Enzymatic RNA synthesis

Abstract

The present invention describes methods for the controlled de novo synthesis of RNA oligonucleotides using enzymatic catalysis. For example, the present invention provides methods for preparing RNA oligonucleotides by the controlled, template-independent addition of nucleotides to the 3'-end of an initiator oligonucleotide via enzymatic catalysis (also referred to as terminal transferase activity). Individual nucleotides can be repeatedly added by a compatible polymerase (e.g., poly(N) polymerase such as poly(U) polymerase) until the desired RNA oligonucleotide sequence is synthesized. Nucleotides and polymerases useful in the methods described herein are also provided.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 745,136, filed Oct. 12, 2018, under 35 U.S.C. § 119(e), the entire content of which is incorporated herein by reference.

[0003] Government Support

[0004] This invention was made with government support under Award No. DE-FG02-02ER63445, awarded by the U.S. Department of Energy. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0006] Synthetic oligonucleotides are crucial for many aspects of biotechnological research in academic and industrial settings. Despite high demand for longer, cheaper, and error-free oligonucleotides, current industry leaders have not addressed many limitations of traditional chemical synthesis methods developed decades ago. This is especially true for de novo RNA oligonucleotide synthesis, which remains largely inaccessible to those investing heavily in enabling genome engineering technologies, RNA-based diagnostics and therapeutics, RNA-based sequencing technologies, high-density nucleic acid-based information storage, and biocomputing. Although there have been some improvements to methods for chemically synthesizing RNA oligonucleotides, overall chemistry has changed little since the late 1970s (Hughes and Ellington 2017). Many complex reaction steps plague chemical synthesis, which require harsh chemical reagents and biologically incompatible organic solvents. These reaction conditions result in depurination of nucleobases, accidental insertions or deletions throughout the sequence, and pre-emptive irreversible capping of oligonucleotides, resulting in undesired truncated products. This greatly increases the overall error rate of synthesis, limits the maximum length of RNA oligonucleotides to less than 120 nucleotides, and requires longer production lead times to obtain appreciable yields of desired products. In addition, chemical synthesis of RNA oligonucleotides is very expensive; compared to the current cost of $0.1 per base for DNA oligonucleotide synthesis (Carlson 2018), RNA synthesis is nearly 100 times higher, not counting the cost of long or complex RNA oligonucleotides. Thus, it is important to address the current limitations of RNA oligonucleotide synthesis. SUMMARY OF THE INVENTION

[0008] This disclosure describes compounds, enzymes, compositions, systems, kits, and methods for the use of enzymatic catalysis for the controlled de novo synthesis of RNA oligonucleotides. For example, provided herein are methods for preparing RNA oligonucleotides by the enzymatic catalysis (also referred to as terminal transferase activity) of the controlled template-independent addition of nucleotides to the 3' end of a primer oligonucleotide. The addition of a single nucleotide can be repeated by a compatible polymerase (e.g., a poly(N) polymerase such as poly(U) polymerase) until the desired RNA oligonucleotide sequence is synthesized.

[0009] This disclosure is based on the discovery that certain polymerases can efficiently catalyze template-independent terminal transferase reactions with a variety of modified and unmodified nucleotides. In one aspect, provided herein are methods for synthesizing RNA oligonucleotides, wherein a poly(N) polymerase incorporates one or more nucleotides at the 3' end of a primer oligonucleotide. It has been found that certain polymerases such as poly(U) and poly(A) polymerases, among others, can catalyze terminal transferase reactions with a variety of nucleotides, including modified nucleotides. After incorporation of one or more nucleotides by the poly(N) polymerase via terminal transferase, this process can be repeated in one or more iterative steps, optionally using different nucleotides, until the desired RNA oligonucleotide sequence is obtained. Also provided herein are novel poly(N) polymerases (e.g., mutant poly(U) polymerases) that can be used in the methods described herein.

[0010] In one aspect, provided herein are methods for preparing RNA oligonucleotides, the method comprising combining a primer oligonucleotide, a poly(N) polymerase (e.g., poly(U) polymerase) with one or more modified nucleotides under conditions sufficient to add at least one modified nucleotide to the 3' terminus of the primer oligonucleotide, thereby synthesizing an RNA oligonucleotide. The method can further comprise adding one or more additional nucleotides (modified or unmodified) to the resulting RNA oligonucleotide in iterative steps until the desired RNA oligonucleotide sequence is obtained. Also provided herein are compounds (e.g., modified nucleotides) that can be used in the methods described herein.

[0011] This disclosure provides other methods for the controlled de novo synthesis of RNA oligonucleotides. For example, in another aspect, the invention provides methods wherein modified nucleotides (i.e., "reversible terminator oligonucleotides", e.g., 2'- or 3'-O-protected nucleotides) are incorporated that reversibly alter the binding affinity of a polymerase (e.g., a poly(N) polymerase such as poly(U)) for the extended primer oligonucleotide. This alteration in binding affinity results in the termination of further nucleotide addition, thereby producing an (n+1) oligonucleotide product that can be further extended after the modified group is restored to its native state (e.g., a 2'- or 3'-OH group) via mild deprotection chemistry. An "(n+1) oligonucleotide" is a product in which a single nucleotide has been added to the primer sequence. These methods are inFigure 2 Illustrated in the general scheme shown in. In certain embodiments, the modified oligonucleotide is a 2'- or 3'-modified reversible terminator oligonucleotide. In certain embodiments, provided herein are methods for synthesizing RNA oligonucleotides, including combining a primer oligonucleotide, a poly(N) polymerase (e.g., poly(U) polymerase), and a reversible terminator nucleotide (e.g., a 2'- or 3'-modified reversible terminator oligonucleotide) under conditions sufficient to add the reversible terminator nucleotide to the 3'-end of the primer oligonucleotide; followed by a step of deprotecting the protected position of the reversible terminator nucleotide of the resulting RNA oligonucleotide. Once deprotected, the resulting (n+1)-extended RNA oligonucleotide can be subjected to subsequent terminal transferase reactions involving one or more modified or unmodified nucleotides until the desired RNA oligonucleotide sequence is obtained. Also provided herein are 2'- or 3'-modified reversible terminator oligonucleotides (e.g., 2'- or 3'-O-protected nucleotides) that can be used in the methods described herein.

[0012] On the other hand, provided herein are methods for synthesizing RNA oligonucleotides using non-hydrolyzable nucleotides. In these methods, the rate at which the polymerase incorporates nucleotides at the 3'-end of the primer oligonucleotide is controlled by introducing non-hydrolyzable nucleotides that compete for the active site of the enzyme. These methods are illustrated in Figure 1 the general scheme shown in. The rate of oligonucleotide synthesis is directly affected by the ratio of hydrolyzable nucleotides to non-hydrolyzable nucleotides through competitive inhibition. In addition to the ratio of these nucleotides, other reaction parameters can be fine-tuned to regulate the reaction rate of the controlled synthesis. In certain embodiments, provided herein are methods for synthesizing RNA oligonucleotides, which include combining a primer oligonucleotide, a poly(N) polymerase (e.g., poly(U) polymerase), one or more nucleotides, and one or more non-hydrolyzable nucleotides under conditions sufficient to add at least one hydrolyzable nucleotide to the 3'-end of the primer oligonucleotide, wherein the concentration of the non-hydrolyzable nucleotides is sufficient to inhibit the rate of addition of one or more nucleotides by the poly(N) polymerase; thereby synthesizing an RNA oligonucleotide. The method can further include adding one or more additional nucleotides (modified or unmodified) to the resulting RNA oligonucleotide until the desired RNA oligonucleotide sequence is obtained. Also provided herein are non-hydrolyzable nucleotides that can be used in the methods described herein.

[0013] In addition, the present disclosure provides methods for joining two oligonucleotides to produce RNA oligonucleotides. In certain embodiments, the method includes providing a first oligonucleotide, wherein the first oligonucleotide comprises a 5'-triphosphate group; providing a second oligonucleotide; providing a poly(U) polymerase; and combining the first and second oligonucleotides with the poly(U) polymerase under conditions sufficient to join the 3'-end of the first oligonucleotide to the second oligonucleotide. This embodiment is feasible because it has been found that 5'-triphosphate nucleotides having an oligonucleotide at the 3'-position are viable substrates for the poly(N) polymerases described herein (e.g., wild-type and mutant poly(U) polymerases).

[0014] Additionally, the RNA oligonucleotides produced by these methods can be reverse transcribed (RT) to produce complementary DNA (e.g., cDNA), which can be amplified by a DNA polymerase via polymerase chain reaction (PCR).

[0015] The present disclosure also provides RNA oligonucleotides and DNA oligonucleotides produced by any of the methods described herein.

[0016] The methods described herein involve enzymatic catalysis. Since enzymatic catalysis occurs under biocompatible reaction conditions, unwanted degradation of RNA molecules currently experienced in chemical synthesis can be eliminated. The methods of the present invention improve the current state of de novo synthesis of RNA oligonucleotides, which is typically carried out with phosphoramidite chemistry under harsh reaction conditions. The harsh reaction conditions for chemical synthesis of RNA oligonucleotides make it difficult to produce long RNA oligonucleotides, such as those greater than 100 nucleotides in length, and are costly. If a substantial yield of long RNA oligonucleotides is produced via chemical synthesis, the error rate of the oligonucleotides may be high. By using the enzymatic methods described herein, many of the problems currently associated with the synthesis of long RNA oligonucleotides are solved. Applications of the methods described herein include the direct synthesis of RNA and the production of materials for nucleic acid nanotechnology, genome engineering technologies, and novel RNA and DNA therapeutics. In certain embodiments, the methods described herein can be miniaturized in a microfluidic format or carried out in a highly parallel manner such as micro-droplet printing. The methods provided herein can also be carried out in the solid phase.

[0017] The present disclosure also provides compositions and kits comprising one or more of the poly(N) polymerases and / or nucleotides described herein.

[0018] Details of certain embodiments of the present invention are set forth in the "Detailed Description of Certain Embodiments" described below. Other features, objects, and advantages of the present invention will be apparent from the definitions, examples, drawings, and claims.

[0019] Definitions

[0020] General Definitions

[0021] As used herein, the term "polymerase" generally refers to an enzyme that can synthesize RNA or DNA oligonucleotides. In some embodiments, the polymerase can synthesize oligonucleotides in a template-dependent manner. In other embodiments, the polymerase can synthesize oligonucleotides in a template-independent manner. In some embodiments, the polymerase is an RNA polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the polymerase is a reverse transcriptase. The polymerase can be derived from any source, such as a recombinant polymerase, a bacterial polymerase. In some embodiments, the polymerase is a poly(N) polymerase. In some embodiments, the polymerase is a poly(U), poly(A), poly(C), or poly(G) polymerase. In some embodiments, the polymerase can add nucleotides, such as nucleotides, to the 3' end of an oligonucleotide, such as a primer oligonucleotide. In some embodiments, the polymerase selectively adds a nucleotide, such as a nucleotide containing a uracil base, to the 3' end of an oligonucleotide, such as a primer oligonucleotide, in the presence of a poly(U) polymerase.

[0022] As used herein, the term "RNA oligonucleotide" generally refers to a polymer of nucleotides, ribonucleotides, or analogs thereof. RNA oligonucleotides can have any sequence. As used herein, RNA oligonucleotides can have any three-dimensional structure and can perform any function known or unknown to those of skill in the art. RNA oligonucleotides can be naturally occurring or synthetic. In some embodiments, the RNA oligonucleotide can be messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, recombinant oligonucleotide, branched oligonucleotide, isolated or synthetic RNA oligonucleotide of any sequence, probe, and / or primer. In some embodiments, the RNA oligonucleotide comprises nucleotides having naturally occurring bases such as adenine or uracil. In some embodiments, the RNA oligonucleotide comprises non-naturally occurring or modified nucleotides, such as nucleotides comprising sugar modifications, base modifications such as purine or pyrimidine modifications. In some embodiments, the RNA oligonucleotide comprises a combination of naturally occurring, non-naturally occurring, and modified nucleotides. In some embodiments, the nucleotide can comprise at least one modified backbone or linkage, such as a phosphorothioate backbone or linkage. In some embodiments, the RNA oligonucleotide is single-stranded. In other embodiments, the RNA oligonucleotide is double-stranded. In some embodiments, the RNA oligonucleotide is synthesized by a template-independent synthesis method. In some embodiments, the RNA oligonucleotide has a length of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides.

[0023] As used herein, the term "DNA oligonucleotide" generally refers to a polymer of DNA nucleotides, deoxyribonucleotides, or analogs thereof. As used herein, a DNA oligonucleotide can have any three-dimensional structure and can perform any function known or unknown to those skilled in the art. DNA oligonucleotides can be naturally occurring or synthetic. In some embodiments, a DNA oligonucleotide can be an exon, intron, cDNA sequence, recombinant oligonucleotide, branched oligonucleotide, plasmid, vector, and / or isolated DNA of any sequence. In some embodiments, a DNA oligonucleotide comprises DNA nucleotides that comprise naturally occurring bases such as adenine, cytosine, guanine, or thymine. In some embodiments, a DNA oligonucleotide comprises non-naturally occurring or modified DNA nucleotides, such as DNA nucleotides comprising sugar modifications, purine, or pyrimidine modifications. In some embodiments, a DNA oligonucleotide comprises a combination of naturally occurring, non-naturally occurring, and modified DNA nucleotides. In some embodiments, a DNA nucleotide can comprise at least one modified backbone or linkage, such as a phosphorothioate backbone or linkage. In some embodiments, a DNA oligonucleotide is single-stranded. In other embodiments, a DNA oligonucleotide is double-stranded. In some embodiments, a DNA oligonucleotide is synthesized by reverse transcription. In some embodiments, a DNA oligonucleotide has a length of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 DNA nucleotides.

[0024] As used herein, the term "nucleotide" or "ribonucleotide" generally refers to a nucleotide monomer comprising ribose, a phosphate group, and a nucleobase. Nucleotides can be naturally occurring, non-naturally occurring, or modified. In some embodiments, a nucleotide comprises a nucleobase or base, such as a purine or pyrimidine base. In some embodiments, the base is a naturally occurring base, such as adenine, cytosine, guanine, thymine, uracil, or inosine. In some embodiments, a nucleotide can comprise a non-naturally occurring nucleobase. In some embodiments, a nucleotide can comprise a modified nucleobase. In some embodiments, a nucleotide can comprise, for example, a ribose modification at the 2' position, such as 2'-F, 2'-O-alkyl, 2'-amino, or 2'-azido. In some embodiments, a nucleotide is a non-hydrolyzable nucleotide, such as can comprise a modified triphosphate group. In certain embodiments, a modified nucleotide is a reversible terminator oligonucleotide, such as a 2'- or 3'-OH-protected nucleotide.

[0025] As used herein, the term "initiator oligonucleotide" generally refers to a short single-stranded RNA oligonucleotide that can initiate template-independent synthesis. In certain embodiments, the initiator oligonucleotide has a length of less than 20 nucleotides. In some embodiments, the initiator oligonucleotide has a length of less than 20, less than 18, less than 15, less than 12, less than 10, less than 8, or less than 5 nucleotides. In some embodiments, the initiator oligonucleotide is labeled at its 5' end, for example, with a fluorophore. In some embodiments, the initiator oligonucleotide is attached to a matrix at its 5' end. In some embodiments, the matrix can be a glass surface, a bead, a biomolecule, or any conceivable matrix suitable for template-independent synthesis.

