquid phase cooler, or heat exchangers. In some aspects, the temperature regulators can also be thermal blankets or insulation sleeves, jacketed reservoirs or chambers (such as any of the reaction chambers, purification chambers, enzyme capture chambers, and / or a reagent reservoirs as described herein), water baths or ice baths. Each of these elements may also be coupled to thermometers to monitor heat or cooling. In some aspects, the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber. In some aspects, the system comprises an in-line temperature regulator that controls the temperature of the solution in at least one of the one or more conduits. Thus in some aspects, the system further comprises a temperature regulator that controls the temperature of the solution in the system. In some aspects, the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber. In some aspects, the temperature regulator is a jacketed chamber, such as a jacketed purification chamber. In some aspects, the temperature regulator is a jacketed reaction chamber. In some aspects, the temperature regulator is a jacketed reagent reservoir. In some aspects, the temperature regulator is a jacketed stir tank.

[0186] Mechanisms of degassing the buffers or solutions may also be incorporated into the systems as described herein. In some aspects, the reagent reservoirs may be operably linked to an inert gas source, such as nitrogen or argon gas tanks to sparge the buffer and / or reagents prior to introduction into the system. Sparging with inert gasses removes oxygen from the buffers, reagents, and / or liquids in the system. In-line degassing apparatuses, such as an in-line vacuum pump) may be optionally included in the systems as disclosed herein. Degassing the buffers and / or solutions used in the system may be useful, for example, for preventing oxidation of oligonucleotides and / or the modified nucleotides used to make oligonucleotides. In some aspects, the systems optionally include one or more in-line degassers for degassing the buffers and / or solutions. In some aspects, the system optionally includes one or more reagent reservoirs that are operably linked to a tank comprising an inert gas (such as nitrogen or argon) such that the contents within the reservoir are sparged prior to introduction into the systems. In some aspects, the system may include one or more in-line spargers for sparging the fluids within the system. In some aspects, the buffers and / or solutions comprise reagents, such as reagents that are useful for template-independent oligonucleotide synthesis.

[0187] In some aspects, the system comprises one or more conduits that connects the first reaction chamber and the second reaction chamber. In some aspects, the conduits comprise an in-line temperature regulator that controls a temperature of the solution in at least one of the one or more conduits. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set ofconduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber. In some implementations, the system for template free synthesis of an oligonucleotide comprises a first reaction chamber, and a second reaction chamber. In some implementations, the system is configured to flow the oligonucleotide in a liquid phase from the first reaction chamber to the second reaction chamber. In some implementations, the system is configured to flow the oligonucleotide in the liquid phase from the second reaction chamber back to the first reaction chamber.

[0188] FIG. 3 illustrates an example of a system in accordance with one implementation, wherein the system comprises a first reaction chamber comprising a transferase 300 and a second reaction chamber 302 comprising a hydrolase. The system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 300 to the second reaction chamber 302 while retaining the transferase in the first reaction chamber 300, and the oligonucleotide can be flowed from the second reaction chamber 302 to the first reaction chamber 300 while retaining the hydrolase in the second reaction chamber 302. Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In this exemplary system, the reaction chambers are batch vessels that are fluidly connected by conduit 304.

[0189] In this system, the oligonucleotide is introduced to the system by addition to first reaction chamber 300. In some implementations, the reservoir comprises an apparatus for mixing solutions in first reaction chamber 300. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to first reaction chamber 300. The solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 304 into second reaction chamber 302. The first reaction chamber 300 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety (such as, for example, an NQP) to the oligonucleotide. The solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 304. In some implementations, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 304, and into second reaction chamber 302 comprises a hydrolase and the transferase is retained in the first reaction chamber 300. Different configurations of the system can be designed to retain the transferase, such as by a membrane positioned near the outlet of first reaction chamber 300. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 300. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of first reaction chamber 300. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 302 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongatedoligonucleotide is then flowed through conduits 304 to first reaction chamber 300. In some implementations, the elongated oligonucleotide is flowed to first reaction chamber 300 and the hydrolase is retained in second reaction chamber 302. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 302. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 302. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 302. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Flowing the elongated oligonucleotide to first reaction chamber 300 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP) The system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into first reaction chamber 300, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may also comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into first reaction chamber 300 or second reaction chamber 302) in the system (e.g., into first reaction chamber 300 or second reaction chamber 302). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir. In some aspects, the system may comprise one or more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchangeand / or concentrate the oligonucleotide. Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir. In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 300 and the second reaction chamber comprising the hydrolase 302). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 300 and the second reaction chamber comprising the hydrolase 302). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system can further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0190] The system may further comprise one or more valves. Valves are used for multiple purposes in a liquid delivery system. Non-limiting examples of valves that can be used in the system include injection or inlet valves that are used to introduce liquid sample into the system, flow path switching valves that may be used to change the flow path of the liquid, column valves for attachment of one or more columns to the system, and outlet valves for waste or sample collection. In some aspects, the one or more valves are multiposition rotary valves. In some aspects, the valves are single position valves. In some aspects, the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system. In some aspects, the system comprises one or more diverter valves. In some aspects, the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system. In some aspects, the systems disclosed herein comprise diverter valves configured to alternatively direct flow of the solution through (i) a first flow pathways comprising flow of the solution from the outlet of the first reaction chamber to an inlet of the purification chamber, and from an outlet of the purification chamber to the inlet of the first reaction chamber, or (ii) a second flow pathway comprising flow of the solution from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from an outlet of the second reaction chamber to the inlet of the first reaction chamber.

[0191] FIG. 4 illustrates an example of a system in accordance with one implementation. The system is an example of a system that comprises a first reaction chamber comprising a transferase 400 and a second reaction chamber 402 comprising a hydrolase. The system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 400 to the second reaction chamber 402 while retaining the transferase in the first reaction chamber 400, and the oligonucleotide can be flowed from the second reaction chamber 402 to the first reaction chamber 400 while retaining the hydrolase in the second reaction chamber 402. Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In the system, thereaction chambers are vessels such as batch vessels that are fluidly connected by conduit 404, but the flow is modulated by control valve 406 .

[0192] FIG. 4 illustrates an example of a system in accordance with one implementation. The oligonucleotide is introduced to the system by addition to a first reaction chamber 400. In this particular embodiment, the solution is flowed from the first reaction chamber 400 to the second reaction chamber 402 via conduit 404. Flow is applied by gravity, and controlled by control valve 406. In some embodiments, the transferase is added to first reaction chamber 400. After oligonucleotide elongation, the oligonucleotide comprising the 3' blocking moiety is flowed to the second chamber through control valve 406 and conduit 404. The transferase is retained in the top chamber, optionally through immobilization, a membrane or a filter. The hydrolase is added to second reaction chamber 402 and the elongated oligonucleotide is deblocked. Product can be separated from the hydrolase by, for example, inverting the chambers and using gravity flow to re-cycle the solution into the first reaction chamber 400. In some aspects, the hydrolase is retained in the second reaction chamber by gravity flow through a filter or a membrane, immobilized enzyme, or any combination thereof.

[0193] The system may further comprise one or more pumps configured to flow the solution or the liquid phase. A pump is a solvent delivery system. A pump can deliver a single solvent or multiple solvents. In some implementations, the pump comprises a mixer that combines multiple solvents into a final solvent that can be delivered to the system. Pumps may be configured to deliver the solvents at a determined flow rate. In some aspects, the one or more pumps deliver solvent at a constant flow rate. In some implementations, the one or more pumps are configured to control a flow rate of the liquid phase. In some implementations, the system further comprises one or more pumps configured to flow the liquid phase from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber. In some aspects, the system further comprises one or more pumps configured to flow the solution from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber. In some aspects, the one or more pumps are configured to control a flow rate of the solution. In some aspects, the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.