[0026] As used herein, the term "template-independent" generally refers to a mode of synthesis of RNA oligonucleotides that does not require a template DNA oligonucleotide. Template-independent synthesis generally involves the use of an initiator oligonucleotide and a polymerase, such as poly(N) polymerase. Oligonucleotides synthesized using a template-independent mode of synthesis, such as RNA oligonucleotides, are generally synthesized by adding nucleotides, such as nucleotides, to the 3' end of an existing oligonucleotide, such as an initiator oligonucleotide.

[0027] Chemical Definitions

[0028] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are determined according to the periodic table of the elements on the inside cover of the 75th edition of the Handbook of Chemistry and Physics, CAS version, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry and specific functional moieties and reactivity are found in the following publications: Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5 thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987。

[0029] The compounds described herein may contain one or more asymmetric centers and can thus exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of a single enantiomer, a diastereomer, or a geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen et al., Tetrahedron 33:2725 (1977), Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further includes compounds in the form of a single isomer substantially free of other isomers or a mixture of various isomers.

[0030] Unless otherwise indicated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, having the structure described in the present invention but replacing hydrogen with deuterium or tritium, replacing 18 F with 19 F or replacing 13 C or 14C substitution 12 Compounds of C are all within the scope of the present disclosure. Such compounds are used, for example, as analytical tools or probes in biological assays.

[0031] When a range of values is listed, it is meant to cover every value and sub-range within that range. For example, "C 1-6 alkyl" means to cover C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0032] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon-based group having 1-10 carbon atoms (C 1-10 alkyl). In some embodiments, the alkyl group has 1-9 carbon atoms (C 1-9 alkyl). In some embodiments, the alkyl group has 1-8 carbon atoms (C 1-8 alkyl). In some embodiments, the alkyl group has 1-7 carbon atoms (C 1-7 alkyl). In some embodiments, the alkyl group has 1-6 carbon atoms (C 1-6 alkyl). In some embodiments, the alkyl group has 1-5 carbon atoms (C 1-5 alkyl). In some embodiments, the alkyl group has 1-4 carbon atoms (C 1-4 alkyl). In some embodiments, the alkyl group has 1-3 carbon atoms (C 1-3 alkyl). In some embodiments, the alkyl group has 1-2 carbon atoms (C 1-2 alkyl). In some embodiments, the alkyl group has 1 carbon atom (C1 alkyl). In some embodiments, the alkyl group has 2-6 carbon atoms (C 2-6 alkyl). C 1-6Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentyl, amyl, neopentyl, 3-methyl-2-butyl, tert-pentyl) and hexyl (C6) (e.g., n-hexyl). Other examples of alkyls include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (unsubstituted alkyl) or substituted by one or more substituents (e.g., halogen such as F) (substituted alkyl). In certain embodiments, the alkyl group is unsubstituted C 1-10 alkyl (e.g., unsubstituted C 1-6 alkyl, e.g., -CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is substituted C 1-10 alkyl (e.g., substituted C 1-6 alkyl, e.g., -CF3, Bn).

[0033] The term "haloalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a halogen such as fluorine, bromine, chlorine or iodine. In some embodiments, the haloalkyl has 1-8 carbon atoms (C 1-8 haloalkyl). In some embodiments, the haloalkyl has 1-6 carbon atoms (C 1-6 haloalkyl). In some embodiments, the haloalkyl has 1-4 carbon atoms (C 1-4 haloalkyl). In some embodiments, the haloalkyl has 1-3 carbon atoms (C 1-3 haloalkyl). In some embodiments, the haloalkyl has 1-2 carbon atoms (C 1-2 haloalkyl). Examples of haloalkyl groups include -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, -CCl3, -CFCl2, -CF2Cl, etc.

[0034] The term "heteroalkyl" refers to an alkyl group that further includes at least one heteroatom selected from oxygen, nitrogen or sulfur (e.g., 1, 2, 3 or 4 heteroatoms) within the parent chain (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal positions of the parent chain.

[0035] The term "alkenyl" refers to a straight-chain or branched hydrocarbon radical group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 2 to 9 carbon atoms (C 2-9 alkenyl). In some embodiments, the alkenyl group has 2 to 8 carbon atoms (C 2-8 alkenyl). In some embodiments, the alkenyl group has 2 to 7 carbon atoms (C 2-7 alkenyl). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (C 2-6 alkenyl). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (C 2-5 alkenyl). In some embodiments, the alkenyl group has 2 to 4 carbon atoms (C 2-4 alkenyl). In some embodiments, the alkenyl group has 2 to 3 carbon atoms (C 2-3 alkenyl). In some embodiments, the alkenyl group has 2 carbon atoms (C2 alkenyl). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). C 2-4 Examples of alkenyl include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. C 2-6 Examples of alkenyl groups include the above C 2-4 alkenyl as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Other examples of alkenyl include heptenyl (C7), octenyl (C8), octatrieneyl (C8), etc. Unless otherwise specified, each example of alkenyl is independently unsubstituted (unsubstituted alkenyl) or substituted by one or more substituents (substituted alkenyl). In certain embodiments, the alkenyl group is unsubstituted C 2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C 2-10 alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., -CH═CHCH3 or ) can be an (E)- or (Z)-double bond.

[0036] The term "heteroalkenyl" refers to an alkenyl group that further includes at least one heteroatom selected from oxygen, nitrogen, or sulfur (e.g., 1, 2, 3, or 4 heteroatoms) within the backbone (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal positions of the backbone.

[0037] The term "alkynyl" refers to a straight-chain or branched hydrocarbon radical group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (C 2-10alkynyl). In some embodiments, the alkynyl group has 2-9 carbon atoms (C 2-9 alkynyl). In some embodiments, the alkynyl group has 2-8 carbon atoms (C 2-8 alkynyl). In some embodiments, the alkynyl group has 2-7 carbon atoms (C 2-7 alkynyl). In some embodiments, the alkynyl group has 2-6 carbon atoms (C 2-6 alkynyl). In some embodiments, the alkynyl group has 2-5 carbon atoms (C 2-5 alkynyl). In some embodiments, the alkynyl group has 2-4 carbon atoms (C 2-4 alkynyl). In some embodiments, the alkynyl group has 2-3 carbon atoms (C 2-3 alkynyl). In some embodiments, the alkynyl group has 2 carbon atoms (C2 alkynyl). One or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). C 2-4 Examples of alkynyl include but are not limited to ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. C 2-6 Examples of alkynyl include the above-mentioned C 2-4 alkynyl as well as pentynyl (C5), hexynyl (C6), etc. Other examples of alkynyl include heptynyl (C7), octynyl (C8), etc. Unless otherwise specified, each example of alkynyl is independently unsubstituted (unsubstituted alkynyl) or substituted by one or more substituents (substituted alkynyl). In certain embodiments, the alkynyl group is unsubstituted C 2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C 2-10 alkynyl.

[0038] The term "heteroalkynyl" refers to an alkynyl group that further includes at least one heteroatom selected from oxygen, nitrogen, or sulfur (e.g., 1, 2, 3, or 4 heteroatoms) within the backbone (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal positions of the backbone.

[0039] The term "carbocyclic group" or "carbocyclic" refers to a non-aromatic cycloalkyl group having 3-14 ring carbon atoms (C 3-14 carbocyclic group) and zero heteroatoms. In some embodiments, the carbocyclic group has 3-10 ring carbon atoms (C 3-10 carbocyclic group). In some embodiments, the carbocyclic group has 3-8 ring carbon atoms (C 3-8 carbocyclic group). In some embodiments, the carbocyclic group has 3-7 ring carbon atoms (C 3-7carbocyclic group). In some embodiments, the carbocyclic group has 3-6 ring carbon atoms (C 3-6 carbocyclic group). In some embodiments, the carbocyclic group has 4-6 ring carbon atoms (C 4-6 carbocyclic group). In some embodiments, the carbocyclic group has 5-6 ring carbon atoms (C 5-6 carbocyclic group). In some embodiments, the carbocyclic group has 5-10 ring carbon atoms (C 5-10 carbocyclic group). Exemplary C 3-6 carbocyclic groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Exemplary C 3-8 carbocyclic groups include, but are not limited to, the above C 3-6 carbocyclic groups and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Exemplary C 3-10 carbocyclic groups include, but are not limited to, the above C 3-8 carbocyclic groups and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ), etc. As shown in the foregoing examples, in certain embodiments, the carbocyclic group is monocyclic (monocyclic carbocyclic group) or polycyclic (e.g., comprising a fused ring, bridged ring or spiro ring system, e.g., a bicyclic system (bicyclic carbocyclic group) or a tricyclic system (tricyclic carbocyclic group)), and can be saturated or can contain one or more carbon-carbon double bonds or triple bonds. "Carbocyclic group" also includes ring systems in which a carbocyclic ring as defined above is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the carbocyclic ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclic group is independently unsubstituted (unsubstituted carbocyclic group) or substituted by one or more substituents (substituted carbocyclic group). In certain embodiments, the carbocyclic group is unsubstituted C 3-14 carbocyclic group. In certain embodiments, the carbocyclic group is substituted C 3-14 carbocyclic group.

[0040] In some embodiments, "carbocyclic group" is a monocyclic saturated carbocyclic group having 3-14 ring carbon atoms (C 3-14cycloalkyl). In some embodiments, the cycloalkyl has 3 to 10 ring carbon atoms (C 3-10 cycloalkyl). In some embodiments, the cycloalkyl has 3 to 8 ring carbon atoms (C 3-8 cycloalkyl). In some embodiments, the cycloalkyl has 3 to 6 ring carbon atoms (C 3-6 cycloalkyl). In some embodiments, the cycloalkyl has 4 to 6 ring carbon atoms (C 4-6 cycloalkyl). In some embodiments, the cycloalkyl has 5 to 6 ring carbon atoms (C 5-6 cycloalkyl). In some embodiments, the cycloalkyl has 5 to 10 ring carbon atoms (C 5-10 "cycloalkyl"). C 5-6 Examples of cycloalkyl include cyclopentyl (C5) and cyclohexyl (C5). C 3-6 Examples of cycloalkyl include the above C 5-6 cycloalkyl and cyclopropyl (C3) and cyclobutyl (C4). C 3-8 Examples of cycloalkyl include the above C 3-6 cycloalkyl and cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of cycloalkyl is independently unsubstituted (unsubstituted cycloalkyl) or substituted by one or more substituents (substituted cycloalkyl). In certain embodiments, the cycloalkyl group is unsubstituted C 3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C 3-14 cycloalkyl.

[0041] The term "heterocyclic group" or "heterocyclic" refers to a 3- to 14-membered non-aromatic ring system group having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (3- to 14-membered heterocyclic group). Where valence permits, in a heterocyclic group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom. The heterocyclic group can be monocyclic (monocyclic heterocyclic group) or polycyclic (e.g., fused, bridged, or spiro ring systems, such as bicyclic systems (bicyclic heterocyclic group) or tricyclic systems (tricyclic heterocyclic group)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. The heterocyclic polycyclic ring system contains one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems where a heterocyclic as defined above is fused to one or more carbocyclic groups, where the point of attachment is on the carbocyclic group or the heterocyclic group ring; or includes ring systems where a heterocyclic as defined above is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclic ring, and in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclic ring system. Unless otherwise specified, each instance of the heterocyclic group is independently unsubstituted (unsubstituted heterocyclic group) or substituted by one or more substituents (substituted heterocyclic group). In certain embodiments, the heterocyclic group is an unsubstituted 3- to 14-membered heterocyclic group. In certain embodiments, the heterocyclic group is a substituted 3- to 14-membered heterocyclic group.

[0042] In some embodiments, the heterocyclic group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5- to 10-membered heterocyclic group). In some embodiments, the heterocyclic group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5- to 8-membered heterocyclic group). In some embodiments, the heterocyclic group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5- to 6-membered heterocyclic group). In some embodiments, the 5- to 6-membered heterocyclic group has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclic group has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclic group has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0043] The term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aryl ring system group (e.g., having 6, 10, or 14 π electrons shared in the ring array) ("C 6-14 aryl"), which has 6 to 14 ring carbon atoms and zero heteroatoms provided in the aryl ring system. In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C 10"Aryl", such as naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 "Aryl", such as anthracenyl). "Aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, where the group or point of attachment is on the aromatic ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aromatic ring system. Unless otherwise specified, each instance of the aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is unsubstituted C 6-14 aryl. In certain embodiments, the aryl group is substituted C 6-14 aryl.

[0044] The term "heteroaryl" refers to a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system group having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring array), where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). Where valence permits, in a heteroaryl group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom. The heteroaryl polycyclic ring system may contain one or more heteroatoms in one or both of the rings. "Heteroaryl" includes ring systems in which a heteroaromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, where the point of attachment is on the heteroaromatic ring, in which case the number of ring members continues to refer to the number of ring members in the heteroaromatic ring system. "Heteroaryl" also includes ring systems in which a heteroaromatic ring as defined above is fused to one or more aryl groups, where the point of attachment is on the aromatic or heteroaromatic ring, in which case the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. For a polycyclic heteroaryl group in which one of the rings does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment may be on either ring, i.e., the ring with the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

[0045] In some embodiments, a heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms provided in an aromatic ring system and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 10-membered heteroaryl”). In some embodiments, a heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms provided in an aromatic ring system and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 8-membered heteroaryl”). In some embodiments, a heteroaryl is a 5- to 6-membered aromatic ring system having ring carbon atoms provided in an aromatic ring system and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 6-membered heteroaryl”). In some embodiments, the 5- to 6-membered heteroaryl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In certain embodiments, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5- to 14-membered heteroaryl.

[0046] Unless otherwise expressly stated, a group is optionally substituted. The term “optionally substituted” means substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups are optionally substituted. “Optionally substituted” means that the group can be substituted or unsubstituted. Generally, the term “substituted” means that at least one hydrogen present on the group is replaced by a permitted substituent, such as a substituent that results in a stable compound upon substitution, such as a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, or other reactions. Unless otherwise specified, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents can be the same or different at each position. The term “substituted” is intended to include substitution by all permitted substituents of organic compounds, including any of the substituents described herein that result in the formation of a stable compound. The present invention contemplates any and all such combinations in order to obtain stable compounds. For the purposes of the present invention, a heteroatom such as nitrogen can have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence requirements of the heteroatom and result in the formation of a stable moiety. The present invention is not intended to be limited in any way by the exemplary substituents described herein.