[0194] FIG. 5 illustrates an example of a system in accordance with one implementation. The system is an example of a system that comprises a first reaction chamber comprising a transferase 500 and a second reaction chamber 502 comprising a hydrolase. The system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 500 to the second reaction chamber 502 while retaining the transferase in the first reaction chamber 500 and the oligonucleotide can be flowed from the second reaction chamber 502 to the first reaction chamber 500 while retaining the hydrolase in the second reaction chamber. Reservoirs and reactionchambers can be, for example, batch vessels, flasks, or in line columns. In the system, the reaction chambers are batch vessels that are fluidly connected by conduits 504 and 504, and the flow is controlled by pump 508.

[0195] In the system of FIG. 5, the oligonucleotide is introduced to the system by addition to first reaction chamber 500. In some implementations, the reservoir comprises an apparatus for mixing solutions in first reaction chamber 500. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to first reaction chamber comprising a transferase 500. The solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 504 into second reaction chamber comprising a hydrolase 502. The movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as pump 506. The first reaction chamber 500 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide. The solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 504. In some implementations, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 504, and into second reaction chamber 502 and the transferase is retained in the first reaction chamber 500. In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 500. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 500. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 500. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 402 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongated oligonucleotide is then flowed through conduits 504 to first reaction chamber 500. In some implementations, the elongated oligonucleotide is flowed to first reaction chamber 500 and the hydrolase is retained in second reaction chamber 502. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 502. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 502. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 502. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Flowing the elongated oligonucleotide to first reactionchamber 500 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP). The system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into first reaction chamber 500, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into first reaction chamber 500 or second reaction chamber 502) in the system (e.g., into first reaction chamber 500 or second reaction chamber 502). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir. In some aspects, the system may comprise one or more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and / or concentrate the oligonucleotide. Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir. In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 500 and the second reaction chamber comprising the hydrolase 502). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the systemcomprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 500 and the second reaction chamber comprising the hydrolase 502). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Nonlimiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with theinterior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0196] FIG. 6 illustrates an example of a system in accordance with one implementation. The system is an example of a system that comprises a first reaction chamber comprising a transferase 600 and a second reaction chamber 602 comprising a hydrolase. The system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 600 to the second reaction chamber 602 while retaining the transferase in the first reaction chamber 600, and the oligonucleotide can be flowed from the second reaction chamber 602 to the first reaction chamber 600 while retaining the hydrolase in the second reaction chamber 602 . Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In the system, the reaction chambers are batch vessels that are fluidly connected by conduits 604 and 606, and the direction of the flow is controlled by control valve 608. The reaction chambers are also fluidly connected by conduits 618 and 620, and the direction of the flow is controlled by control valve 616. In some aspects, the flow can be unidirectional through each of these conduits such that the flow path for flowing the solution from first reaction chamber 600 to second reaction chamber 602 is not the same flow path for flowing the solution from second reaction chamber 602 to first reaction chamber 600. In some aspects, the pathways can be modified using control valves 616 and 608 such that the solution can cycle through a reaction chamber (e.g., the first reaction container 600 or second reaction container 602) before advancing to the other reaction chamber.

[0197] In the system of FIG. 5, the oligonucleotide is introduced to the system by addition to first reaction chamber 600. In some implementations, the reservoir comprises an apparatus for mixing solutions in first reaction chamber 600. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to first reaction chamber comprising a transferase 600. The solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 604 into second reaction chamber comprising a hydrolase 602. The movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as pump 610. The first reaction chamber 600 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide. The solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 604 and 606. In some implementations, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 604 and 606 through control valve 608, and into second reaction chamber 602 and the transferase is retained in the first reaction chamber 600. In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 600. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 600. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations,the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 600. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 602 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongated oligonucleotide is then flowed through conduits 604 to first reaction chamber 600. In some implementations, the elongated oligonucleotide is flowed to first reaction chamber 600 and the hydrolase is retained in second reaction chamber 602. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 602. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 602. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 602. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Flowing the elongated oligonucleotide to first reaction chamber 600 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP) The system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into first reaction chamber 600, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into first reaction chamber 600 or second reaction chamber 602) in the system (e.g., into first reaction chamber 600 or second reaction chamber 602). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir. In some aspects, the system may comprise oneor more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and / or concentrate the oligonucleotide.Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir. In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 600 and the second reaction chamber comprising the hydrolase 602). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 600 and the second reaction chamber comprising the hydrolase 602). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or moreanalytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0198] In some aspects, the system further comprises a third reaction chamber comprising an enzyme. In some aspects, the system comprises a third reaction chamber that comprises a transferase. In some aspects, the system comprises a third reaction chamber that comprises a second transferase. In some aspects, the system further comprises a third reaction chamber comprising a second transferase, wherein the system is further configured to selectively flow said oligonucleotide in the solution from the second chamber to the third reaction chamber while retaining the hydrolase in the second reaction chamber, and flow the oligonucleotide in the solution from the third reaction chamber to the second reaction chamber while retaining the second transferase in the third reaction chamber. In some aspects, the transferase and the second transferase are different types of transferase. In some aspects, wherein the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system. In some aspects, the third reaction chamber comprises a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase.

[0199] Thus in some aspects, provided herein is a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reactionchamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase.

[0200] In some aspects, the third reaction chamber comprises a ssRNA ligase wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the ssRNA ligase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the ssRNA ligase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the ssRNA ligase is retained in the third reaction chamber. In some aspects, the reaction chamber comprising the ssRNA ligase comprises a filter that prevents passage of ssRNA ligase and allows passage of the ligated oligonucleotide.

[0201] Thus in some aspects, provided herein is a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a ssRNA ligase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the ssRNA ligase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the ssRNA ligase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the ssRNA ligase is retained in the third reaction chamber comprising the ssRNA ligase.

[0202] In some aspects, the third reaction chamber comprises a primase wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the primase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprisingthe primase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the primase is retained in the third reaction chamber.

[0203] Thus in some aspects, provided herein is a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a primase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the primase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the primase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the primase is retained in the third reaction chamber comprising the primase. In some aspects, the reaction chamber comprising the primase comprises a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.

[0204] FIG. 7 illustrates an example of a system in accordance with one implementation. The system comprises a first reaction chamber 700 comprising a transferase and a second reaction chamber 702 comprising a hydrolase that is fluidly connected by conduits 704, 706, and 708. Flow path is controlled through conduits 704, 706, and 708 by control valves 710 and 712. Second reaction chamber comprising a hydrolase 702 is fluidly connected back to first reaction chamber 700 via conduits 714, 716, and 718, and flow path is controlled by control valves 720 and 724. The flow is controlled by pump 726. The system further comprises a third reaction chamber comprising a second transferase 726. Third reaction chamber comprising a second transferase 726 is fluidly connected to first reaction chamber comprising a first transferase via conduits 704 and 728 through valve 710, and via conduits 730 and 718 through valve 724. Third reaction chamber comprising a second transferase 726 is fluidly connected to second reaction chamber comprising a hydrolase via conduits 706 and 708 through valve 712, and via conduits 814 and 816 through valves 720 and 724. Pump 726 is fluidly connected to the system using conduits 732 and 734. In some aspects, the enzyme in the third reaction chamber is a primase. In some aspects, the enzyme in the third reaction chamber is a ssRNA ligase.

[0205] In the system of FIG. 7, the oligonucleotide is introduced to the system by addition to first reaction chamber 700. In some implementations, the first reaction chamber comprises an apparatus for mixing solutions in first reaction chamber 700. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to first reaction chamber comprising a transferase 700. The solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 704 into second reaction chamber comprising a hydrolase 702. Thesolution is then flowed from the second reaction chamber 702 through conduits 714, 716 and 718 back to first reaction chamber comprising transferase. Alternatively, the solution can be flowed to the third reaction chamber comprising a second transferase via conduits 714 and 716 through valves 720 and 724. The movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as control valves 720, 725, 710, 712, and pump 726. After each addition of nucleotide, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 804, 806, and 808 or 806 and 808 through control valves 728 or 728 and 712 into second reaction chamber 702.

[0206] The system may further comprise one or more reagent reservoirs. In some aspects, the system comprises a reagent reservoir comprising an inlet and an outlet; and wherein the system is configured to flow the oligonucleotide in the solution from an outlet of one of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from the outlet of the second reaction chamber to an inlet of the reagent reservoir. In some aspects, the reagent reservoir comprises an impeller. In some aspects, the reagent reservoir is replaceable or cleanable. In some aspects, the system further comprising a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the first reaction chamber. In some aspects, the product reservoir comprises an impeller. In some aspects, the product reservoir is replaceable or cleanable.

[0207] In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 700. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 700. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 700. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 702 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongated oligonucleotide is then flowed through conduits 704 to first reaction chamber 700. In some implementations, the elongated oligonucleotide is flowed to first reaction chamber 700 and the hydrolase is retained in second reaction chamber 702. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 702. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 702. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix orsupport, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 702. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Flowing the elongated oligonucleotide to first reaction chamber 800 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP) the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into first reaction chamber 700, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into first reaction chamber 700 or second reaction chamber 702) in the system (e.g., into first reaction chamber 800 or second reaction chamber 702). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir. In some aspects, the system may comprise one or more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and / or concentrate the oligonucleotide. Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir. In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that isfluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 700 and the second reaction chamber comprising the hydrolase 702). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 700 and the second reaction chamber comprising the hydrolase 702). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Nonlimiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressuresensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0208] FIG. 8 is a system in accordance with one implementation. In the system, the reaction chamber comprising transferase 800 is fluidly connected to a second reaction chamber comprising hydrolase 802 by conduits 804 and 806 and valve 808. Second reaction chamber is fluidly connected back to first reaction chamber comprising transferase 800 by conduits 818 and 820 by valve 816. A reagent reservoir / product reservoir 810 is also fluidly connected to the system by valves 822 and 816, and conduits 822 and 824.

[0209] In the system of FIG. 8, the oligonucleotide is introduced to the system by addition to first reaction chamber 800. In some implementations, the reservoir comprises an apparatus for mixing solutions in first reaction chamber 800. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to first reaction chamber comprising a transferase 800. The solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 804 into second reaction chamber comprising a hydrolase 802. The solution is then flowed from the second reaction chamber 802 through conduits 818 or 806 back to the reagent reservoir which may optionally act as a product reservoir via control valves 816 or 822, respectively. From the reagent reservoir / product reservoir, the solution can be cycled through to the first reaction chamber 800. The movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as reagent reservoir 810. The first reaction chamber 800 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide. The solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 804 and 806. In some implementations, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 804 and 806 through control valve 808 into reagent reservoir 810 via conduit 822, mixed with new nucleotide comprising a 3' blocking moiety, and then flowed into second reaction chamber 802 using control valve 816 through conduit 818, while transferase is retained in the first reaction chamber 800. FIG. 9 is further embodiment of the system of FIG. 8, wherein there are one or more reagent reservoirs (e.g., shown here as 822-834, but may comprise more or less reagent reservoirs) optionally connected to reagent reservoir / product reservoir 810 by conduits 836 and 838. In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 800. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 800. In some implementations the transferase is retained by immobilizing thetransferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 800. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 802 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongated oligonucleotide is then flowed through conduits 804 to first reaction chamber 800. In some implementations, the elongated oligonucleotide is flowed to first reaction chamber 800 and the hydrolase is retained in second reaction chamber 802. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 802. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 802. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 802. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Flowing the elongated oligonucleotide to first reaction chamber 800 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP) the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into first reaction chamber 800, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into first reaction chamber 800 or second reaction chamber 802) in the system (e.g., into first reaction chamber 800 or second reaction chamber 802). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas forsparging the contents of the reservoir. In some aspects, the system may comprise one or more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and / or concentrate the oligonucleotide. Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir. In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 800 and the second reaction chamber comprising the hydrolase 802). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 800 and the second reaction chamber comprising the hydrolase 802). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices,memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0210] The system may further comprise a purification chamber. In some implementations, the system comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide. In some implementations, the purification chamber comprises a column. In some implementations, the column is a liquid chromatography column. In some implementations, the purification chamber comprises size exclusion chromatography resin. In some implementations, the purification chamber comprises ion-exchange resin. In some implementations, the purification chamber comprises reverse phase resin. In some aspects, the purification chamber is configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide. In some aspects, the purification chamber is configured to capture incidental enzymes such as free transferase or free hydrolase that was not retained by the chamber.

[0211] FIG. 10 illustrates an example of a system in accordance with one implementation. The system is an example of a system that comprises a first reaction chamber 1010 and a second reaction chamber 1018. Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns, in the system, the reaction chambers are in line columns. Reagent reservoir 1000 is fluidly connected to a first reaction chamber 1010 comprising a transferase via conduits 1004 and 1008, optionally including pump 1006. First reaction chamber 1010 is fluidly connected to a second reaction chamber 1018 comprising a hydrolase by conduits 1012 and 1016, optionally an AT 1014 that is in line between first reaction chamber 1010. Second reaction chamber 1018 is fluidly connected topurification chamber 1026 by conduits 1020 and 1024, optionally including AT 1022. Purification chamber 1028 is fluidly connected to product reservoir 1034 that comprises impeller 10106. Product reservoir 1034 is fluidly connected to reagent reservoir 1000 by conduit 1040. Product reservoir also is fluidly connected to conduit 10108. Optionally, in some implementations, the reagent reservoir 1000 and product reservoir are the same reservoir.

[0212] In the system of FIG. 10, the oligonucleotide is introduced to the system by addition into reagent reservoir 1000. In some implementations, the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1000, such as impeller 1002. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to reagent reservoir 1000. Impeller 1002 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1004 and 1008 via pump 1006 into a first reaction chamber 1010. The first reaction chamber 1010 comprises a transferase. The transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide. The solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1012 and 1016, passing through AT 1014 into a second reaction chamber 1018. In some implementations, the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduits 1012 and 1016, and into second reaction chamber 1018 and the transferase is retained in the first reaction chamber 1010. In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1010. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 1010. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1010. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 1018 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. The elongated oligonucleotide is flowed through conduits 1020 and 1024, and optionally via AT 1012, and into purification chamber 1026. In some implementations, the elongated oligonucleotide is flowed to purification chamber 1026 and the hydrolase is retained in second reaction chamber 1018. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1018. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 1018. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1018. The in-line column comprises resinor beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. In some implementations, the elongated oligonucleotide is flowed through conduits 1020 and 1024, and into purification chamber 1026 and the hydrolase is retained in the second reaction chamber 1018. The elongated oligonucleotide is optionally purified in purification chamber 1026, then flowed through conduits 1028 and 1032, and optionally via AT 1030 into product reservoir 1034. Product reservoir 1034 comprises impeller 1036, which is used to stir the solution comprising the elongated oligonucleotide. The elongated oligonucleotide can exit the system using conduit 1038 or can be flowed to reagent reservoir 1000 through conduit 1040. Flowing the elongated oligonucleotide to reagent reservoir 1000 reintroduces an oligonucleotide into the system. Upon introduction of another preselected nucleotide (e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP) the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide. In some implementations, upon entry into reagent reservoir 1000, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system. In some implementations, the conduits, reservoirs and / or chambers as described above in the system may also comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and / or cofactors) at defined locations (e.g., into reagent reservoir 1000, first reaction chamber 1010 or second reaction chamber 1018) in the system (e.g., into first reaction chamber 1010 or second reaction chamber 1018). Optionally, the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and / or reservoirs. For example, the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and / or reservoirs. In some aspects, the degasser can be integrated downstream of a reagent reservoir comprising buffers and / or reagents such that the degasser is fluidly connected to the system, and the buffer and / or reagents are degassed prior to introduction into the system. In some aspects, the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system. In some aspects, the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir. In some aspects, the system may comprise one or more in-line spargers for sparging the solutions within the system. In some aspects, the system described herein may comprise one or more purification chambers. The systems described herein may comprise two purification chambers. In some aspects, the purification chambers are different types of purification chambers. As a non-limiting example, the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and / or concentrate the oligonucleotide. Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.In some aspects, the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety. In some aspects, the enzymatic conjugation reaction can be performed in batch. In some aspects, the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1010 and the second reaction chamber comprising the hydrolase 1018). In some aspects, the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1010 and the second reaction chamber comprising the hydrolase 1018). In some aspects, the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein. In some aspects, the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated. For example, the upon introduction into reagent reservoir 1000 of the system, the oligonucleotide is flowed through the system using a pre-determined flow rate until it reaches product reservoir 1034. In some implementations, the flow for the system is automated. In some implementations, the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters. The flow rate can be manually or automatically adjusted. In some implementations, the flow rate is adjusted by a device, such as a device that is connected to the system. In some implementations, the system is connected to a device. In some implementations, the flow rate can be adjusted by a device that is connected to the system. In some implementations, the device is a host computer connected to a network. In some implementations, the host computer is a client computer or a server. In some implementations, the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and / or one or more mass spectrometers). Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system. In some implementations, the system is connected to an analytical system. The analytical system can be used to assess properties of the oligonucleotide that is flowed through the system. Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and / or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and / or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system. Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber. In some implementations, the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and / or chambers. In some implementations, the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and / or detect changes in the solution.