[0047] Exemplary substituents include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )3, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(Raa )3-C(=S)N(R bb )2,-C(=O)SR aa ,-C(=S)SR aa ,-SC(=S)SR aa ,-SC(=O)SR aa ,-OC(=O)SR aa ,-SC(=O)OR aa ,-SC(=O)R aa ,-P(=O)(R aa )2,-P(=O)(OR cc )2,-OP(=O)(R aa )2,-OP(=O)(OR cc )2,-P(=O)(N(R bb )2)2,-OP(=O)(N(R bb )2)2,-NR bb P(=O)(R aa )2,-NR bb P(=O)(OR cc )2,-NR bb P(=O)(N(R bb )2)2,-P(R cc )2,-P(OR cc )2,-P(R cc )3 + X - ,-P(OR cc )3 + X - ,-P(R cc )4,-P(OR cc )4,-OP(R cc )2,-OP(R cc )3 + X - ,-OP(OR cc )2,-OP(OR cc )3 + X - ,-OP(R cc )4,-OP(OR cc )4,-B(R aa )2,-B(OR cc )2,-BR aa (OR cc ),C 1-10 Alkyl,C 1-10 Perhaloalkyl,C 2-10 Alkenyl,C 2-10 Alkynyl,Hetero-C1-10 Alkyl, hetero-C 2-10 Alkenyl, hetero-C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3- to 14-membered heterocyclic group, C 6-14 Aryl, and 5- to 14-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic group, heterocyclic group, aryl, and heteroaryl is independently substituted by 0, 1, 2, 3, 4, or 5 R dd groups; wherein X - is a counterion;

[0048] Or two geminal hydrogens on a carbon atom are replaced by the following groups: =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc ;

[0049] Each instance of R aa is independently selected from C 1-10 alkyl, C 1-10 perhaloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, hetero-C 1-10 alkyl, hetero-C 2-10 alkenyl, hetero-C 2-10 alkynyl, C 3-10 carbocyclic group, 3- to 14-membered heterocyclic group, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R aa groups are joined to form a 3- to 14-membered heterocyclic group or a 5- to 14-membered heteroaryl ring;

[0050] Each instance of R bb is independently selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2ORcc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)(R aa )2, -P(=O)(OR cc )2, -P(=O)(N(R cc )2)2, C 1-10 alkyl, C 1-10 perhaloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, hetero-C 1-10 alkyl, hetero-C 2-10 alkenyl, hetero-C 2-10 alkynyl, C 3-10 carbocyclic group, 3- to 14-membered heterocyclic group, C 6-14 aryl and 5- to 14-membered heteroaryl, or two R bb groups are linked to form a 3- to 14-membered heterocyclic group or a 5- to 14-membered heteroaryl ring;

[0051] R cc each instance of which is independently selected from hydrogen, C 1-10 alkyl, C 1-10 perhaloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, hetero-C 1-10 alkyl, hetero-C 2-10 alkenyl, hetero-C 2-10 alkynyl, C 3-10 carbocyclic group, 3- to 14-membered heterocyclic group, C 6-14 aryl and 5- to 14-membered heteroaryl, or two R cc groups are linked to form a 3- to 14-membered heterocyclic group or a 5- to 14-membered heteroaryl ring;

[0052] R dd each instance of which is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X -, -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH2, -NHC(=NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2(C 1-6 alkyl), -SO2O(C 1-6 alkyl), -OSO2(C 1-6 alkyl), -SO(C 1-6 alkyl), -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3 - C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6(alkyl), C(=S)NH2, -C(=O)S(C 1-6 (alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)(OC 1-6 (alkyl)2, -P(=O)(C 1-6 (alkyl)2, -OP(=O)(C 1-6 (alkyl)2, -OP(=O)(OC 1-6 (alkyl)2, C 1-6 alkyl, C 1-6 perhaloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, hetero-C 1-6 alkyl, hetero-C 2-6 alkenyl, hetero-C 2-6 alkynyl, C 3-10 carbocyclic group, C 6-10 aryl, 3- to 10-membered heterocyclic group, 5- to 10-membered heteroaryl;

[0053] or two geminal R dd substituents may be joined together to form =O or =S.

[0054] The term "halo" or "halogen" means fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) or iodine (iodo, -I).

[0055] The term "hydroxy group" or "hydroxy" means the group -OH. Extensively, the term "substituted hydroxy group" or "substituted hydroxy" means a hydroxy group in which the oxygen atom directly attached to the parent molecule is replaced by a group other than hydrogen, including groups selected from: -OR aa , -ON(R bb )2, -OC(=O)SR aa , -OC(=O)R aa , -OCO2R aa , -OC(=O)N(R bb )2, -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -OC(=NR bb )N(R bb )2, -OS(=O)R aa , -OSO2R aa , -OSi(R aa )3, -OP(R cc )2, -OP(R cc )3 + X -, -OP(OR cc )2, -OP(OR cc )3 + X - , -OP(=O)(R aa )2, -OP(=O)(OR cc )2 and -OP(=O)(N(R bb )2)2, where X - 、R aa 、R bb and R cc are as defined herein.

[0056] The term "amino" refers to the group -NH2. Extending this, the term "substituted amino" refers to a mono-substituted amino, a di-substituted amino, or a tri-substituted amino. In certain embodiments, "substituted amino" is a mono-substituted amino or a di-substituted amino. The term "mono-substituted amino" refers to an amino in which the nitrogen atom directly attached to the parent molecule is substituted by one hydrogen and one group other than hydrogen, including groups selected from: -NH(R bb ), -NHC(=O)R aa , -NHCO2R aa , -NHC(=O)N(R bb )2, -NHC(=NR bb )N(R bb )2, -NHSO2R aa , -NHP(=O)(OR cc )2 and -NHP(=O)(N(R bb )2)2, where R aa 、R bb and R cc are as defined herein, and where the R bb of the -NH(R bb ) group is not hydrogen. The term "di-substituted amino" refers to an amino in which the nitrogen atom directly attached to the parent molecule is substituted by two groups other than hydrogen, including groups selected from: -N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -NR bb SO2R aa , -NR bb P(=O)(OR cc)2, and -NR bb P(=O)(N(R bb )2)2, where R aa , R bb and R cc are as defined herein, provided that the nitrogen atom directly attached to the parent molecule is not replaced by hydrogen. The term "trisubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is substituted by three groups, including groups selected from -N(R bb )3 and -N(R bb )3 + X - , where R bb and X - are as defined herein.

[0057] The term "thio" or "thiol" refers to the -SH group. Extending this, the term "substituted thio" or "substituted thiol" refers to a thiol group in which the sulfur atom directly attached to the parent molecule is substituted by a group other than hydrogen. In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a "thiol protecting group"). Sulfur protecting groups include, but are not limited to: -R aa , -N(R bb )2, -C(=O)SR aa , -C(=O)R aa , -CO2R aa , -C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -S(=O)R aa , -SO2R aa , -Si(R aa )3, -P(R cc )2, -P(R cc )3 + X - , - P(OR cc )2, -P(OR cc )3 + X - , -P(=O)(R aa )2, -P(=O)(OR cc )2 and -P(=O)(N(R bb )2)2, where R aa , R bb and R cc are as defined herein.

[0058] The term "acyl" refers to a group having the general formula: -C(=O)R X1 , -C(=O)OR X1 , -C(=O)-O-C(=O)R X1 , -C(=O)SR X1 , -C(=O)N(R X1 )2, -C(=S)R X1 , -C(=S)N(R X1 )2, -C(=S)O(R X1 ), -C(=S)S(R X1 ), -C(=NR X1 )R X1 , -C(=NR X1 )OR X1 , -C(=NR X1 )SR X1 and -C(=NR X1 )N(R X1 )2, where R X1 is hydrogen; halogen; substituted or unsubstituted hydroxy; substituted or unsubstituted mercapto; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic substituted or unsubstituted branched or straight-chain aliphatic group; cyclic or acyclic substituted or unsubstituted branched or straight-chain heteroaliphatic group; cyclic or acyclic substituted or unsubstituted branched or straight-chain alkyl; cyclic or acyclic substituted or unsubstituted branched or straight-chain alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphatic oxy, heteroaliphatic oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphatic amino, mono- or di-heteroaliphatic amino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino or mono- or di-heteroarylamino; or two R X1The groups together form a 5- or 6-membered heterocycle. Exemplary acyl groups include aldehyde (-CHO), carboxylic acid (-CO2H), ketone, acyl halide, ester, amide, imine, carbonate, carbamate, and urea. Acyl substituents include, but are not limited to, any substituent described herein that results in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, hydroxy, mercapto, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, etc., each of which may or may not be further substituted).

[0059] The term "amino acid" refers to a molecule containing both an amino group and a carboxyl group. Amino acids include α-amino acids and β-amino acids, the structures of which are shown below. In certain embodiments, the amino acid is an α-amino acid.

[0060]

[0061] Suitable amino acids include, but are not limited to, natural α - amino acids, such as D - and L - isomers of the 20 common naturally occurring α - amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as shown below), unnatural α - amino acids, natural β - amino acids (e.g., β - alanine) and unnatural β - amino acids. Exemplary natural α - amino acids include L - alanine (A), L - arginine (R), L - asparagine (N), L - aspartic acid (D), L - cysteine (C), L - glutamic acid (E), L - glutamine (Q), glycine (G), L - histidine (H), L - isoleucine (I), L - leucine (L), L - lysine (K), L - methionine (M), L - phenylalanine (F), L - proline (P), L - serine (S), L - threonine (T), L - tryptophan (W), L - tyrosine (Y) and L - valine (V). Exemplary unnatural α - amino acids include D - arginine, D - asparagine, D - aspartic acid, D - cysteine, D - glutamic acid, D - glutamine, D - histidine, D - isoleucine, D - leucine, D - lysine, D - methionine, D - phenylalanine, D - proline, D - serine, D - threonine, D - tryptophan, D - tyrosine, D - valine, divinyl, α - methyl - alanine (Aib), α - methyl - arginine, α - methyl - asparagine, α - methyl - aspartic acid, α - methyl - cysteine, α - methyl - glutamic acid, α - methyl - glutamine, α - methyl - histidine, α - methyl - isoleucine, α - methyl - leucine, α - methyl - lysine, α - methyl - methionine, α - methyl - phenylalanine, α - methyl - proline, α - methyl - serine, α - methyl - threonine, α - methyl - tryptophan, α - methyl - tyrosine, α - methyl - valine, norleucine, terminally unsaturated α - amino acids and bis - α - amino acids (e.g., modified cysteine, modified lysine, modified tryptophan, modified serine, modified threonine, modified proline, modified histidine, modified alanine, etc.). There are many known unnatural amino acids, any of which can be included in the peptides of the present invention. See, for example, S. Hunt, The Non–Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by G.C. Barrett, Chapman and Hall, 1985.

[0062] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen - protecting group (also referred to herein as an “amino - protecting group”). Nitrogen - protecting groups include, but are not limited to: - OH, - OR aa , - N(R cc)2, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , C 1-10 alkyl (such as aralkyl, heteroaralkyl), C 2-10 alkenyl, C 2-10 alkynyl, hetero C 1-10 alkyl, hetero C 2-10 alkenyl, hetero C 2-10 alkynyl, C 3-10 carbocyclic group, 3- to 14-membered heterocyclic group, C 6-14 aryl and 5- to 14-membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic group, heterocyclic group, aralkyl, aryl, and heteroaryl is independently substituted by 0, 1, 2, 3, 4, or 5 R dd groups, where R aa , R bb , R cc and R dd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3 rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0063] For example, protecting groups (such as nitrogen or oxygen protecting groups) such as amide groups (such as -C(=O)R aa)including, but not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoyl-phenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitroacetanilide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzoxyacetamidoamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

[0064] Protecting groups (such as nitrogen or oxygen protecting groups) such as carbamate groups (such as -C(=O)OR aa)including but not limited to methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, methyl 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]carbamate (DBD-Tmoc), 4-methoxybenzoylmethyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, tert-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, ethyl 2-(p-toluenesulfonyl)carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonoethyl carbamate (Peoc), 2-triphenylphosphonylisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acetoxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-phenylisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, tert-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacryloyl vinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, bis(2-pyridyl)methyl carbamate, 2-furylmethyl carbamate, 2-iodoethyl carbamate, isoboryl carbamate, isobutyl carbamate, isonicotinamido carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate and 2,4,6-trimethylbenzyl carbamate.,

[0065] Protecting groups (e.g., nitrogen or oxygen protecting groups) such as sulfonamide groups (e.g., -S(=O)2R aa) including but not limited to p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4’,8’-dimethoxynaphthalenemethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethanesulfonamide and benzoylmethylsulfonamide.

[0066] Other protecting groups (such as nitrogen or oxygen protecting groups) include but are not limited to phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothiocarbonyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-bis(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamine (Fcm), N-2-methylpyridineamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneimine, N-diphenylmethyleneamine, N-[(2-pyridyl)isopropylidene]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)benzylideneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboric acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amine N-oxides, diphenylphosphoramidite (Dpp), dimethylthiophosphoramidite (Mpt), diphenylthiophosphoramidite (Ppt), dialkylphosphoramidites, dibenzylphosphoramidites, diphenylphosphoramidite, benzenesulfonamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfonamide, 2-nitro-4-methoxybenzenesulfonamide, triphenylmethylsulfenamide and 3-nitropyridinesulfenamide (Npys).In certain embodiments, the protecting group (e.g., nitrogen or oxygen protecting group) is benzyl (Bn), tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), trifluoroacetyl, trityl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethoxycarbonyl (Troc), triphenylmethyl (Tr), p-toluenesulfonyl (Ts), p-bromobenzenesulfonyl (Bs), p-nitrobenzenesulfonyl (Ns), methanesulfonyl (Ms), trifluoromethanesulfonyl (Tf), or dansyl (Ds).

[0067] As used herein, the term "salt" refers to any and all salts, including pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to those salts that are within the scope of sound medical judgment and are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, and the article is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by reacting an amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with an organic acid such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4 alkyl)4 -- Salts. Representative alkali metal salts or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cation salts formed using counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. Brief Description of the Drawings

[0069] The drawings forming a part of this specification illustrate several embodiments of the present invention and, together with the description, are used to explain the principles of the present invention.

[0070] Figure 1 . Schematic illustration of the reaction for the controlled incorporation of natural ribonucleoside triphosphates (rNTPs) at the 3'-end of a primer oligonucleotide under hindered reaction conditions. The rNTP incorporation rate (K n ) is controlled by the addition of non-hydrolyzable or incompatible nucleotides, which act as competitive inhibitors of the incorporation of hydrolyzable nucleotides, such as those nucleotides having α-, β-, or γ-phosphate modifications of the triphosphate. The number of incorporation events (where n = zero to several hundred) is determined by the percentage (0%-100%) of non-hydrolyzable or incompatible nucleotides present in the extension reaction, such that a higher percentage of competitive inhibitor nucleotides limits the length of the RNA oligonucleotide produced by the reaction. When the primer oligonucleotide is attached to a surface via a cleavable covalent linkage (X), the base composition of the RNA oligonucleotide can be varied by a rapid switch in the reaction conditions.

[0071] Figure 2 . Schematic illustration of the reaction for the incorporation of modified rNTPs at the 3'-end of a primer oligonucleotide, which limits the extension reaction to the addition of only 1 nucleotide. The incorporation of modified rNTPs reversibly prevents further extension events until the extended oligonucleotide is treated with a mild RNA-tolerant deprotecting agent to generate a native hydroxyl group. When the primer oligonucleotide is attached to a surface via a cleavable covalent linkage (X), the growing RNA oligonucleotide can be extended repeatedly without purification to remove nucleotides from previous incorporation events. In addition to modifications at other sites on the nucleotide (such as the nucleobase), the reversible terminator rNTPs can also have non-native chemical domains, for example, at the 2'-, 3'-, or 2'- and 3'- positions (R and R') of the nucleotide. Each modification can be further derivatized to include a linker and a fluorophore in order to optically verify the (n + 1) incorporation event after enzymatic catalysis prior to mild deprotection treatment and subsequent extension.

[0072] Figures 3A to 3C . Figure 3ABar graph showing initial activity screening of polymerase μR387K using various divalent cations and divalent cations at reduced concentrations in combination with 200 μM dNTP. Denaturing gel electrophoresis analysis of dATP incorporation ( Figure 3B ) and rATP incorporation ( Figure 3C ) with nucleotide concentrations of dATP and rATP being 200 - 50 μM and 5.0 - 0.62 mM, respectively. 0.25 mM Mn 2+ and Mg 2+ were supplemented in these reactions. Control reactions consisted of all reaction components except nucleotides. For all reactions, the primer oligonucleotide was HPLC-purified poly dT-15-mer.

[0073] Figure 4A and 4B . Figure 4A Denaturing gel electrophoresis analysis showing incorporation of modified nucleotides (2'-amino-rATP, 2'-O-methyl-rATP, 2'-F-rATP & 2'-azido-rATP) by Saccharomyces cerevisiae poly(A) polymerase. The final concentration of all 2'-modified ribonucleotides was 2.5 mM and incubated at 37 °C for 60 minutes. Control reactions consisted of all reaction components except nucleotides. Figure 4B Denaturing gel electrophoresis analysis showing incorporation of the reversible terminator 2'-O-allyl-ATP by Saccharomyces cerevisiae poly(A) polymerase under a series of nucleotide concentration ranges (250 μM to 4000 μM) incubated at 37 °C for 60 minutes. Negative reactions consisted of all reaction components except nucleotides.