[0213] FIG. 11 illustrates an example of a system in accordance with one implementation. The system is an example of a system that comprises a first reaction chamber 1114 and a second reaction chamber 1122. Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In the system, the reaction chambers are in line columns. Reagent reservoir 1100 comprises impeller 1102, and is fluidly connected to a first reaction chamber 1114 comprising a transferase via conduits 1104 and 1108, optionally including pump 1106. First reaction chamber 1114 is fluidly connected to a second reaction chamber l l22 comprising a hydrolase by conduits 1116, 1138, 1104, 1108, 1110, 1120, valves 1112, 1120, 1118, pump 1106, and reservoir 1110. Second reaction chamber 1122 is fluidly connected to purification chamber 1128 1116, 1138, 1104, 1108, 1110, 1120, 1126, 1136, valves 1112, 1120, 1118, 1132, pump 1106, and reservoir 1110. Product reservoir 1034 is fluidly connected to reagent reservoir 1000 by conduit 1040. Optionally, in some implementations, the reagent reservoir 1000 and product reservoir are the same reservoir.

[0214] In the system of FIG. 11, the oligonucleotide is introduced to the system by addition into reagent reservoir 1100. In some implementations, the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1100, such as impeller 1102. Preselected nucleotide (e.g., a nucleotide with a 3' blocking moiety such as a NQP) can also be added to reagent reservoir 1100. Impeller 1102 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1104 and 1108 via pump 1106 into a diverter valve 1108. From the diverter valve, the solution is flowed through conduit 1110, through two diverter valves 1112 and 1120. The solution is routed to the first reaction chamber 1114 via conduit 1124, then flowed through conduit 1116 to reach another diverter valve 1118. Diverter valve 1118 flows the solution back to the reagent reservoir 1100 by conduit 1138, and further rerouted through conduits 1104, 1108, and diverter valve 1118. The first reaction chamber 1114 comprises a transferase. The transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide. Thesolution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1104, 1108, and 1110 through diverter valves 1112 and 1120 into a second reaction chamber 1122 via conduit 1128. Conduit 1130 routes the solution through diverter valve 1118 and conduit 1138 for return back to reagent chamber 1100. This cycle between first reaction and second reaction chamber can be reiterated any number of times. The oligonucleotide can optionally be purified between extension cycles or at the end of the reaction. From the reagent reservoir 1100, the solution is flowed through conduits 1104 and 1108 and 1110 by pump 1106 and through conduits 1122 and 1120 through a purification chamber 1128. Conduit 1130 flows the solution to diverter valve 1132, where the purified oligonucleotide can return to reaction chamber 1100 for further extension / deblocking using conduit 1136, or exit the system using conduits 1134 and 1138 through diverter valve 1118. In some implementations, the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1114. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 1114. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1114. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange. Second reaction chamber 1122 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide. In some implementations, the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1122. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 1122. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1122. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, ...

Claims

CLAIMSWhat is claimed is:

1. A method of template- free synthesis of an oligonucleotide, comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.

2. The method of claim 1, wherein the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.

3. The method of claim 1 or 2, wherein the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.

4. The method of claim 3, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

5. The method of claim 3 or 4, further comprising separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase.

6. The method of any one of claims 1-5, further comprising repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.

7. The method of claim 6, wherein a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.

8. The method of claim 6 or 7, wherein at least two different types of transferases are used in separate cycles.

9. The method of any one of claims 1-8, wherein the elongating occurs in a first reaction chamber.

10. The method of any one of claims 1-9, wherein the removing the 3' blocking moiety occurs in a second reaction chamber.

11. A method of template- free synthesis of an oligonucleotide, comprising: in a first reaction chamber, elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the second reaction chamber into the first reaction chamber.

12. The method of claim 11, wherein the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.

13. The method of claim 11 or 12, wherein the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.

14. The method of claim 13, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

15. The method of any one of claims 13-14, wherein the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber.

16. The method of any one of claims 11-15, further comprising repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.

17. The method of claim 16, wherein a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.

18. The method of claim 16 or 17, wherein at least two different types of transferases are used in separate cycles.

19. The method of any one of claims 11-18, wherein the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits.

20. The method of any one of claims 11-19, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps.

21. The method of any one of claims 11-20, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves.

22. The method of any one of claims 11-21, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the first reaction chamber.

23. The method of any one of claims 11-22, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the second reaction chamber.

24. The method of any one of claims 11-23, wherein the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the second reaction chamber.

25. The method of any one of claims 11-24, wherein the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the first reaction chamber.

26. The method of any one of claims 11-25, wherein flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into the first reaction chamber comprises: flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into one or more reservoirs, and flowing the elongated oligonucleotide without the 3' blocking moiety from the one or more reservoirs into the first reaction chamber.

27. A method of template- free synthesis of an oligonucleotide, comprising, in a plurality of reaction chambers: in at least a first reaction chamber, elongating an oligonucleotide in solution by attaching afirst nucleotide comprising a 3' blocking moiety to the oligonucleotide using a first transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution from the at least the first reaction chamber into at least a second reaction chamber, wherein the first transferase is retained in the at least the first reaction chamber; in the at least the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the at least the second reaction chamber into at least a third reaction chamber comprising a second transferase; and in the at least the third reaction chamber, elongating the elongated oligonucleotide without the 3' blocking moiety the solution by attaching a second nucleotide comprising a 3' blocking moiety to the elongated oligonucleotide without the 3' blocking moiety using the second transferase to make a further elongated oligonucleotide comprising the 3' blocking moiety of the second nucleotide.

28. The method of claim 27, wherein the first transferase and the second transferase are different types of transferases.

29. The method of claim 27 or 28, wherein the first nucleotide and the second nucleotide are preselected types of nucleotides, and / or wherein the first nucleotide and the second nucleotide are different types of nucleotides.

30. The method of any one of claims 27-29, wherein the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.

31. The method of claim 30, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

32. The method of claim 30 or 31, wherein the hydrolase is retained in the at least the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows from the second reaction chamber into the at least the third reaction chamber.

33. The method of any one of claims 27-32, wherein the first reaction chamber, the second reaction chamber, and the third reaction chamber are connected through a plurality of conduits, wherein: the elongated oligonucleotide comprising the 3' blocking moiety flows from the at least the first reaction chamber into at least a second reaction chamber through a first portion of the plurality of conduits; and the elongated oligonucleotide without the 3' blocking moiety flows from the at least thesecond reaction chamber into at least a third reaction chamber through a second portion of the plurality of conduits.

34. The method of any one of claims 27-33, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more pumps.

35. The method of any one of claims 27-34, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more valves.

36. The method of any one of claims 1-35, wherein the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.

37. The method of claim 36, wherein the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.

38. The method of any one of claims 3-10, 13-26, and 30-37, wherein the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution.

39. The method of any one of claims 3-10, 13-26, and 30-38, wherein the hydrolase removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution.

40. The method of claim 39, wherein the hydrolase removes three 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution.

41. The method of any one of claims 3-10, 13-26, and 30-40, wherein the hydrolase is immobilized on a solid support.

42. The method of any one of claims 1-41, wherein the transferase comprises a polymerase from the DNA polymerase X family; a template independent transferase, preferably a terminal deoxynucleotidyl transferase (TdT); or an ssRNA ligase.

43. The method of any one of claims 1-42, wherein the transferase is immobilized on a solid support.

44. The method of any one of claims 1-43, wherein elongating produces an inorganic pyrophosphate byproduct.

45. The method of claim 44, further comprising degrading the inorganic pyrophosphate using a pyrophosphatase, preferably using an inorganic pyrophosphatase.

46. The method of claim 45, wherein the pyrophosphatase is immobilized on a solid support.

47. The method of claim 45 or 46, wherein the elongating and the degrading occur within the same reaction chamber.

48. The method of claim 47, wherein the transferase and the pyrophosphatase are fused together, or wherein the transferase and the pyrophosphatase are immobilized on the same solid support, or wherein the transferase and the pyrophosphatase are immobilized on different solid supports.

49. The method of claim 45 or 47, wherein the transferase is immobilized on a solid support and the pyrophosphatase is in the solution.

50. The method of claim 49, wherein the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.

51. The method of any one of claims 45-50, wherein the elongating and the degrading occur within different reaction chambers.

52. The method of claim 51, comprising flowing the elongated oligonucleotide comprising the 3' blocking moiety and the inorganic pyrophosphate byproduct, in the solution, from a reaction chamber comprising the transferase into a reaction chamber comprising the pyrophosphatase, followed by flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reaction chamber comprising the pyrophosphatase into a reaction chamber in which the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety.

53. The method of any one of claims 10-52, wherein the first reaction chamber or the second reaction chamber is a column.

54. The method of claim 53, wherein the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support; or a fluidized bed comprising the transferase immobilized on a solid support; or a filter that prevents passage of the transferase and allows passage of the oligonucleotide.

55. The method of claim 53 or 54, wherein the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support; or a fluidized bed comprising the hydrolase immobilized on a solid support; or a filter that prevents passage of hydrolase and allows passage of the oligonucleotide.

56. The method of any one of claims 10-52, wherein the first reaction chamber or the second reaction chamber is a batch reaction chamber.

57. The method of claim 56, wherein the batch reaction chamber comprises an impeller.

58. The method of claim 55, wherein the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.

59. The method of any one of claims 56-58, wherein the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support.

60. The method of any one of claims 1-58, further comprising separating unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide.

61. The method of claim 60, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography, preferably size exclusion chromatography, reverse phase chromatography, or ion exchange chromatography.

62. The method of claim 60, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis or tangential flow filtration.

63. The method of any one of claims 60-62, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides, or only after elongating the oligonucleotide by four or more nucleotides.

64. The method of any one of claims 1-63, wherein the 3' blocking moiety is a phosphate moiety.

65. The method of any one of claims 1-64, wherein the nucleotide comprising the 3' blocking moiety comprises a ribonucleotide or a deoxyribonucleotide, or further comprises a 2' modification, preferably 2'-F or 2'-O-methyl.

66. The method of any one of claims 1-65, wherein the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.

67. A method of template-free synthesis of an oligonucleotide, comprising: flowing an oligonucleotide, in a solution, from a reagent reservoir into a first reaction chamber comprising a transferase; in the first reaction chamber, elongating the oligonucleotide by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using the transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the nucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber comprising a hydrolase, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety in the solution to make an elongatedoligonucleotide without the 3' blocking moiety; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, into the reagent reservoir.

68. The method of claim 67, comprising mixing the oligonucleotide and the nucleotides comprising a 3 '-blocking moiety in the reservoir.

69. The method of claim 67 or 68, further comprising flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, in the solution, through a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety before flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety into the reagent reservoir.

70. The method of any one of claims 67-69, further comprising flowing the oligonucleotide without the 3' blocking moiety into a product reservoir before flowing the oligonucleotide without the 3' blocking moiety into the reagent reservoir.

71. The method of any one of claims 67-70, further comprising replacing or cleaning the reagent reservoir before flowing the elongated oligonucleotide without the 3' blocking moiety into the reagent reservoir.

72. The method of any one of claims 67-71, comprising repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.

73. The method of claim 72, further comprising flowing buffer without the oligonucleotide through the system between cycles.

74. A method of template-free synthesis of an oligonucleotide, comprising:(a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and nucleotides comprising a 3' blocking moiety from a reagent reservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir; and(b) removing the 3' blocking moiety from the oligonucleotide in the solution to make an elongated oligonucleotide without the 3' blocking moiety, comprising subjecting the oligonucleotide to one or more deblocking flow pathway cycles comprising: flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reagentreservoir to a second reaction chamber comprising a hydrolase, and flowing the elongated oligonucleotide from the second reaction chamber to the reagent reservoir.

75. A method of template-free synthesis of an oligonucleotide, comprising:(a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and 3 '-blocked nucleotides from a reagent reservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir;(b) flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir to a second reaction chamber comprising a hydrolase; and(c) removing the 3' blocking moiety from the oligonucleotide in the solution to make an elongated oligonucleotide without the 3' blocking moiety, comprising subjecting the oligonucleotide to one or more deblocking flow pathway cycles comprising: flowing the elongated oligonucleotide comprising the 3' blocking moiety from the second reaction chamber to a product reservoir, and flowing the elongated oligonucleotide from the product reservoir to the second reaction chamber.

76. The method of claim 74 or 75, wherein the elongating comprises subjecting the oligonucleotide to a plurality of elongation flow path cycles.

77. The method of any one of claims 74-76, wherein the removing comprises subjecting the oligonucleotide to a plurality of deblocking flow pathway cycles.

78. The method of any one of claims 74-77, comprising separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety.

79. The method of claim 78, wherein the separating comprises subjecting the oligonucleotide to one or more purification flow pathway cycles comprising: flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir or the product reservoir to a purification chamber configured to separate the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety or the elongatedoligonucleotide comprising the 3' blocking moiety from the purification chamber to the reagent reservoir or the product reservoir.

80. The method of claim 78 or 79, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography, preferably size exclusion chromatography, reverse phase chromatography, or ion exchange chromatography.

81. The method of claim 78 or 79, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis or tangential flow filtration.

82. The method of any one of claims 78-81, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides, or only after elongating the oligonucleotide by four or more nucleotides.

83. The method of any one of claims 74-81, comprising repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.

84. A method of template-free synthesis of an oligonucleotide, comprising: in first reaction chamber, elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the an elongated oligonucleotide comprising the 3' blocking moiety in the solution using a hydrolase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide without the 3' blocking moiety; and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, into the first reaction chamber.

85. The method of claim 84, comprising separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety.

86. The method of claim 85, wherein: the separating comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety from the first reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety; or the separating comprises flowing the elongated oligonucleotide without the 3' blockingmoiety from the second reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety.

87. The method of any one of claims 84-86, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography.

88. The method of claim 87, wherein the liquid chromatography comprises size exclusion chromatography, reverse phase chromatography, or ion exchange chromatography.

89. The method of any one of claims 84-86, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis, or tangential flow filtration.

90. The method of any one of claims 84-89, wherein the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides, or only after elongating the oligonucleotide by four or more nucleotides.