[0074] Figure 5A and 5B . Figure 5A Denaturing gel electrophoresis analysis showing incorporation of natural ribonucleotides by Schizosaccharomyces pombe poly(U) polymerase. The final concentration of all natural ribonucleotides was 1.0 mM and incubated at 37 °C for 30 minutes. Control reactions consisted of all reaction components except nucleotides. Figure 5B Kinetics analysis showing incorporation of natural ribonucleotides by Schizosaccharomyces pombe poly(U) polymerase; measuring the function of the total concentration of single-stranded RNA changing with time. Error bars represent standard deviation from the mean, N = 3.

[0075] Figure 6 . Denaturing gel electrophoresis analysis of incorporation of modified ribonucleotide (2'-O-methyl) by Schizosaccharomyces pombe poly(U) polymerase. The final concentration of all modified ribonucleotides was 2.5 mM and incubated at 37 °C for 60 minutes. Control reactions consisted of all reaction components except nucleotides.

[0076] Figure 7A and 7B. Figure 7A Denaturing gel electrophoresis analysis of the incorporation of natural ribonucleotides by Schizosaccharomyces pombe poly(U) polymerase in the presence of two different initiator oligonucleotides (5'-FAM-rA-15-mer and 5'-Cy5-rU-15-mer). The final concentration of all four natural ribonucleotides was 1.0 mM and the incubation was carried out at 37 °C for 30 minutes. The control reaction consisted of all reaction components except the nucleotides used for each initiator oligonucleotide. Figure 7B Denaturing gel electrophoresis analysis showing the incorporation of reversible terminator 2'-O-allyl-ATP or -UTP via strong hairpin formation using initiator oligonucleotides with secondary structure. The sequences of each oligonucleotide were similar, differing only in the position of the hairpin relative to the 3'-end, giving rise to the following: 1 base from the 3'-end (H1), 5 bases from the 3'-end (H5), 10 bases from the 3'-end (H10) and 20 bases from the 3'-end. The sequence base compositions are shown. To ensure correct hairpin formation of the oligonucleotides prior to enzymatic extension, the oligonucleotides were heated to 95 °C and then slowly cooled to 15 °C at a rate of 0.1 °C / min on a thermal cycler in an appropriate enzymatic reaction buffer. After cooling, the remaining reaction components were added to the hairpin initiator oligonucleotides and the extension reaction was carried out at 37 °C for 5 minutes.

[0077] Figures 8A to 8C . Figure 8A Denaturing gel electrophoresis analysis of the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction in the presence (+) and absence (-) of inorganic pyrophosphatase (PPi-ase) for each natural ribonucleotide. The control reaction consisted of all reaction components except the nucleotides. Kinetic analysis showed that in the presence of PPi-ase, Schizosaccharomyces pombe poly(U) polymerase increased the incorporation rate of ATP ( Figure 8B ) and UTP ( Figure 8C ).

[0078] Figure 9A and 9B . Figure 9A Denaturing gel electrophoresis analysis showing the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction using unmodified rUTP and base-modified pseudouridine (PsUTP) at different nucleotide concentrations. The reaction was incubated at 37 °C for 30 minutes. Figure 9BDenaturing gel electrophoresis analysis of the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction showing the use of a series of nucleoside triphosphates with modified adenosine, uridine, cytidine, guanosine, or the universal base inosine. The listed numbers shown correspond to the gel lanes in which the activity of the poly(U) polymerase was analyzed in the presence of 1 mM of each nucleotide. For comparison purposes, native nucleoside triphosphates were incubated and analyzed in parallel. Control reactions are labeled "C" on the gel, representing those reactions using all RNA synthesis reaction components except the nucleoside triphosphates. The initiator oligonucleotide is a poly-rU-15mer with a 5'-Cy5 fluorophore. All reactions were incubated at 37 °C for 30 minutes.

[0079] Figure 10 .Denaturing gel electrophoresis analysis of the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction incubated with increasing concentrations of the non-hydrolyzable ribonucleotide uridine 5'-[(α,β)-imino] triphosphate and UTP. The reactions were incubated at 37 °C for 30 minutes, and the control reactions consisted of all reaction components except the non-hydrolyzable ribonucleotide.

[0080] Figures 11A to 11E . Figure 11A Denaturing gel electrophoresis analysis of the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction incubated with 1 mM concentration of 2'-O-allyl-ATP, a 2'-blocked reversible terminator, with poly-rU-15-mer or poly-rA-15-mer initiator oligonucleotides. To demonstrate enzyme activity, a control reaction supplemented with 2'-O-methyl-ATP (previously confirmed to be incorporated by the Schizosaccharomyces pombe poly(U) polymerase) is shown. Additionally, a negative control reaction is included on the denaturing gel, which was supplemented with all reaction components except the nucleoside triphosphates. For the reversible terminator reaction, the second band shows positive incorporation compared to the negative control reaction, resulting in an (n+1) extension event. All RNA synthesis reactions were incubated with 10 pmol of the initiator oligonucleotide at 37 °C for 30 minutes. Figure 11B Denaturing gel electrophoresis analysis showing the kinetics of incorporation of the 2'-O-allyl-ATP reversible terminator by the Schizosaccharomyces pombe poly(U) polymerase. The reactions were incubated at 37 °C for 0.5, 5, 10, 30, and 60 minutes. A negative control was included for each time point, which had all reaction components except the 2'-O-allyl-ATP reversible terminator and was labeled with a minus sign (-) on the gel. The reactions incubated with the 2'-O-allyl-ATP reversible terminator were labeled with a plus sign (+) on the gel. For all reactions, the initiator oligonucleotide was a poly-rU-15mer labeled with a 5'-Cy5 fluorophore. Figure 11CDenaturing gel electrophoresis analysis optimizing buffer composition and pH in biocompatible deblocking of incorporated 2'-O-allyl-ATP reversible terminator. The deblocking reaction was incubated at 50 °C for 10 minutes, then the oligonucleotide material was purified, concentrated, and then further extended using optimal reaction conditions for poly(U) polymerase. For each buffer, a negative control was included, which had all reaction components except the 2'-O-allyl-ATP reversible terminator and was marked with a minus sign (-) on the gel. The reaction incubated with the 2'-O-allyl-ATP reversible terminator was marked with a plus sign (+) on the gel. The starting material, labeled (S), was included to visualize the entire synthesis cycle. High-resolution denaturing gel electrophoresis analysis of the synthesis cycle from the starting material (S or n+0) to (n+2) is shown in Figure 11D as before, a negative control was included, which had all reaction components except the 2'-O-allyl-ATP reversible terminator and was marked with a minus sign (-) on the gel. Figure 11E Denaturing gel electrophoresis analysis of (n+5) oligonucleotide synthesis using Schizosaccharomyces pombe poly(U) polymerase and 2'-O-allyl-ATP reversible terminator is shown. Each cycle consisted of a bulk solution extension reaction for 1 minute at 37 °C and a bulk solution deblocking reaction for 10 minutes at 50 °C using optimized conditions. After each cycle, a small aliquot of the material was set aside for gel analysis. The (n+0) starting material was 20-nt in length and the (n+5) final product was 25-nt in length.

[0081] Figure 12A and 12B . Figure 12A Denaturing gel electrophoresis analysis of (n+1) incorporation of 2'-O-allyl-ATP, -UTP, -CTP, and -GTP reversible terminator nucleoside triphosphates using Schizosaccharomyces pombe poly(U) polymerase is shown. All extension reactions were similarly treated and incubated with 1 mM nucleotides at 37 °C for 1 minute. The control reaction contained all reaction components except the nucleotides. Figure 12B Denaturing gel electrophoresis analysis of binary (n+2) synthesis using a combination of 2'-O-allyl-ATP and -UTP reversible terminators is shown. The following combinations were tested using optimized enzymatic extension and deblocking reaction conditions in bulk solution: (n+2)A-A, (n+2)A-U, (n+2)U-A, and (n+2)U-U. For comparison, the (n+1) reaction with 2'-O-allyl-ATP and -UTP reversible terminators is shown.

[0082] Figure 13A and 13B . Figure 13A Shows His at the N-terminus 6Denaturing gel electrophoresis analysis of the expression and purification of Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His-tag, as shown by the bright band in the square under the lane labeled "purified". The expressed N-terminal His- 6 tagged Schizosaccharomyces pombe poly(U) polymerase has an expected molecular weight of approximately 45 kDa. Figure 13B Shows denaturing gel electrophoresis analysis of the activity of Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His- 6 tag by incorporation of 2'-O-allyl-ATP reversible terminator nucleoside triphosphates. Reactions were supplemented with increasing amounts of initiator oligonucleotide (from 20 pmol to 1000 pmol) to determine the relative conversion rate by the concentrated N-terminal His- 6 tagged Schizosaccharomyces pombe poly(U) polymerase as a function of initiator oligonucleotide material. All reaction volumes were 10 μL and the initiator oligonucleotide was a 5'-Cy5 fluorophore-labeled poly-rU-15-mer. Reactions were incubated at 37 °C for 30 seconds. Control reactions contained all reaction components except the reversible terminator nucleotide (20 pmol of initiator oligonucleotide).

[0083] Figure 14A and 14B . Figure 14A Shows the development of a solid support system for an enzymatic RNA oligonucleotide system. The 5'-amine initiator oligonucleotide was labeled with a biotin-PEG-NHS linker, which allows the initiator oligonucleotide to be anchored to the streptavidin surface in a container such as a microplate, bead, slide, etc. Denaturing gel electrophoresis analysis of the oligonucleotide labeling is shown and quality control was performed using streptavidin-functionalized beads. The bound oligonucleotide includes an intrinsic Cy5 dye, which can be visualized using fluorescence microscopy. Figure 14B Shows denaturing gel electrophoresis analysis of solid-phase RNA oligonucleotide synthesis using Schizosaccharomyces pombe poly(U) polymerase. Synthesis was performed on beads in separate reaction vessels, one each for (n+1), (n+2), and (n+3). In the (n+3) example, the synthesized sequence was +ACU. The extension reaction was carried out at 37 °C for 1 minute and the deprotection reaction was carried out at 50 °C for 10 minutes. The beads were washed with 10 mM Tris-HCl (pH 6.5) between extension and deprotection.

[0084] Figures 15A to 15D . Figure 15ADescribes an exemplary protocol for the generation and use of a reusable solid-phase support system for enzymatic RNA oligonucleotide synthesis. Briefly, a solid support such as a bead, well, or slide is covalently derivatized with a suitable linker that binds to an initiator oligonucleotide preferably containing ribosylinosine (rI) or deoxyinosine (dI) at the 3'-end. Solid-phase enzymatic RNA oligonucleotide synthesis is carried out to generate the desired product, and then endonuclease V is allowed to incubate with the intact oligonucleotide (initiator + product). This cleaves the oligonucleotide product from the solid support, while leaving the intact ribosylinosine (rI) or deoxyinosine (dI) on the solid support for reuse in future synthesis reactions. If desired, the 2'-O-allyl form of this nucleobase can be used to introduce ribosylinosine (rI) into the 3'-end of the anchored initiator oligonucleotide using Schizosaccharomyces pombe poly(U) polymerase. Figure 15B Shows a denaturing gel electrophoresis analysis of endonuclease V cleavage of an initiator RNA oligonucleotide containing a large amount of deoxyinosine (dI) and the surface from amine-functionalized silica beads. The exemplary oligonucleotide initiator sequence and the bis-NHS-PEG linker used to covalently anchor the 5'-amine oligonucleotide to the surface of amine silica beads are shown. Endonuclease V cleavage was carried out at 37 °C for 1 hour using an appropriate buffer and then immediately run on a denaturing gel. Figure 15C Shows a denaturing gel electrophoresis analysis of Schizosaccharomyces pombe poly(U) polymerase-controlled and uncontrolled extensions using reversible terminator 2'-O-allyl-ATP and rNTP mixture on a fresh, uncleaved solid-phase support and an endonuclease V-cleaved solid-phase support, respectively. The extension reactions were supplemented with 1 mM nucleotide or nucleotide mixture and incubated at 37 °C for 15 minutes. Before endonuclease V cleavage, the beads were washed with 10 mM Tris-HCl (pH 6.5). Endonuclease V cleavage was carried out at 37 °C for 1 hour using an appropriate buffer and then immediately run on a denaturing gel. Figure 15DDenaturing gel electrophoresis analysis showing the controlled (n+2) product synthesis by Schizosaccharomyces pombe poly(U) polymerase using a covalently attached endonuclease V-cleavable initiator oligonucleotide and 2'-O-allyl-ATP reversible terminator nucleoside triphosphates. The extension reaction was supplemented with 1 mM nucleotide and incubated at 37 °C for 15 minutes. The deblocking reaction was carried out at 50 °C for 10 minutes. The beads were washed with 10 mM Tris-HCl (pH 6.5). Endonuclease V cleavage was carried out at 37 °C for 1 hour using an appropriate buffer and then immediately run on a denaturing gel. The control reaction was extended to (n+2) and incubated in the presence of endonuclease V but contained an anchored Cy5 initiator oligonucleotide without riboinosine (rI) or deoxyinosine (dI). This was used to demonstrate that the oligonucleotide did not leech during endonuclease V cleavage. After each synthesis cycle, the beads with the Cy5 initiator oligonucleotide remained visibly blue.

[0085] Figure 16A and 16B . Figure 16A Depicts a prototype enzymatic RNA oligonucleotide synthesizer that has the ability to simultaneously synthesize 4× oligonucleotides at a large reaction scale using a solid support such as silica or magnetic beads derivatized with an initiator oligonucleotide. The syringe barrel is loaded with sufficient solid support to meet the scale requirements for the synthesis run. Before loading the solid support, a filter is placed at the bottom of the syringe barrel and glued in place. This filter holds the solid support in place while allowing the removal of liquid from the syringe barrel. A typical synthesis cycle consists of an extension reaction, a wash step, a deblocking reaction, and then a final wash step. This process is repeated until the desired oligonucleotide is completed. To cleave the final product from the solid support, a chemical or biological cleavage reagent is added to each syringe barrel, incubated for a predetermined time if needed, eluted, and collected via filtration. If the solid support is to be reused, for example if endonuclease V is used to cleave the final oligonucleotide product, it can be left in the syringe barrel and primed for the next synthesis run. To heat the reactor, the syringe barrel can be removed, capped at both ends, and placed in an incubator for the required time. Alternatively, a heating jacket can be placed around the syringe barrel. The major advantage of enzyme-based RNA oligonucleotide synthesis is the recycling of materials (enzymes, nucleotides, etc.). Figure 16B Depicts a dual-valve system that can be controlled to direct the liquid in the syringe barrel to waste collection or recycle collection. The recycled components can be directly applied to the next oligonucleotide synthesis cycle or purified and stored for future synthesis runs. Similar setups and synthesis reactors can be scaled up to accommodate industrial-level oligonucleotide manufacturing.

[0086] Figure 17 . Figure 17 Depicts an exemplary scheme for the synthesis of the reversible terminator nucleoside triphosphate 2'-O-allyl-ATP. The starting material nucleoside can be exchanged for any natural base (U, T, G, C) and / or desired modified base. The triphosphate can also be exchanged for a phosphorothioate at the α-phosphate.