91. The method of any one of claims 84-89, comprising repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.

92. The method of any one of claims 67-91, wherein the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.

93. The method of any one of claims 67-92, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

94. The method of any one of claims 67-93, wherein the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber.

95. The method of any one of claims 67-94, wherein the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits.

96. The method of any one of claims 67-95, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps.

97. The method of any one of claims 67-96, wherein the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and / or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves.

98. The method of any one of claims 67-97, wherein the nucleotide comprising the 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.

99. The method of claim 98, wherein the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.

100. The method of any one of claims 67-99, wherein the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution.

101. The method of any one of claims 67-100, wherein the hydrolase removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution.

102. The method of claim 101, wherein the hydrolase removes three 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution.

103. The method of any one of claims 67-102, wherein the hydrolase is immobilized on a solid support.

104. The method of any one of claims 67-103, wherein the transferase is a polymerase from the DNA polymerase X family; a template independent transferase, preferably a terminal deoxynucleotidyl transferase (TdT); or an ssRNA ligase.

105. The method of any one of claims 67-104, wherein the transferase is immobilized on a solid support.

106. The method of any one of claims 67-105, wherein elongating produces an inorganic pyrophosphate byproduct.

107. The method of claim 106, further comprising degrading the inorganic pyrophosphate using a pyrophosphatase, preferably an inorganic pyrophosphatase.

108. The method of claim 107, wherein the pyrophosphatase is immobilized on a solid support.

109. The method of any one of claims 107-108, wherein the elongating and the degrading occur within the same reaction chamber.

110. The method of claim 109, wherein the transferase and the inorganic pyrophosphatase are fused together, or wherein the transferase and the pyrophosphatase are immobilized on the same solid support or different solid supports.

111. The method of claim 107 or 109, wherein the transferase is immobilized on a solid support and the pyrophosphatase is in the solution.

112. The method of claim 111, wherein the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.

113. The method of any one of claims 107-112 wherein the elongating and the degrading occur within different reaction chambers.

114. The method of any one of claims 67-113, wherein the first reaction chamber or the second reaction chamber is a column.

115. The method of claim 114, wherein the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support, or comprises a fluidized bed comprising the transferase immobilized on a solid support, or comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide.

116. The method of claim 114 or 115, wherein the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support or a fluidized bed comprising the hydrolase immobilized on a solid support, or a filter that prevents passage of hydrolase and allows passage of the oligonucleotide.

117. The method of any one of claims 67-106, wherein the first reaction chamber or the second reaction chamber is a batch reaction chamber.

118. The method of claim 117, wherein the batch reaction chamber comprises an impeller or a rotating bed reactor comprising the transferase immobilized on a solid support.

119. The method of claim 117 or 118, wherein the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support.

120. The method of any one of claims 67-119, wherein the 3' blocking moiety is a phosphate moiety.

121. The method of any one of claims 67-120 wherein the nucleotide comprising the 3' blocking moiety is a ribonucleotide or a deoxyribonucleotide, or further comprises a 2' modification, preferably 2'-F or 2'-O-methyL122. The method of any one of claims 67-121, wherein the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.

123. The method of any one of claims 67-122, wherein the oligonucleotide comprises a 5' modification.

124. A method of template-free synthesis of an oligonucleotide, comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase in the solution to make an elongated oligonucleotidecomprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.

125. The method of claim 124, wherein separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase comprises flowing the solution through a column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety.

126. The method of claim 125, wherein the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a liquid chromatography column.

127. The method of claim 125 or 126, wherein the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column, or comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide comprising the 3' blocking moiety.

128. The method of any one of claims 125-127, comprising flowing the oligonucleotide and the transferase, in the solution, from a reagent reservoir to the column that retains the transferase, and flowing the oligonucleotide comprising the 3' blocking moiety, in the solution, from the column that retains the transferase to the reagent reservoir.

129. The method of any one of claims 124-128, wherein removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety comprises adding a hydrolase to the solution after separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase.

130. The method of claim 129, further comprising separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.

131. The method of claim 130, wherein separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety from the hydrolase comprises flowing the solution through a column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.

132. The method of claim 131, wherein the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column.

133. The method of claim 131 or 132, wherein the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column, or comprises a filter that prevents passage of the hydrolase and allows passage of the elongated oligonucleotide without the 3' blocking moiety.

134. The method of any one of claims 129-133, comprising flowing the elongated oligonucleotide without the 3' blocking moiety and the hydrolase, in the solution, from a reagent reservoir to the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety to the reagent reservoir.

135. The method of any one of claims 129-134, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

136. The method of any one of claims 125-135, comprising replacing or cleaning the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety or the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.

137. The method of claim 136, wherein the cleaning comprises flowing a buffer through the column to elute the transferase or the hydrolase.

138. The method of any one of claims 124-137, further comprising separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.

139. The method of claim 138, wherein separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety comprises flowing the solution through a column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.

140. The method of claim 139, wherein the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column, preferably a size exclusion column, a reverse phase column, or an ion exchange column.

141. The method of claim 138, wherein the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using dialysis or tangential flow filtration.

142. The method of any one of claims 138-141, wherein the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety after elongating the oligonucleotide by four or more nucleotides, or only after elongating the oligonucleotide by four or more nucleotides.

143. The method of any one of claims 124-142, wherein the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.

144. The method of any one of claims 124-143, wherein the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.

145. The method of claim 144, wherein the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.

146. The method of any one of claims 125-145, wherein the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution, and / or removes one or more 5' phosphate moieties and / or 5' phosphate analogs from unreacted nucleotides in the solution.

147. The method of any one of claims 124-146, wherein the transferase is a polymerase from the DNA polymerase X family; a template independent transferase, preferably a terminal deoxynucleotidyl transferase (TdT); or an ssRNA ligase.

148. The method of any one of claims 124-147, wherein elongating produces an inorganic pyrophosphate byproduct.

149. The method of claim 124-148, further comprising degrading the inorganic pyrophosphate using a pyrophosphatase, preferably an inorganic pyrophosphatase.

150. The method of claim 149 or 149, wherein the transferase and the inorganic pyrophosphatase are fused together.

151. The method of any one of claims 139-149, wherein the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety further separates the pyrophosphatase from the elongated oligonucleotide comprising the 3' blocking moiety.

152. The method of any one of claims 124-151, wherein the 3' blocking moiety is a phosphate moiety.

153. The method of any one of claims 124-152, wherein the nucleotide comprising the 3' blocking moiety is a ribonucleotide or a deoxyribonucleotide.

154. The method of any one of claims 124-153, wherein the nucleotide comprising the 3' blocking moiety comprises a 2' modification, preferably 2'-F or 2'-O-methyl.

155. The method of any one of claims 124-154, wherein the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.

156. The method of any one of claims 124-155, wherein the oligonucleotide comprises a 5' modification.

157. The method of any one of claims 1-66, wherein the oligonucleotide comprises a 5' modification.

158. The method of any one of claims 1-157, wherein the oligonucleotide is at least 4 nucleotides in length.

159. The method of any one of claims 1-158, comprising synthesizing the oligonucleotide prior to elongating the oligonucleotide with the transferase.

160. The method of claim 159, wherein synthesizing the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.

161. The method of claim 160, wherein the nucleotide acceptor is a nucleotide monomer.

162. The method of claim 160, wherein the nucleotide acceptor is a nucleotide polymer, preferably a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer.

163. The method of any one of claims 160-162, wherein the nucleotide donor is a nucleotide monomer.

164. The method of any one of claims 160-163, wherein the nucleotide donor comprises a 3'-blocking moiety.

165. The method of claim 164, wherein the 3'-blocking moiety of the nucleotide donor is a phosphate.

166. The method of claim 1 4 or 165, comprising removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.

167. The method of claim 166, wherein the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase.

168. The method of any one of claims 159-167, wherein making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor havinga 3'-0H group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.

169. The method of any one of claims 42, 104, or 144, wherein when the transferase comprises a single strand RNA ligase, the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.

170. The method of claim 169, wherein nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combination thereof.

171. The method of claim 169 or 170, wherein the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety, or a 5'-OH.

172. The method of any one of claims 1-171, wherein the oligonucleotide is elongated under an inert atmosphere, preferably argon or nitrogen gas.

173. The method of any one of claims 1-172, wherein elongating the oligonucleotide is performed between 35 °C and 45 °C.

174. The method of any one of claims 1-173, wherein removing the 3' blocking moiety is performed between 45 °C and 55 °C.

175. The method of any one of claims 1-174, wherein the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and / or concentrated using a desalting chamber.

176. A composition comprising the oligonucleotide made according to the method of any one of claims 1-175.

177. The composition of claim 176, wherein the oligonucleotide is substantially free of depurination or depyrimidination impurities.

178. The composition of claim 176 or 177, wherein the oligonucleotide is substantially free of N-3- cyanoethylthymine (CNET) impurities.

179. The composition of any one of claims 176-178, wherein the oligonucleotide is substantially free of N(2)-actyl-2,6-diaminopurine and / or an isobutyryl diaminopurine impurities.

180. The composition of any one of claims 176-179, wherein the oligonucleotide is substantially free of methylcytosine.

181. The composition of any one of claims 176-180 is substantially free of Class IV impurities, preferably no detectable level of Class IV impurities.

182. A system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase; wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber.

183. The system of claim 182, further comprising a temperature regulator that controls a temperature of the solution in the system.

184. The system of claim 183, wherein the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber.

185. The system of any one of claims 182-184, comprising one or more conduits that connects the first reaction chamber and the second reaction chamber.

186. The system of claim 185, comprising an in-line temperature regulator that controls a temperature of the solution in at least one of the one or more conduits.

187. The system of claim 185 or 186, wherein the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.

188. The system of any one of claims 182-187, comprising one or more pumps configured to flow the solution from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber.

189. The system of claim 188, wherein the one or more pumps are configured to control a flow rate of the solution.

190. The system of any one of claims 182-189, further comprising a third reaction chamber comprising a second transferase, wherein the system is further configured to selectively flow said oligonucleotide in the solution from the second chamber to the third reaction chamber while retaining the hydrolase in the second reaction chamber, and flow the oligonucleotide in the solution from the third reaction chamber to the second reaction chamber while retaining the second transferase in the third reaction chamber.

191. The system of claim 190, wherein the transferase and the second transferase are different types of transferase.

192. The system of any one of claims 182-191, wherein the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.

193. The system of any one of claims 182-192, wherein the hydrolase can remove a 3' blocking moiety from the oligonucleotide in the solution.

194. The system of any one of claims 182-193, wherein the hydrolase is a phosphatase, preferably an alkaline phosphatase.

195. The system of any one of claims 182-194, wherein the transferase can react a nucleotide triphosphate (NTP) comprising a 3' blocking moiety, or an analog thereof comprising a 5' phosphate analog, with the oligonucleotide to elongate the oligonucleotide.

196. The system of claim 195, wherein the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.

197. The system of claim 195 or 196, wherein the NTP comprising the 3' blocking moiety or the analog thereof is a ribonucleotide or a deoxyribonucleotide, or further comprises a 2' modification, preferably 2'-F or 2'-O-methyl.

198. The system of any one of claims 195-197, wherein the 3' blocking moiety of the NTP or the analog thereof is a phosphate moiety.

199. The system of any one of claims 182-198, wherein the hydrolase can remove a 3'-blocking moiety from unreacted nucleotides comprising a 3' blocking moiety in the solution.

200. The system of claim 199, wherein the unreacted nucleotides are ribonucleotides or deoxyribonucleotides, or comprise a 2' modification, or preferably 2'-F or 2'-O-methyl.

201. The system of claim 199 or 200, wherein the 3' blocking moiety of the unreacted nucleotides is a phosphate moiety or a 5' phosphate analog.

202. The system of claim 201, wherein the 5' phosphate analog of the unreacted nucleotides is a 5'-(a-P-thio)phosphate moiety.

203. The system of any one of claims 182-202, wherein the hydrolase can remove one or more 5' phosphate moieties and / or 5'-(a-P-thio)phosphate moieties from the unreacted nucleotides in the solution.

204. The system of any one of claims 182-203, wherein the hydrolase is immobilized on a solid support.

205. The system of any one of claims 182-204, wherein the transferase comprises a polymerase from the DNA polymerase X family; a template independent transferase, preferably a terminal deoxynucleotidyl transferase (TdT); or an ssRNA ligase.

206. The system of any one of claims 182-205, wherein the transferase is immobilized on a solid support.

207. The system of any one of claims 182-206, wherein the first reaction chamber further comprises a pyrophosphatase, preferably an inorganic pyrophosphatase.

208. The system of any one of claims 182-207, further comprising a third reaction chamber comprising a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase.

209. The system of claim 207 or 208, wherein the pyrophosphatase is immobilized on a solid support.

210. The system of claim 207, wherein the transferase and phosphatase are fused together.

211. The system of claim 207, wherein the transferase and phosphatase are immobilized on the same solid support or are immobilized on different solid supports.

212. The system of any one of claims 182-211, wherein the first reaction chamber or the second reaction chamber is a column.

213. The system of claim 212, wherein the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support; or a fluidized bed comprising the transferase immobilized on a solid support; or comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide.

214. The system of claim 212 or 213, wherein the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support or a fluidized bed comprising the hydrolase immobilized on a solid support or a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide.

215. The system of any one of claims 182-211, wherein the first reaction chamber or the second reaction chamber is a batch reaction chamber, optionally wherein the batch reaction chamber comprises an impeller.

216. The system of claim 215, wherein: the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support; and / or the second reaction chamber comprises a rotating bed reactor comprising the hydrolase immobilized on a solid support.

217. The system of any one of claims 182-216, further comprising a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide.

218. The system of claim 217, wherein the purification chamber comprises a column, preferably a liquid chromatography column, a size exclusion column, an ion exchange column, or a reverse phase column.

219. The system of claim 217, wherein the purification chamber is part of a tangential flow filtration system.

220. The system of any one of claims 182-219, wherein the first reaction chamber or the second reaction chamber are cleanable or replaceable.

221. The system of any one of claims 182-220, wherein: the first reaction chamber comprises an inlet and an outlet; the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet; and wherein the system is configured to flow the oligonucleotide in the solution from an outlet of one of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from the outlet of the second reaction chamber to an inlet of the reagent reservoir.

222. The system of claim 221, wherein the reagent reservoir comprises an impeller.

223. The system of claim 221 or 222, wherein the reagent reservoir is replaceable or cleanable.

224. The system of any one of claims 221-223, further comprising a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the reagent reservoir.

225. The system of any one of claims 221-223, further comprising a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the first reaction chamber.

226. The system of claim 224 or 225, wherein the product reservoir comprises an impeller.

227. The system of any one of claims 224-226, wherein the product reservoir is replaceable or cleanable.

228. The system of any one of claims 221-227, wherein the reagent reservoir comprises the oligonucleotide and a nucleotide comprising a 3' blocking moiety.

229. The system of any one of claims 221-228, further comprising the purification chamber, wherein the purification chamber comprises an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution, in order, from the outlet of the second reaction chamber to the inlet of the purification chamber, and from the outlet of the purification chamber to the inlet of the reagent reservoir.

230. The system of any one of claims 221-229, wherein the system comprises the purification chamber, wherein the system is configured to flow the oligonucleotide, in the solution, from the outlet of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, from the outlet of the second reaction chamber to the inlet of the purification chamber, from the outlet of the purification chamber to the inlet of the product reservoir, and from the outlet of the product reservoir to the inlet of the reagent reservoir.