[0087] Figures 18A to 18D Gel electrophoresis analysis showing the ability of the H336 mutant to incorporate the natural nucleotides GTP "-G" and CTP "-C". The blank reaction was supplemented with all components except the enzyme and nucleotides. All reactions were incubated with 1 mM nucleotide, 5 pmol primer oligonucleotide, and 1 μg enzyme at 37 °C for 30 minutes. Extension reactions were analyzed using a 15% TBE-urea denaturing gel.

[0088] Figures 19A to 19F Gel electrophoresis analysis showing the ability of the poly(U) polymerase mutant H336R to incorporate a series of natural nucleotides and nucleotide analogs compared to wild-type poly(U) polymerase. Figure 19A 、 19D Depicts the extension results for wild-type and H336R mutants for ATP-based nucleotides, respectively. Figure 19B 、 19E Depicts the extension results for wild-type and H336R mutants for UTP- and ITP-based nucleotides, respectively. Figure 19C 、 19F Depicts the extension results for wild-type and H336R mutants for CTP- and GTP-based nucleotides, respectively. All reactions were incubated with 1 mM nucleotide, 5 pmol primer oligonucleotide, and 1 μg enzyme at 37 °C for 30 minutes. Extension reactions were analyzed using a 15% TBE-urea denaturing gel.

[0089] Figure 20 Shows non-discriminating incorporation of 2'-O-methyl-adenosine triphosphate (2'-O-Me-ATP) by various Schizosaccharomyces pombe poly(U) polymerase mutants, particularly at position H336; a single mutant is shown here for comparison with wild-type (WT). The blank reaction contained all components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0090] Figure 21 Shows non-discriminating incorporation of 2'-fluoro-adenosine triphosphate (2'-F-ATP) by various Schizosaccharomyces pombe poly(U) polymerase mutants, particularly at position N171; a single mutant is shown here for comparison with mutant H336R. The blank reaction contained all reaction components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0091] Figure 22 Shows controlled incorporation (capping) of 3'-methoxy-ATP (3'-O-Me-ATP) by various Schizosaccharomyces pombe poly(U) polymerase mutants, especially at position N171; a single mutant is shown here for comparison with mutants H336R and wild type. The upper band shows the (n+1) product. Note: The wild-type sample shows positive incorporation but significant pyrophosphorolysis occurs. The negative reaction contains all reaction components except the enzyme. Samples were analyzed using a 15% TBE-urea gel under denaturing conditions.

[0092] Figure 23 Shows controlled incorporation of the reversible terminator 3'-O-allyl-ATP (3'-(O-allyl)-ATP) by various Schizosaccharomyces pombe mutants. The negative reaction contains all reaction components except the enzyme. Samples were analyzed using a 15% TBE-urea gel under denaturing conditions.

[0093] Figure 24 Shows controlled incorporation of the reversible terminator 3'-O-allyl carbonate dATP (3'-(O-allyl carbonate)-dATP) by the poly(U) polymerase double mutant H336R-N171A. The gel image shows that as the amount of purified enzyme stock solution increases (2 μL, 4 μL, and 6 μL), the input amount of the initiator oligonucleotide varies (2 pmol / rxn, 5 pmol / rxn, and 10 pmol / rxn). The upper band shows the (n+1) product. The blank reaction contains all components except the enzyme. Samples were analyzed using a 15% TBE-urea gel under denaturing conditions.

[0094] Figure 25 Shows reaction calibration of the controlled incorporation of the reversible terminator 3'-O-azidomethyl carbonate dTTP (3'-(O-azidomethyl carbonate)-dTTP) by the Schizosaccharomyces pombe poly(U) polymerase single mutant H336R. The reaction was incubated for 30 minutes at 37 °C with different concentrations of the enzyme stock solution and nucleotide concentration. The letter "E" represents the amount of purified enzyme stock solution added, in μL, and the letter "N" represents the final concentration of the nucleotide in the extension reaction, in mM. The control reaction contains all components except the nucleotide. After incubation of the reaction, samples were analyzed using a 15% TBE-urea gel under denaturing conditions. The lower band shows the unextended starting material and the upper band shows the positively extended oligonucleotide.

[0095] Figure 26Shows the reaction calibration assessment of a purified poly(U) polymerase stock solution H336R with the reversible terminator 3'-O-allyl adenosine triphosphate (3'-(O-allyl)-ATP). The gel shows the response of (n+1) extension with increasing amounts of initiator oligonucleotide input. The reaction was supplemented with 1 mM reversible terminator nucleotide and 1 μL of the purified enzyme stock solution. The reaction was incubated at 37 °C for 5 minutes. The upper band shows the (n+1) product. The blank reaction contained all components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions. This is an example of reaction scalability.

[0096] Figure 27 Shows an example of controlled enzymatic synthesis using the poly(U) polymerase mutant H336R with the reversible terminator 3'-O-allyl adenosine triphosphate (3'-O-allyl-ATP) in bulk solution. Shown here is the (n+5) synthesis in bulk solution. After synthesis, the reaction was analyzed on a 15% TBE-urea gel under denaturing conditions.

[0097] Figure 28 Shows an example structure of a 3'-reversible terminator nucleotide for enzymatic incorporation. Various examples of protecting groups for the 3'-hydroxyl group. As labeled, these can be removed by redox chemistry, optically, fluoride anion, and catalysts.

[0098] Figure 29 Shows the selection of 3'-protecting groups in the case where the furanyl ring carries oxygen. The 2'- can be natural ribose, deoxy, or various moieties that promote binding, pharmacokinetics, pharmacodynamics, general stability, and probe labeling.

[0099] Figure 30 Shows examples of additional 3'-protecting groups that are irreversible (capping) terminators and esterase-sensitive terminators for both non-bridged and bridged nucleoside triphosphates. In the non-bridged case, the 2'- can be natural ribose, deoxy, or various moieties that promote binding, pharmacokinetics, pharmacodynamics, general stability, and probe labeling. These 3'-protecting groups can be further derivatized with other important moieties such as amino acids, oligonucleotides, or large chemical domains, which can confer additional functionality to the 3'-end of the synthesized oligonucleotide and, if insensitive to any known deprotection methods, can be used as a final irreversible cap.

[0100] Figure 31 Shows an example protocol for preparing 3'-azidomethyl ethers for nucleoside triphosphates, where the 2'- can be natural OH or various modifications such as -F, -OMe, -OCH2CH2CH3, or other modifications that have been shown to be beneficial for the biological activity of the target oligomer or contribute to broader scientific impact.

[0101] Figure 32An exemplary scheme for preparing 3'-azidomethyl ethers for locking triphosphonucleotides is shown.

[0102] Figure 33 An exemplary scheme for preparing 3'-allyl ethers for triphosphonucleotides is shown, where the 2' can be a native OH or various modifications such as F, OMe, OCH2CH2CH3 or other modifications that have been shown to be beneficial for the biological activity of the target oligomer or contribute to other applications.

[0103] Figure 34 An exemplary scheme for preparing 3'-azidomethyl ethers for locking triphosphonucleotides is shown.

[0104] Detailed description of certain embodiments

[0105] Methods for de novo synthesis of RNA oligonucleotides using enzymatic catalysis are described herein. For example, methods for synthesizing RNA oligonucleotides are provided herein, in which a terminal transferase (e.g., poly(N) polymerase) incorporates one or more nucleotides onto a primer oligonucleotide. For example, methods for preparing RNA oligonucleotides are provided herein, in which poly(U) polymerase incorporates one or more modified nucleotides onto a primer oligonucleotide via a terminal transferase.

[0106] In one aspect, methods are provided herein in which modified nucleotides (i.e., 2'- or 3'-modified reversible terminator oligonucleotides) that reversibly alter the binding affinity of a polymerase (e.g., poly(U) polymerase) for an extended primer oligonucleotide are incorporated, thereby producing an (n + 1)-extended RNA oligonucleotide, which can be deprotected and then further extended.

[0107] In another aspect, methods for synthesizing RNA oligonucleotides are provided herein, in which non-hydrolyzable nucleotides are used to control the rate at which a polymerase (e.g., poly(U) polymerase) incorporates hydrolyzable nucleotides onto a primer oligonucleotide.

[0108] In another aspect, methods are provided herein for ligating two oligonucleotides using a poly(N) polymerase described herein (e.g., a wild-type or mutant poly(U) polymerase described herein) to produce a desired RNA oligonucleotide.

[0109] Additionally, the RNA oligonucleotides produced by these methods can be reverse transcribed (RT) to produce complementary DNA (e.g., cDNA), which can be amplified by polymerase chain reaction (PCR) using a high-fidelity DNA polymerase. RNA oligonucleotides and DNA oligonucleotides produced by any of the methods described herein are also provided herein.

[0110] Modified nucleotides that can be used in the methods described herein, and poly(N) polymerases (e.g., mutant poly(U) polymerases) that can be used in the methods described herein are also provided herein.

[0111] The present disclosure also provides compositions and kits that include one or more of the poly(N) polymerases and / or nucleotides described herein. On the other hand, the present disclosure provides reaction mixtures and systems for performing the methods described herein.

[0112] RNA oligonucleotide synthesis

[0113] The present disclosure provides methods for synthesizing RNA oligonucleotides, in which a poly(N) polymerase incorporates one or more modified nucleotides onto a primer oligonucleotide by a terminal transferase (e.g., a poly(N) polymerase). In certain embodiments, the present disclosure provides methods for template-independent synthesis of RNA oligonucleotides, the methods comprising:

[0114] (a) providing a primer oligonucleotide, wherein the primer oligonucleotide is single-stranded RNA;

[0115] (b) providing a poly(N) polymerase;

[0116] (c) combining the primer oligonucleotide, the poly(N) polymerase, and one or more modified nucleotides under conditions sufficient to add at least one modified nucleotide to the 3'-end of the primer oligonucleotide.

[0117] In certain embodiments, the poly(N) polymerase is poly(U) polymerase. Thus, in certain embodiments, the present disclosure provides methods for template-independent synthesis of RNA oligonucleotides, the methods comprising:

[0118] (a) providing a primer oligonucleotide, wherein the primer oligonucleotide is single-stranded RNA;

[0119] (b) providing poly(U) polymerase;

[0120] (c) combining the primer oligonucleotide, the poly(U) polymerase, and one or more modified nucleotides under conditions sufficient to add at least one modified nucleotide to the 3'-end of the primer oligonucleotide.

[0121] Once one or more modified nucleotides are added to the primer oligonucleotide, one or more additional nucleotides (modified or unmodified) can then be added to synthesize the desired RNA oligonucleotide. Thus, in certain embodiments, the method further comprises adding one or more natural or modified nucleotides to the 3'-end of the resulting RNA oligonucleotide (i.e., the RNA oligonucleotide formed in step (c)) until the desired RNA sequence is obtained. In certain embodiments, one or more additional modified nucleotides are added. In certain embodiments, the method further comprises:

[0122] (d) Repeat steps (a)-(c) until the desired RNA sequence is obtained.

[0123] Poly(N) polymerase

[0124] As described herein, the enzyme that incorporates one or more nucleotides is an RNA polymerase, such as a Poly(N) polymerase. Poly(N) polymerases that can be used in the methods described herein are provided, such as mutant (i.e., mutated) Poly(U) polymerases.

[0125] In certain embodiments, the Poly(N) polymerase is a Poly(U) polymerase, a Poly(A) polymerase, a Poly(C) polymerase, or a Poly(G) polymerase. The RNA polymerase can be a wild-type polymerase, or a mutant (i.e., mutated), variant, or homolog thereof. In certain embodiments, the Poly(N) polymerase is a wild-type polymerase. In certain embodiments, the polymerase is a mutant of the Poly(N) polymerase. In certain embodiments, the polymerase is a variant of the Poly(N) polymerase. In certain embodiments, the mutant or variant has about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the wild-type polymerase. In certain embodiments, the polymerase is a homolog of the Poly(N) polymerase.

[0126] In certain embodiments, the Poly(N) polymerase is a Poly(U) polymerase. In certain embodiments, the Poly(U) polymerase is a wild-type Schizosaccharomyces pombe Poly(U) polymerase, or a mutant or homolog thereof. In certain embodiments, the Poly(U) polymerase is a wild-type Schizosaccharomyces pombe Poly(U) polymerase. In certain embodiments, the Poly(U) polymerase is a mutant of the Schizosaccharomyces pombe Poly(U) polymerase. In certain embodiments, the Poly(U) polymerase is a variant of the Schizosaccharomyces pombe Poly(U) polymerase. In certain embodiments, the Poly(U) polymerase is a homolog of the Schizosaccharomyces pombe Poly(U) polymerase.

[0127] In certain embodiments, the Poly(N) polymerase is a Poly(A) polymerase. In certain embodiments, the Poly(A) polymerase is a wild-type Saccharomyces cerevisiae Poly(A) polymerase or a mutant thereof. In certain embodiments, the Poly(N) polymerase is a wild-type Saccharomyces cerevisiae Poly(A) polymerase. In certain embodiments, the Poly(N) polymerase is a mutant of the Saccharomyces cerevisiae Poly(A) polymerase. In certain embodiments, the Poly(N) polymerase is a variant of the Saccharomyces cerevisiae Poly(A) polymerase. In certain embodiments, the Poly(N) polymerase is a homolog of the Saccharomyces cerevisiae Poly(A) polymerase.

[0128] Poly(U) polymerase mutant

[0129] As described herein, in certain embodiments, the poly(N) polymerase is a mutant of the poly(N) polymerase (i.e., a mutated poly(N) polymerase). In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase that contains a mutation at one or more positions selected from H336, N171, and T172.

[0130] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase that contains an H336 mutation (i.e., where the amino acid H at position 336 is replaced by another amino acid). In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase that contains an H336 mutation selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W. In certain embodiments, the H336 mutation is the only mutation. In certain embodiments, the poly(N) polymerase contains one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes an H336 mutation selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W.

[0131] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase that contains an H336R mutation. In certain embodiments, the H336R mutation is the only mutation. In certain embodiments, the poly(N) polymerase contains one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes one mutation: H336R.

[0132] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising the N171 mutation. In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising an N171 mutation selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K. In certain embodiments, the N171 mutation is the only mutation. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes an N171 mutation selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K.

[0133] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising the N171A mutation. In certain embodiments, the N171A mutation is the only mutation. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes one mutation: N171A.

[0134] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising the N171T mutation. In certain embodiments, the N171T mutation is the only mutation. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes one mutation: N171T.

[0135] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising the T172 mutation. In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising a T172 mutation selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K. In certain embodiments, the T172 mutation is the only mutation. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that are about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes one T172 mutation selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K.

[0136] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336 and N171 mutations. In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336 mutation and N171 mutation: the H336 mutation is selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; the N171 mutation is selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K. In certain embodiments, the H336 and N171 mutations are the only mutations. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes one H336 mutation selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; and one N171 mutation selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K.

[0137] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336R and N171A mutations. In certain embodiments, the H336R and N171A mutations are the only mutations. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes two mutations: H336R and N171A.

[0138] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising the H336R and N171T mutations. In certain embodiments, the H336R and N171T mutations are the only mutations. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3 but includes two mutations: H336R and N171T.

[0139] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336 and T172 mutations. In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336 and T172 mutations, wherein the H336 mutation is selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; and the T172 mutation is selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K. In certain embodiments, the H336 and T172 mutations are the only mutations. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3, but includes an H336 mutation and a T172 mutation, wherein the H336 mutation is selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; and the T172 mutation is selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K. In certain embodiments, the H336 mutation is H336R.

[0140] In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising H336, N171, and T172 mutations. In certain embodiments, the poly(N) polymerase is Schizosaccharomyces pombe poly(U) polymerase comprising an H336 mutation, an N171 mutation, and a T172 mutation: the H336 mutation is selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; the N171 mutation is selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K; the T172 mutation is selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K. In certain embodiments, the H336, N171, and T172 mutations are the only mutations. In certain embodiments, the poly(N) polymerase comprises one or more additional mutations that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the poly(N) polymerase is identical to SEQ ID NO:3, but includes an H336 mutation selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S, and H336W; an N171 mutation selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H, and N171K; and a T172 mutation selected from the group consisting of: T172E, T172L, T172Q, T172S, T172M, T172D, T172G, T172C, T172A, T172W, T172T, T172I, T172V, T172P, T172R, T172H, and T172K.In certain embodiments, the H336 mutation is H336R. In certain embodiments, the N171 mutation is N171A or N171T.

[0141] Modified RNA nucleotide

[0142] As described herein, one or more modified nucleotides can be incorporated into an oligonucleotide to synthesize a desired RNA oligonucleotide. Modified nucleotides can be incorporated to prepare customized RNA or DNA oligonucleotides. In other embodiments, modified nucleotides can be incorporated to block the incorporation of subsequent nucleotides (i.e., by using the "reversible terminators" described herein). Modified nucleotides are provided herein that can be used in the methods described herein as well as other applications (e.g., chemical oligonucleotide synthesis, therapeutic applications, etc.).

[0143] "Modified nucleotide" includes one or more non-naturally modified nucleotide monomers (e.g., containing a ribose, phosphate group, and nucleobase). In certain embodiments, for example, a modified nucleotide is a structural equivalent of a naturally occurring RNA or DNA nucleotide (i.e., guanine (G), uracil (U), adenine (A), cytosine (C)), but contains one or more non-natural modifications. In certain embodiments, a modified nucleotide is an equivalent of a naturally occurring nucleotide in which one or more positions are substituted, or in which one or more substituents or groups are removed or replaced. In certain embodiments, a modified nucleotide contains a modified sugar, a modified base, a modified phosphate, or any combination thereof. "Modified nucleotide" includes the 2'- and 3'-reversible terminator nucleotides described herein.

[0144] The following formula is intended to illustrate possible modification sites on a nucleotide. Other modifications are contemplated. In certain embodiments, a modified nucleotide has the following formula:

[0145]

[0146] or a salt thereof, wherein:

[0147] "Base" (also referred to herein as "B") is a natural or non-natural nucleobase; and

[0148] R and R' are independently hydrogen or natural or non-natural sugar substituents.

[0149] In certain embodiments, a modified nucleotide has the following formula:

[0150]

[0151] or a salt thereof, wherein:

[0152] X is O or S;

[0153] Y is O or S;

[0154] A "base" (also referred to herein as "B") is a natural or unnatural nucleobase; and

[0155] R and R' are independently hydrogen or a natural or unnatural sugar substituent.

[0156] In certain embodiments, Y is O. In certain embodiments, Y is S. In certain embodiments, X is O. In certain embodiments, X is S.

[0157] In certain embodiments, the modified nucleotide is a base-modified nucleotide. "Base-modified" encompasses nucleotides in which a G, U, A, or C base is substituted or modified, or in which a G, U, A, or C base is replaced by a different group (such as hypoxanthine).

[0158] Non-limiting examples of modified bases include, but are not limited to, 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouridine, 3-methyluridine, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine, 5-alkyluridine, 5-halouridine, 6-azapyrimidine, 6-alkylpyrimidine, propyne, queosine, 2-thiouridine, 4-thiouridine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxouridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, and threonine derivatives.

[0159] Other non-limiting examples of bases include, but are not limited to, natural or unnatural pyrimidines or purines; which may include, but are not limited to, N 1 -methyladenine, N 6 -methyladenine, 8'-azidoadenine, N,N-dimethyladenosine, aminoallyl-adenosine, 5'-methyluridine, pseudouridine, N 1- -methylpseudouridine, 5'-hydroxymethyluridine, 2'-thiouridine, 4'-thiouridine, hypoxanthine, xanthine, 5'-methylcytidine, 5'-hydroxymethylcytidine, 6'-thioguanine, and N 7 -methylguanine.

[0160] In certain embodiments, the base-modified nucleotide is selected from the group consisting of: N 1-Methyladenosine-5'-triphosphate, N 6 -Methyladenosine-5'-triphosphate, N 6 -Methyl-2-aminoadenosine-5'-triphosphate, 5-methyluridine-5'-triphosphate, N 1 -Methylpseudouridine-5'-triphosphate, pseudouridine-5'-triphosphate, 5-hydroxymethyluridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-hydroxymethylcytidine-5'-triphosphate, N 7 -Methylguanosine triphosphate, 8'-adizoadenisone-5'-triphosphate, inosine 5'-triphosphate, 2-thiouridine-5'-triphosphate, 6-thioguanosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and xanthine 5'-triphosphate.

[0161] In certain embodiments, the modified nucleotide is a sugar-modified nucleotide. "Sugar-modified" nucleotides include nucleotides in which the ribose or deoxyribose moiety is substituted, or in which the ribose or deoxyribose is replaced by a different sugar moiety. In certain embodiments, the ribose or deoxyribose is modified (e.g., substituted) at the 1', 2', 3', 4' and / or 5' positions. In some embodiments, the nucleotide may be modified at the 2' position. In some embodiments, the nucleotide may be modified at the 3' position.

[0162] In certain embodiments, the 2' and / or 3' positions of the sugar are substituted with a natural or non-natural "sugar substituent" R or R'. In certain embodiments, R and R' are independently selected from hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl group, optionally substituted hydroxyl group, optionally substituted amino group or optionally substituted thiol group.

[0163] In certain embodiments, R and / or R' are independently -OR P wherein each instance of R P is independently an oxygen protecting group, an optionally substituted acyl group or an amino acid. In certain embodiments, R and / or R' comprise a reactive component for bioconjugation (e.g., a click chemistry handle, e.g., an azide or an alkyne), a fluorophore, a catalytic protein, an oligonucleotide or a reporter label.

[0164] In some embodiments, the 2' position of a sugar such as ribose can be modified with a group such as: a halogen, e.g., a fluorine group; an alkyl group, e.g., a methyl or ethyl group; a methoxy group; an amino group; a thiol group; an aminopropyl group; a dimethylaminoethyl group; a dimethylaminopropyl group; a dimethylaminoethoxyethyl group; an azide group; a silyl group; a cycloalkyl group; or an N-methylacetamide group.

[0165] In certain embodiments, the 2'-position of a sugar such as ribose is modified with a group such as a hydroxyl group (-OH), hydrogen (-H), fluorine (-F), amine (-NH3), azide (-N3), thiol (-SH), methoxy (-OCH3), or methoxyethanol (-OCH2CH2OCH3).

[0166] In certain embodiments, the 2'-position can also be substituted with a component such as a redox-active component, a fluorescent or intrinsically fluorescent component, natural and unnatural amino acids, peptides, proteins, monosaccharides or oligosaccharides, functional / ligand-binding glycans, and polymers or macromolecules such as polyethylene glycol (PEG).

[0167] In some embodiments, the 3'-position of a sugar such as ribose can be modified with a group such as a halogen, such as a fluorine group; an alkyl group, such as a methyl or ethyl group; a methoxy group; an amino group; a thiol group; an aminopropyl group; a dimethylaminoethyl group; a dimethylaminopropyl group; a dimethylaminoethoxyethyl group; an azide group; a silyl group; a cycloalkyl group; or an N-methylacetamide group.

[0168] In certain embodiments, the 3'-position of a sugar such as ribose is modified with a hydroxyl group (-OH), hydrogen (-H), fluorine (-F), amine (-NH3), azide (-N3), thiol (-SH), methoxy (-OCH3), or methoxyethanol (-OCH2CH2OCH3).

[0169] In certain embodiments, the 3'-position can also be substituted with a component such as a redox-active component, a fluorescent or intrinsically fluorescent component, natural and unnatural amino acids, peptides, proteins, monosaccharides or oligosaccharides, functional / ligand-binding glycans, and polymers or macromolecules such as polyethylene glycol (PEG).

[0170] In certain embodiments, the sugar-modified nucleotide is modified at the 2'-position. For example, in certain embodiments, the sugar-modified nucleotide is a 2'-F, 2'-O-alkyl, 2'-amino, or 2'-azide-modified nucleotide.

[0171] In certain embodiments, the sugar-modified nucleotide is a 2'-F-modified nucleotide. In certain embodiments, the sugar-modified nucleotides are selected from 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, and 2'-fluoro-2'-deoxyuridine-5'-triphosphate.

[0172] In certain embodiments, the sugar-modified nucleotide is a 2'-O-alkyl-modified nucleotide. In certain embodiments, the sugar-modified nucleotides are selected from 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, and 2'-O-methylinosine-5'-triphosphate.

[0173] In certain embodiments, the sugar-modified nucleotide is a 2'-O-amino-modified nucleotide. In certain embodiments, the sugar-modified nucleotides are selected from 2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate, 2'-amino-2'-deoxyadenosine-5'-triphosphate, and 2'-amino-2'-deoxyguanosine-5'-triphosphate.

[0174] In certain embodiments, the sugar-modified nucleotide is a 2'-O-azido-modified nucleotide. In certain embodiments, the sugar-modified nucleotides are selected from 2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate, 2'-azido-2'-deoxyadenosine-5'-triphosphate, and 2'-azido-2'-deoxyguanosine-5'-triphosphate.

[0175] In certain embodiments, the modified nucleoside triphosphate is an irreversible terminator, also known as a capped nucleotide, such as 3'-O-methyl-NTP, 3'-O-methyl-dNTP, 3'-azido-dNTP, 3'-azido-NTP, 3'-amine-dNTP, 3'-amine-NTP, etc.

[0176] In certain embodiments, the sugar-modified nucleotide is a 2'-modified reversible terminator RNA nucleotide (e.g., a 2'-O-protected reversible terminator nucleotide). 2'-modified reversible terminator nucleotides are described herein. In certain embodiments, the 2'-modified reversible terminator nucleotides also comprise a modified base moiety.

[0177] In certain embodiments, the sugar-modified nucleotide is a 3'-modified reversible terminator RNA nucleotide (e.g., a 3'-O-protected reversible terminator nucleotide). 3'-modified reversible terminator nucleotides are described herein. In certain embodiments, the 3'-modified reversible terminator nucleotides also comprise a modified base moiety.

[0178] Encompasses other modifications of sugars. These modifications include, but are not limited to, replacing the oxygen of the ring with sulfur. In certain embodiments, a bridge is introduced between the 2'-carbon and the 4'-carbon (e.g., to restrict the ring conformation). In some embodiments, the modified nucleotide is a bridged nucleotide, such as locked nucleic acid (LNA); constrained ethyl nucleotide (cEt) or ethylene-bridged nucleic acid (ENA) nucleotide.

[0179] In some embodiments, nucleotides can, for example, comprise modified phosphate groups, such as phosphorothioates. Non-limiting examples of modified phosphate groups include phosphorothioates, phosphotriesters, methylphosphonates, alkyls, heterocycles, amides, morpholinos, peptide nucleic acids (PNA) and other known phosphorus-containing groups. In certain embodiments, the modification is an α-phosphate modification of the triphosphate. In certain embodiments, the nucleotide is an (α)-phosphorothioate. In certain embodiments, it is a β- and / or γ-phosphate modification of the triphosphate.

[0180] In certain embodiments, nucleotides modified with fluorophores can be used to verify the success of each repetitive incorporation event, thereby producing, in some embodiments, virtually error-free RNA oligonucleotides. In certain embodiments, the modified nucleotide comprises a fluorophore.

[0181] Modified nucleotides can comprise more than one modification. For example, a modified nucleotide can comprise a base modification and a sugar modification.

[0182] Synthesis of RNA oligonucleotides with reversible terminators

[0183] Also provided herein is a method for synthesizing RNA oligonucleotides using reversible terminator nucleotides. A "reversible terminator nucleotide" is a nucleotide that comprises a non-natural chemical moiety at the 2'- and / or 3'-position that can be removed. After adding a reversible terminator nucleotide to a primer oligonucleotide, the non-natural chemical moiety at the 2'- and / or 3'-position blocks the incorporation of a second nucleotide, for example, by interfering with the binding of the oligonucleotide to the polymerase. The non-natural chemical moiety at the 2'- and / or 3'-position can then be removed, leaving the 3'-position open for the addition of additional nucleotides. In certain embodiments, the method allows for the controlled addition of one nucleotide at a time, also referred to as "(n + 1)" addition. In certain embodiments, the reversible terminator nucleotide is protected at the 2'- and / or 3'-hydroxyl group (i.e., "2'- and / or 3'-O-protected reversible terminator nucleotide").

[0184] Provided herein is a template-independent method for synthesizing RNA oligonucleotides, the method comprising:

[0185] (a) providing a primer oligonucleotide, wherein the primer oligonucleotide is single-stranded RNA;

[0186] (b) Provide poly(N) polymerase;

[0187] (c) Combine the initiator oligonucleotide, poly(N) polymerase, and reversible terminator nucleotide under conditions sufficient to add the reversible terminator nucleotide to the 3' end of the initiator oligonucleotide;

[0188] (d) Deprotect the RNA oligonucleotide formed in step (c) at the protecting positions (e.g., 2' and / or 3' positions) of the reversible terminator nucleotide; and

[0189] (e) Optionally, repeat steps (a)-(c) until the desired RNA sequence is obtained.

[0190] In certain embodiments, the poly(N) polymerase is poly(U) polymerase. Provided herein is a method for template-independent synthesis of RNA oligonucleotides, the method comprising:

[0191] (a) Provide an initiator oligonucleotide, wherein the initiator oligonucleotide is single-stranded RNA;

[0192] (b) Provide poly(U) polymerase;

[0193] (c) Combine the initiator oligonucleotide, poly(U) polymerase, and 2'- and / or 3'-O-protected reversible terminator nucleotide under conditions sufficient to add the 2'- and / or 3'-O-protected reversible terminator nucleotide to the 3' end of the initiator oligonucleotide;

[0194] (d) Deprotect the RNA oligonucleotide formed in step (c) at the protected 2'- and / or 3'-O-positions of the 2'- and / or 3'-O-protected reversible terminator nucleotide;

[0195] (e) Optionally, repeat steps (a)-(c) until the desired RNA sequence is obtained.

[0196] As described herein, 2'-O-protected reversible terminator nucleotides can also be used. In certain embodiments, the poly(N) polymerase is poly(U) polymerase. Provided herein is a method for template-independent synthesis of RNA oligonucleotides, the method comprising:

[0197] (a) Provide an initiator oligonucleotide, wherein the initiator oligonucleotide is single-stranded RNA;

[0198] (b) Provide poly(U) polymerase;

[0199] (c) Combine the initiator oligonucleotide, poly(U) polymerase, and 2'-O-protected reversible terminator nucleotide under conditions sufficient to add the 2'-O-protected reversible terminator nucleotide to the 3' end of the initiator oligonucleotide;

[0200] (d) Deprotect the RNA oligonucleotide formed in step (c) at the protected 2'-O-position of the 2'-O-protected reversible terminator nucleotide;

[0201] (e) Optionally, repeat steps (a)-(c) until the desired RNA sequence is obtained.