231. The system of any one of claims 182-220, wherein: the first reaction comprises an inlet and an outlet; the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet, and one or more diverter valves configured to control a flow pathway of the solution; wherein the system is configured to controllably flow the oligonucleotide in the solution through the flow pathway selected from a plurality of flow pathways comprising (i) a first flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the first reaction chamber without flowing through the second reaction chamber, and (ii) a second flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the second reaction chamber without flowing through the first reaction chamber.

232. The system of claim 231, wherein the system is configured to automatically select the flow pathway.

233. The system of claim 231 or 232, wherein: the first flow pathway comprises flow of the solution from the outlet of the first reaction chamber to the inlet of the reagent reservoir without flowing through the second reaction chamber;the second flow pathway comprises flow of the solution from the outlet of the second reaction chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber.

234. The system of any one of claims 231-233, wherein the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a third flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent reservoir to the inlet of the purification chamber without flowing through the first reaction chamber or the second reaction chamber.

235. The system of claim 234, wherein the third flow pathway further comprises flow of the solution from the outlet of the purification chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber or the second reaction chamber.

236. The system of any one of claims 231-235, wherein the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a fourth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the first reaction chamber, from an outlet of the first reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir.

237. The system of any one of claims 231-236, wherein the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a fifth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the second reaction chamber, from an outlet of the second reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir.

238. The system of any one of claims 231-237, wherein the reagent reservoir comprises an impeller.

239. The system of any one of claims 182-220, wherein the transferase in the first reaction chamber is immobilized on a solid support, and the first reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the transferase; and and the hydrolase in the second reaction chamber is immobilized on a solid support, and the second reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the hydrolase.

240. The system of any one of claims 182-220, wherein the transferase in the first reaction chamber is immobilized on a solid support within arotating bed reactor, and the first reaction chamber comprises an inlet and an outlet; and and the hydrolase in the second reaction chamber is immobilized on a solid support within a rotating bed reactor, and the second reaction chamber comprises an inlet and an outlet.

241. The system of claim 239 or 240, comprising one or more diverter valves configured to alternatively direct flow of the solution through (i) a first flow pathways comprising flow of the solution from the outlet of the first reaction chamber to an inlet of the purification chamber, and from an outlet of the purification chamber to the inlet of the first reaction chamber, or (ii) a second flow pathway comprising flow of the solution from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from an outlet of the second reaction chamber to the inlet of the first reaction chamber.

242. A system for template-free synthesis of an oligonucleotide comprising: a reagent reservoir; a first column configured to substantially separate a transferase from an oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety; wherein the system is configured to (i) flow a solution comprising an oligonucleotide comprising the 3' blocking moiety and a transferase from the reagent reservoir to the first column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the transferase in the first column, (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the first column to the reagent reservoir, (iv) flow the solution comprising an oligonucleotide without the 3' blocking moiety and a hydrolase to the second column, (v) substantially separate the oligonucleotide without the 3' blocking moiety from the hydrolase in the second column, and (vi) flow the solution comprising the oligonucleotide without the 3' blocking moiety from the second column to the reagent reservoir.

243. The system of claim 242, comprising a plurality of conduits that connects the first column, the second column, and the reagent reservoir.

244. The system of claim 242 or 243, further comprising a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety.

245. The system of claim 244, further comprising one or more conduits that connects the purification chamber and the reagent reservoir.

246. The system of claim 244 or 245, wherein the purification chamber comprises a column, preferably a size exclusion column, an ion exchange column, or a reverse phase column, or wherein the purification chamber is part of a tangential flow filtration system.

247. The system of any one of claims 244-246, wherein the system is further configured to (vii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the reagent reservoir to the purification chamber, (viii) separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety in the purification chamber, and (ix) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the purification chamber to the reagent reservoir.

248. The system of any one of claims 242-247, comprising one or more diverter valves configured to controllably flow the solution from the reagent reservoir to the first column or the second column.

249. The system of claim 248, wherein the one or more diverter valves is configured to select a flow path for the solution, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the solution from the reagent reservoir to the first column and from the first column to the reagent reservoir and (ii) flow of the solution from the reagent reservoir to the second column and from the second column to the reagent reservoir.

250. The system of any one of claims 244-249, wherein system comprises the purification chamber, and the one or more diverter valves is further configured to controllably flow the solution from the reagent reservoir to the purification chamber.

251. The system of claim 249 or 250, wherein the plurality of flow paths further comprises (iii) flow of the solution from the reagent reservoir to the purification chamber and from the purification chamber to the reagent reservoir.

252. The system of any one of claims 242-251, further comprising one or more wash buffer reservoirs connected to the first column, the second column, or the purification chamber.

253. The system of claim 252, wherein the one or more diverter valves is configured to controllably flow wash buffer from the wash buffer reservoir to the first column, the second column, or the purification chamber.

254. The system of claim 252 or 253, wherein the one or more diverter valves is configured to select a wash buffer flow path for wash buffer in the one or more wash buffer reservoirs, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the wash buffer from the one or more wash buffer reservoirs to the first column and from the first column to a systemwaste outlet and (ii) flow of the wash buffer from the one or more wash buffer reservoirs to the second column and from the second column to the system waste outlet.

255. The system of claim 254, wherein the system comprises the purification chamber, and wherein the plurality of flow paths further comprises (iii) flow of the wash buffer from the one or more wash buffer reservoirs to the purification chamber and from the purification chamber to the system waste outlet.

256. The system of any one of claims 242-255, further comprising a temperature regulator that controls a temperature of the solution in the system.

257. The system of claim 256, wherein the temperature regulator is configured to control the temperature of the solution in the first column or the second column.

258. The system of claim 256 or 257, comprising an in-line temperature regulator that controls a temperature of the solution in one or more conduits of the system.

259. The system of any one of claims 242-258, comprising one or more pumps configured to flow the solution from the reagent reservoir to the first column or from the reagent reservoir to the second column.

260. The system of claim 259, wherein the system comprises the purification chamber, and the one or more pumps are further configured to flow the solution from the reagent reservoir to the purification chamber.

261. The system of claim 259 or 260, wherein the one or more pumps are configured to control a flow rate of the solution.

262. The system of any one of claims 242-261, wherein the first column is further configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety.

263. The system of any one of claims 242-261, further comprising a third column configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety, wherein the system is further configured to flow a solution comprising the oligonucleotide comprising the 3' blocking moiety and the pyrophosphatase from the reagent reservoir to the third column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the pyrophosphatase in the third column, and (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the third column to the reagent reservoir.

264. The system of any one of claims 242-263, wherein the first column comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide; or a resin that binds the transferase oran ion exchange column, an affinity column, a size exclusion column, or reverse phase column.

265. The system of any one of claims 242-264, wherein the second column comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide; or a resin that binds the transferase; or an ion exchange column, an affinity column, a size exclusion column, or reverse phase column.

266. The system of any one of claims 242-265, wherein the reagent reservoir comprises an impeller.

267. The system of any one of claims 242-266, wherein the reagent reservoir comprises a reagent port.

268. The system of any one of claims 242-267, wherein the first column or the second column are cleanable or replaceable.

269. The system of any one of claims 182-268, wherein the system comprises a degassing system or a sparging system.

270. The system of claim 269, wherein the sparging system is an in-line sparging system or is configured to sparge liquids in a reservoir, and / or is configured to sparge using an inert gas.

271. The system of claim 269 wherein the degasser system is configured to remove oxygen from the system.

272. The system of claim 183 or 256, wherein the temperature regulator is a jacketed stir tank.

273. The system of any one of claims 217-219, 229, 230, 234-237, 241, 244-255, and 260, wherein the purification chamber is configured to concentrate the elongated oligonucleotide comprising the 3' blocking moiety.

274. The system of any one of claims 217-219, 229, 230, 234-237, 241, 244-255, 260, and 273, wherein the purification chamber comprises a membrane with a molecular weight cutoff of about 500 kDa to about 5000 kDa.

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