[0202] As described herein, 3'-O-protected reversible terminator nucleotides can also be used. Provided herein are template-independent methods for the synthesis of RNA oligonucleotides, the methods comprising:

[0203] (a) Providing a primer oligonucleotide, wherein the primer oligonucleotide is single-stranded RNA;

[0204] (b) Providing poly(U) polymerase;

[0205] (c) Combining the primer oligonucleotide, poly(U) polymerase, and 3'-O-protected reversible terminator nucleotide under conditions sufficient to add the 3'-O-protected reversible terminator nucleotide to the 3'-end of the primer oligonucleotide;

[0206] (d) Deprotect the RNA oligonucleotide formed in step (c) at the protected 3'-O-position of the 3'-O-protected reversible terminator nucleotide;

[0207] (e) Optionally, repeat steps (a)-(c) until the desired RNA sequence is obtained.

[0208] Any poly(N) polymerase described herein can be used in the above reversible terminator methods. In certain embodiments, the mutant poly(U) polymerase described herein is used to incorporate reversible terminator nucleotides. In certain embodiments, the mutant poly(U) polymerase described herein is used to incorporate the 3'-reversible terminator nucleotides described herein.

[0209] RNA oligonucleotides of any specific sequence can be synthesized using the methods described herein.

[0210] Reversible terminator RNA nucleotides

[0211] Some of the methods described herein employ reversible terminator RNA oligonucleotides. A "reversible terminator nucleotide" is a modified nucleotide that contains a non-native chemical moiety at the 2'- and / or 3'-position that can be removed. In certain embodiments, the reversible terminator nucleotide is protected at the 2'-O- and / or 3'-O-position with an oxygen protecting group. Also provided herein are new reversible terminator nucleotides (e.g., 2'-modified reversible terminator nucleotides and 3'-modified reversible terminator nucleotides).

[0212] In certain embodiments, the 2'-modified reversible terminator nucleotide is protected at the 2'-O-position with an oxygen protecting group ("2'-O-protected reversible terminator nucleotide"). In certain embodiments, the 3'-modified reversible terminator nucleotide is protected at the 3'-O-position with an oxygen protecting group ("3'-O-protected reversible terminator nucleotide").

[0213] For example, in certain embodiments, the reversible terminator nucleotide (i.e., 2'- and / or 3'-O-protected reversible terminator nucleotide) has the formula:

[0214]

[0215] or a salt thereof, wherein:

[0216] Each instance of R P is hydrogen, an oxygen protecting group, an optionally substituted acyl group, or an amino acid, or two Rs P are joined together with an intervening atom to form an optionally substituted heterocyclic group; provided that at least one R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid; and

[0217] "Base" (also referred to herein as "B") is a natural or unnatural nucleotide (e.g., modified) base. Other portions of the nucleotide can be modified as described above and herein.

[0218] For example, in certain embodiments, the reversible terminator nucleotide (i.e., 2'- and / or 3'-O-protected reversible terminator nucleotide) has the formula:

[0219]

[0220] or a salt thereof, wherein:

[0221] Y is O or S;

[0222] X is O or S;

[0223] RR P Each instance of is hydrogen, an oxygen protecting group, an optionally substituted acyl group or an amino acid, or two Rs P are joined together with an intervening atom to form an optionally substituted heterocyclic group; provided that at least one R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid; and

[0224] "Base" (also referred to herein as "B") is a natural or unnatural nucleotide (e.g., modified) base.

[0225] In certain embodiments, the 3'-modified reversible terminator nucleotide (i.e., 3'-O-protected reversible terminator nucleotide) has the formula:

[0226]

[0227] or a salt thereof, wherein:

[0228] Y is O or S;

[0229] X is O or S;

[0230] R P is an oxygen protecting group;

[0231] R is hydrogen or a natural or unnatural sugar substituent as described herein; and

[0232] "Base" (also referred to herein as "B") is a natural or unnatural nucleotide (e.g., modified) base.

[0233] Optionally, in certain embodiments, the linking group links the 2'-carbon to the 4'-carbon (e.g., via group R').

[0234] In certain embodiments, the 3'-modified reversible terminator nucleotide is a locked nucleotide or a bridged nucleotide. In certain embodiments, the 3'-modified reversible terminator nucleotide (i.e., the 3'-O-protected reversible terminator nucleotide) has the following formula:

[0235]

[0236] or a salt thereof, wherein:

[0237] Y is O or S;

[0238] X is O or S;

[0239] R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid;

[0240] R is hydrogen or a natural or unnatural sugar substituent as described herein; "Base" (also referred to herein as "B") is a natural or unnatural nucleotide (e.g., modified) base.

[0241] In certain embodiments, Y is O. In certain embodiments, Y is S. In certain embodiments, X is O. In certain embodiments, X is S.

[0242] As described herein, a "base" (also referred to herein as "B") can be any naturally occurring or non-naturally occurring nucleobase. Naturally occurring bases include G, U, A, and C. Non-natural (e.g., modified) bases include substituted or modified variants of G, U, A, and C. Non-limiting examples of modified bases include, but are not limited to, 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine, 5-alkyluridine, 5-halouridine, 6-azapyrimidine, 6-alkylpyrimidine, propyne, queosine, 2-thiouridine, 4-thiouridine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, and threonine derivatives.

[0243] Other non-limiting examples of bases include, but are not limited to, natural or non-natural pyrimidines or purines; may include, but are not limited to, N 1 -methyladenine, N 6 -methyladenine, 8'-azidoadenine, N,N-dimethyladenosine, aminoallyl-adenosine, 5'-methyluracil, pseudouracil, N 1 -methylpseudouracil, 5'-hydroxymethyluracil, 2'-thiouridine, 4'-thiouridine, hypoxanthine, xanthine, 5'-methylcytidine, 5'-hydroxymethylcytidine, 6'-thioguanine, and N 7 -methylguanine.

[0244] In certain embodiments, the nucleotide sugar is replaced by a natural or non-natural "sugar substituent" R. In certain embodiments, R is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl group, optionally substituted hydroxy group, optionally substituted amino group, or optionally substituted thiol group. In certain embodiments, R is hydrogen. In certain embodiments, R is halogen. In certain embodiments, R is -CN. In certain embodiments, R is -NO2. In certain embodiments, R is -N3. In certain embodiments, R is optionally substituted alkyl. In certain embodiments, R is optionally substituted alkenyl. In certain embodiments, R is optionally substituted alkynyl. In certain embodiments, R is optionally substituted aryl. In certain embodiments, R is optionally substituted heteroaryl. In certain embodiments, R is optionally substituted carbocyclic group. In certain embodiments, R is optionally substituted heterocyclic group. In certain embodiments, R is optionally substituted acyl group. In certain embodiments, R is optionally substituted hydroxy group. In certain embodiments, R is optionally substituted amino group. In certain embodiments, R is optionally substituted thiol group.

[0245] In certain embodiments, R is -OR P , wherein R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid.

[0246] In certain embodiments, R comprises a reactive moiety for bioconjugation (e.g., a click chemistry handle such as an azide or an alkyne), a fluorophore, a catalytic protein, an oligonucleotide, or a reporter label.

[0247] In some embodiments, R is halogen, such as fluoro; alkyl, such as methyl or ethyl; methoxy; amino; thio; aminopropyl; dimethylaminoethyl; dimethylaminopropyl; dimethylaminoethoxyethyl; azido; silyl; cycloalkyl; or N-methylacetamido group.

[0248] In certain embodiments, R is hydroxy (-OH), hydrogen (-H), fluoro (-F), amine (-NH3), azido (-N3), thiol (-SH), methoxy (-OCH3), or methoxyethanol (-OCH2CH2OCH3).

[0249] In certain embodiments, R includes a redox-active moiety, a fluorescent or intrinsically fluorescent moiety, natural and non-natural amino acids, peptides, proteins, monosaccharides or oligosaccharides, functional / ligand-binding glycans, or polymers or macromolecules such as polyethylene glycol (PEG).

[0250] As defined herein, each RP is independently an oxygen protecting group, an optionally substituted acyl group, or an amino acid. In certain embodiments, R P is an oxygen protecting group. In certain embodiments, R P is an optionally substituted acyl group. In certain embodiments, R P is an amino acid. In certain embodiments, R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid that can be cleaved by an esterase.

[0251] In certain embodiments, the reversible terminator nucleotide can be deprotected under photochemical conditions. Thus, in certain embodiments, the reversible terminator RNA oligonucleotide is protected with a photo-labile oxygen protecting group at the 2'-O- and / or 3'-O- position. In certain embodiments, the 2'-modified reversible terminator nucleotide is protected with a photo-labile protecting group at the 2'-O position. In certain embodiments, the 3'-modified reversible terminator nucleotide is protected with a photo-labile protecting group at the 3'-O position.

[0252] In certain embodiments, the 2'- or 3'-O-protecting group (e.g., R P ) has one of the following formulas:

[0253]

[0254] In certain embodiments, the 2'- or 3'-O-protecting group (e.g., R P ) has one of the following formulas:

[0255]

[0256] In certain embodiments, the 2'- or 3'-O-protecting group (e.g., R P ) has one of the following formulas:

[0257]

[0258] In certain embodiments, the 2'- or 3'-O-protecting group (e.g., R P ) has the following formula:

[0259]

[0260] In certain embodiments, the 2'- or 3'-O-protecting group (e.g., R P ) is an amino acid having the following formula:

[0261]

[0262] In certain embodiments, R PEach instance is independently an alkyl group, a silyl group, an allyl group, an azidomethyl group, a benzyl group, a coumarin group, or a carbonate group.

[0263] In certain embodiments, the 2'-modified reversible terminator nucleotide is a nucleotide modified with 2'-O-alkyl, 2'-O-silyl, 2'-O-allyl, 2'-O-azidomethyl, 2'-O-benzyl, 2'-O-coumarin, or 2'-O-carbonate. In certain embodiments, the 2'-modified reversible terminator nucleotide is a 2'-O-carbonate modified nucleotide selected from 2'-O-allyloxycarbonyl and 2'-O-(2-oxo-2H-chromen-4-yl)methoxycarbonyl.

[0264] In certain embodiments, the 2'-O-protected reversible terminator is 2'-O-allyl-NTP or 2'-O-azidomethyl-NTP.

[0265] In certain embodiments, the 3'-modified reversible terminator nucleotide is a nucleotide modified with 3'-O-alkyl, 3'-O-silyl, 3'-O-allyl, 3'-O-azidomethyl, 3'-O-benzyl, 3'-O-coumarin, or 3'-O-carbonate. In certain embodiments, the 3'-modified reversible terminator nucleotide is a 3'-O-carbonate modified nucleotide selected from 3'-O-allyloxycarbonyl and 3'-O-(2-oxo-2H-chromen-4-yl)methoxycarbonyl.

[0266] In certain embodiments, the 3'-O-protected reversible terminator is 3'-O-allyl-NTP, 3'-O-azidomethyl-NTP, 3'-O-allyl carbonate-NTP, 3'-O-allyl carbonate-dNTP, 3'-O-azidomethyl carbonate-NTP, or 3'-O-azidomethyl carbonate-dNTP.

[0267] In certain embodiments, the 3'-O-protected reversible terminator is 3'-O-allyl-NTP, 3'-(O-allyl carbonate)-dNTP (e.g., 3'-(O-allyl-carbonate)-dATP, etc.), 3'-(O-azidomethyl carbonate)-dNTP, 3'-(O-acetate)-dNTP, 3'-(O-acyl amino acid)-dNTP, 3'-(O-3-methylcoumarin)-dNTP, 3'-(O-(4-methylcoumarin carbonate)-dNTP, 3'-(O-(2-nitrobenzyl)-dNTP, 3'-(O-(2-nitrobenzyl carbonate)-dNTP, 3'-(O-TMS)-dNTP, or 3'-(O-Teoc)-dNTP.

[0268] Figures 28 - 34Shows certain other embodiments of reversible terminator nucleotides, including certain embodiments of 3'-protected nucleotides.

[0269] As described herein, reversible terminator oligonucleotides can be protected with oxygen protecting groups (e.g., R P group). Oxygen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3 rdThose described in detail in Edition, John Wiley & Sons, 1999. Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenoxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methylbenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylseleno)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridylmethyl, 4-pyridylmethyl, 3-methyl-2-pyridylmethyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, bis(p-methoxyphenyl)phenylmethyl, tris(p-methoxyphenyl)methyl, 4-(4'-bromobenzoyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxy)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinylidene acetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, tert-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N’,N’-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylthiophosphino, alkyl 2,4-dinitrophenylsulfonate, sulfate ester, methanesulfonate (mesylate), benzylsulfonate, and toluenesulfonate (Ts). In certain embodiments, the oxygen protecting group is a silyl group. In certain embodiments, the oxygen protecting group is tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsilyloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxyethyl (EE), 2-methoxy-2-propyl (MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP), trityl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl, p-methoxybenzyl (PMB), tert-butyl, benzyl (Bn), allyl, or pivaloyl (Piv).

[0270] In certain embodiments, the 3'-reversible terminator is a 3'-O-amino acid (e.g., including any standard or non-standard amino acid). In certain embodiments, the amino acid can be removed using an esterase.

[0271] As generally defined herein, R” is hydrogen, halogen, -CN, -NO2, -N3-, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl, optionally substituted hydroxy, optionally substituted amino, or optionally substituted thiol. In certain embodiments, R” is hydrogen. In certain embodiments, R” is halogen. In certain embodiments, R” is -CN. In certain embodiments, R” is -NO2. In certain embodiments, R” is -N3. In certain embodiments, R” is optionally substituted alkyl. In certain embodiments, R” is optionally substituted alkenyl. In certain embodiments, R” is optionally substituted alkynyl. In certain embodiments, R” is optionally substituted aryl. In certain embodiments, R” is optionally substituted heteroaryl. In certain embodiments, R” is optionally substituted carbocyclic group. In certain embodiments, R” is optionally substituted heterocyclic group. In certain embodiments, R” is optionally substituted acyl. In certain embodiments, R” is optionally substituted hydroxy. In certain embodiments, R” is optionally substituted amino. In certain embodiments, R” is optionally substituted thiol.

[0272] In certain embodiments, R''' comprises a reactive component for bioconjugation (e.g., a click chemistry handle such as an azide or an alkyne), a fluorophore, a catalytic protein, an oligonucleotide, or a reporter label.

[0273] As generally defined herein, R''' is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl, optionally substituted hydroxy, optionally substituted amino, optionally substituted thiol, or an oxygen protecting group. In certain embodiments, R''' is hydrogen. In certain embodiments, R''' is halogen. In certain embodiments, R''' is -CN. In certain embodiments, R''' is -NO2. In certain embodiments, R''' is -N3. In certain embodiments, R''' is optionally substituted alkyl. In certain embodiments, R''' is optionally substituted alkenyl. In certain embodiments, R''' is optionally substituted alkynyl. In certain embodiments, R''' is optionally substituted aryl. In certain embodiments, R''' is optionally substituted heteroaryl. In certain embodiments, R''' is optionally substituted carbocyclic group. In certain embodiments, R''' is optionally substituted heterocyclic group. In certain embodiments, R''' is optionally substituted acyl. In certain embodiments, R''' is optionally substituted hydroxy. In certain embodiments, R''' is optionally substituted amino. In certain embodiments, R''' is optionally substituted thiol.

[0274] In certain embodiments, R''' comprises a reactive component for bioconjugation (e.g., a click chemistry handle such as an azide or an alkyne), a fluorophore, a catalytic protein, an oligonucleotide, or a reporter label.

[0275] As generally defined herein, R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group. In certain embodiments, R N is hydrogen. In certain embodiments, R N is optionally substituted alkyl. In certain embodiments, R N is optionally substituted acyl. In certain embodiments, R N is a nitrogen protecting group.

[0276] In certain embodiments, R N comprises a reactive component for bioconjugation (e.g., a click chemistry handle such as an azide or an alkyne), a fluorophore, a catalytic protein, an oligonucleotide, or a reporter label.

[0277] Synthesis of RNA oligonucleotides having non-hydrolyzable RNA nucleotides

[0278] The present invention also provides a method for synthesizing RNA oligonucleotides using non-hydrolyzable nucleotides. As described herein, by introducing non-hydrolyzable nucleotides that compete for the active site of the enzyme, the ratio of nucleotides (i.e., hydrolyzable nucleotides) incorporated by the polymerase at the 3'-end of the initiator oligonucleotide can be controlled. The non-hydrolyzable nucleotides are not incorporated, and the incorporation rate of the hydrolyzable nucleotides is directly affected by the ratio of hydrolyzable nucleotides to non-hydrolyzable nucleotides through competitive inhibition. Ultimately, the number of nucleotides incorporated is determined by the concentration of non-hydrolyzable nucleotides in the reaction mixture. After the poly(N) polymerase incorporates one or more nucleotides via terminal transferase, the process can be repeated in one or more iterative steps, optionally using different nucleotides, until the desired RNA oligonucleotide sequence is obtained.

[0279] The present invention provides a template-independent method for synthesizing RNA oligonucleotides, the method comprising:

[0280] (a) providing an initiator oligonucleotide, wherein the initiator oligonucleotide is single-stranded RNA;

[0281] (b) providing a poly(N) polymerase;

[0282] (c) combining the initiator oligonucleotide, the poly(N) polymerase, one or more nucleotides, and one or more non-hydrolyzable nucleotides under conditions sufficient to add at least one hydrolyzable nucleotide to the 3'-end of the initiator oligonucleotide, wherein the concentration of the non-hydrolyzable nucleotides is sufficient to inhibit the ratio of the poly(N) polymerase from adding one or more nucleotides.

[0283] As described herein, in certain embodiments, the poly(N) polymerase is poly(U) polymerase. The present invention provides a template-independent method for synthesizing RNA oligonucleotides, the method comprising:

[0284] (a) providing an initiator oligonucleotide, wherein the initiator oligonucleotide is single-stranded RNA;

[0285] (b) providing poly(U) polymerase;

[0286] (c) combining the initiator oligonucleotide, poly(U) polymerase, one or more nucleotides, and one or more non-hydrolyzable nucleotides under conditions sufficient to add at least one hydrolyzable nucleotide to the 3'-end of the initiator oligonucleotide, wherein the concentration of the non-hydrolyzable nucleotides is sufficient to inhibit the ratio of poly(U) polymerase from adding one or more nucleotides.

[0287] In certain embodiments, the concentration of the non-hydrolyzable nucleotide is such that 1-100 nucleotides are incorporated thereby. In certain embodiments, the concentration of the non-hydrolyzable nucleotide is such that 1-50 nucleotides are incorporated thereby. In certain embodiments, the concentration of the non-hydrolyzable nucleotide is such that 1-20 nucleotides are incorporated thereby. In certain embodiments, the concentration of the non-hydrolyzable nucleotide is such that fewer than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3 or fewer than 2 hydrolyzable nucleotides are incorporated thereby.

[0288] Once one or more nucleotides are added to the initiator oligonucleotide, one or more additional nucleotides can then be added to synthesize the desired RNA oligonucleotide. Thus, in certain embodiments, the method further comprises adding one or more natural or modified nucleotides to the 3' end of the resulting RNA oligonucleotide (i.e., the RNA oligonucleotide formed in step (c)) until the desired RNA sequence is obtained. In certain embodiments, the method further comprises:

[0289] (d) repeating steps (a)-(c) until the desired RNA sequence is obtained.

[0290] The methods provided herein employ non-hydrolyzable nucleotides. A "non-hydrolyzable" nucleotide is a nucleotide that can bind to RNA polymerase but cannot undergo enzymatic addition (i.e., a terminal transferase reaction) to the initiator oligonucleotide (e.g., added to the 3' end of the initiator oligonucleotide). In certain embodiments, the non-hydrolyzable nucleotide is a phosphate ester-modified nucleotide (i.e., comprising a modified triphosphate group). Also provided herein are non-hydrolyzable nucleotides that can be used in the methods described herein.

[0291] For example, in certain embodiments, the non-hydrolyzable nucleotide has the following formula:

[0292]

[0293] or a salt thereof, wherein:

[0294] each Y is independently -O-, -NR N -, -C(R C )2- or -S-; provided that at least one Y is not -O-;

[0295] R and R' are independently hydrogen or a sugar substituent as defined herein;

[0296] "Base" is a natural or non-natural (e.g., modified) nucleobase as defined herein;

[0297] R N is hydrogen, optionally substituted alkyl or a nitrogen protecting group;

[0298] RC Each instance of -NR is independently hydrogen, a halogen, or an optionally substituted alkyl group. In certain embodiments, -NR N - is -NH-. In certain embodiments, -C(R C )2- is -CH2-.

[0299] In certain embodiments, the non-hydrolyzable nucleotide comprises a modified triphosphate group. In certain embodiments, the non-hydrolyzable nucleotide is selected from the group consisting of uridine-5'-[(α,β)-imido]triphosphate, adenosine-5'-[(α,β)-imido]triphosphate, guanosine-5'-[(α,β)-methyleno]triphosphate, cytidine-5'-[(α,β)-methyleno]triphosphate, adenosine-5'-[(β,γ)-imido]triphosphate, guanosine-5'-[(β,γ)-imido]triphosphate, and uridine-5'-[(β,γ)-imido]triphosphate. The triphosphate group may comprise any other modification.

[0300] In certain embodiments, the non-hydrolyzable nucleotide is a 3'-modified nucleotide. In certain embodiments, the non-hydrolyzable nucleotide is selected from 3'-O-methyladenosine-5'-triphosphate and 3'-O-methyluridine-5'-triphosphate.

[0301] The non-hydrolyzable nucleotide may further comprise any other nucleotide modification described above and herein.

[0302] RNA oligonucleotide synthesis reaction

[0303] The terminal transferase reaction described herein (i.e., step (c) of any method described herein) is carried out in the presence of a polymerase (such as poly(N) polymerase). In certain embodiments, step (c) is carried out in the presence of one or more other enzymes. In certain embodiments, step (c) is carried out in the presence of a mixture of two or more different enzymes. The mixture of enzymes may comprise more than one different poly(N) polymerase (e.g., 2 or 3 different poly(N) polymerases). The mixture of poly(N) polymerases may include wild-type and mutant poly(N) polymerases (such as the mutant poly(U) polymerase provided herein).

[0304] In certain embodiments, step (c) is carried out in the presence of one or more other phosphatases in addition to the poly(N) polymerase. In certain embodiments, step (c) is carried out in the presence of yeast inorganic pyrophosphatase (PPI enzyme) in addition to the poly(N) polymerase.

[0305] In certain embodiments, the terminal transferase reaction in step (c) is carried out in the presence of one or more other additives. In certain embodiments, step (c) is carried out in the presence of a crowding agent. In certain embodiments, the crowding agent is polyethylene glycol (PEG) or Ficoll. In certain embodiments, the crowding agent is polyethylene glycol (PEG). In certain embodiments, step (c) is carried out in the presence of an RNase inhibitor. In certain embodiments, step (c) is carried out in the presence of non-hydrolyzable nucleotides.

[0306] initiator oligonucleotide

[0307] The methods described herein use an initiator oligonucleotide. The initiator oligonucleotide can have any sequence and can have any length of nucleotides. In certain embodiments, the initiator oligonucleotide has a length of 20 nucleotides or less. In certain embodiments, the initiator oligonucleotide has a length of 5 - 20 nucleotides. In certain embodiments, the initiator oligonucleotide has a length greater than 20 nucleotides.

[0308] In certain embodiments, the initiator oligonucleotide is a poly-rN oligonucleotide. In certain embodiments, the initiator oligonucleotide is poly-rU, poly-rC, poly-rG, or poly-rA.

[0309] The initiator oligonucleotide can also be covalently linked to a solid support. In certain embodiments, after obtaining the desired RNA oligonucleotide sequence, the oligonucleotide is cleaved from the solid phase support. Thus, in certain embodiments, the initiator oligonucleotide is covalently linked to the solid support via a cleavable linker.

[0310] The initiator oligonucleotide can contain other modifications, such as a fluorophore. In certain embodiments, the initiator oligonucleotide contains a 5'-fluorophore. In certain embodiments, the fluorophore is Cy5 or FAM. The initiator oligonucleotide can also contain one or more other functional groups or handles for bioconjugation. In certain embodiments, the initiator oligonucleotide is functionalized with biotin.

[0311] In certain embodiments, the initiator oligonucleotide contains a 5'-phosphate (e.g., 5'-mono, di, or triphosphate). In certain embodiments, the initiator oligonucleotide contains a 5'-monophosphate. In certain embodiments, the initiator oligonucleotide contains a 5'-diphosphate. In certain embodiments, the initiator oligonucleotide contains a 5'-triphosphate.

[0312] In certain embodiments, the initiator oligonucleotide contains a 5'-capping group (i.e., 5'-capping).

[0313] In certain embodiments, the 5'-capping can be a mononucleotide (1-nt), dinucleotide (2-nt), trinucleotide (3-nt), or N-nucleotide (i.e., any available oligonucleotide length). The 5'-capping can also comprise a combination of one or more natural and unnatural (e.g., modified) nucleobases, including those described herein.

[0314] In certain embodiments, the 5'-capping is guanine capping. In certain embodiments, the 5'-capping is 7-methylguanosine capping (m 7 G). In certain embodiments, the guanine or m 7 G capping comprises a guanine nucleotide linked to the oligonucleotide via a 5'-5' triphosphate bond. In certain embodiments, the 5'-capping comprises methylation of the 2'-hydroxyl of the first and / or second ribose of the 5'-end of the oligonucleotide.

[0315] In certain embodiments, the 5'-capping is 5'-trimethylguanosine capping or 5'-monomethylphosphate capping. In other embodiments, the 5'-capping is NAD + , NADH, or 3'-dephospho coenzyme A capping.

[0316] In certain embodiments, the initiator oligonucleotide comprises a primer site for reverse transcription synthesis of an RNA oligonucleotide. In certain embodiments, the initiator oligonucleotide comprises a primer site for PCR amplification.

[0317] Splice an RNA fragment together with a 5'-triphosphorylated oligonucleotide

[0318] In certain embodiments, the methods provided herein can be used to splice (i.e., ligate) oligonucleotide fragments together using a 5'-triphosphate group by a template-independent polymerase to produce long RNA (e.g., length >100-nt) molecules.

[0319] A 5'-triphosphate oligonucleotide, synthesized as an initiator oligonucleotide or as a product of a controlled template-independent enzymatic synthesis (e.g., the methods described herein), can be a substrate for a polymerase such as poly(U) polymerase or a mutant variant thereof (e.g., the mutant variants described herein). In certain embodiments, poly(U) polymerase or a mutant variant thereof accepts large 3'-modifications. In some cases, the 3'-modification is a series of nucleic acids (i.e., oligonucleotides) rather than a single nucleoside triphosphate or protecting group.

[0320] Accordingly, provided herein are methods for synthesizing RNA oligonucleotides, the methods comprising:

[0321] (a) providing a first oligonucleotide, wherein the first oligonucleotide comprises a 5'-triphosphate group;

[0322] (b) Provide a second oligonucleotide;

[0323] (c) Provide poly(U) polymerase;

[0324] (d) Combine the first oligonucleotide, the second oligonucleotide, and poly(U) polymerase under conditions sufficient to ligate the first oligonucleotide to the 3' end of the second oligonucleotide.

[0325] In certain embodiments, the second oligonucleotide is a 3'-OH oligonucleotide.

[0326] In certain embodiments, the 5'-triphosphate oligonucleotide is modified to include a phosphorothioate in the α-phosphate.

[0327] In certain embodiments, the first oligonucleotide (e.g., a 5'-triphosphorylated oligonucleotide) includes one or more modifications to the nucleobase, sugar, or backbone of the oligonucleotide. In certain embodiments, the second oligonucleotide includes one or more modifications to the nucleobase, sugar, or backbone of the oligonucleotide.

[0328] In certain embodiments, template-independent ligation occurs under reaction conditions that enhance ligation activity, such as adding a crowding agent as described herein.

[0329] Reverse transcription and amplification of RNA oligonucleotides

[0330] The methods provided herein can be applied to the synthesis of DNA oligonucleotides. After obtaining the desired RNA oligonucleotide by the methods described herein, one or more additional reverse transcription and / or amplification steps can be performed to generate DNA (e.g., cDNA, ssDNA, double-stranded DNA). The end result is a method for controlling template-independent DNA oligonucleotide synthesis.

[0331] Thus, in certain embodiments, the methods provided in this aspect further include the following steps:

[0332] (f) Use a reverse transcription priming site, a primer, and reverse transcriptase to perform reverse transcription on the resulting RNA oligonucleotide to produce a complementary single-stranded DNA oligonucleotide. In certain embodiments, the reverse transcriptase is a high-fidelity reverse transcriptase.

[0333] In certain embodiments, the methods provided herein further include the following steps:

[0334] (g) Use DNA polymerase to amplify the complementary single-stranded DNA oligonucleotide or cDNA produced by reverse transcription from the synthetic RNA oligonucleotide in step (f) to produce double-stranded DNA. In certain embodiments, the DNA polymerase is a high-fidelity DNA polymerase. Examples

[0335] Introduction

[0336] Oligonucleotide-based therapies are an emerging form in the era of rational design and personalized post-genomic medicine (Khvorova et al. 2017). Composed of short sequences of natural and / or non-natural modified nucleic acid building blocks, oligonucleotide therapeutics can be specifically tailored to affect targets with maximal efficacy while retaining an optimal pharmacokinetic profile (Deleavey et al. 2012). This depends largely on the chemical and structural architecture of the oligonucleotide therapeutic, which can include a carefully selected combination of modifications to the sugar ring, nucleobase, and phosphate backbone, as well as the overall three-dimensional structure of the oligonucleotide (Cummins et al. 1995, Eckstein 2014, Watts et al. 2008, Wilson et al. 2006). Both the chemical composition and the sequence of the nucleic acid building blocks confer the overall properties of the oligonucleotide therapeutic; slight rearrangements or different chemical moieties can potentially improve its therapeutic capabilities (Khvorova et al. 2017, Koch et al. 2014, Bohr et al. 2017). This is clearly a key advantage compared to traditional small molecule therapies, where major redesigns may be required for optimization. Despite the wide variety of successful oligonucleotide therapeutics, such as short (<50 nt) antisense oligonucleotides (ASO) (Goyal et al. 2018, Uhlmann et al. 1990), short interfering RNAs (siRNA) (Dana et al. 2017), microRNAs (miRNA) (Rupaimoole et al. 2017), etc., as well as longer (>100 nt) messenger RNAs (mRNA) (Pardi et al. 2018) and long non-coding RNAs (lncRNA) (Arun et al. 2018), a consistent problem is their production, especially on a large scale, which is severely limited by current oligonucleotide synthesis technologies (Ma et al. 2012).

[0337] Since the 1970s, the chemical synthesis of DNA and RNA oligonucleotides using phosphoramidite chemistry has been a mainstay of scientific research (Beaucage et al. 1992). For the synthesis of short and simple DNA oligonucleotides composed of the four natural nucleobases, phosphoramidite chemistry is extremely reliable and inexpensive. However, aside from the advent of large-scale parallel synthesis and automation technologies, there have been only minor incremental improvements to the core methods of phosphoramidite-based oligonucleotide synthesis (Beaucage et al. 1992, Kosuri et al. 2014). This is especially true for the chemical synthesis of RNA and highly modified oligonucleotides, which remains very expensive, low-yielding, and often requires multiple purifications after synthesis, greatly increasing the lead time for isolating large amounts of the desired product (Baronti et al. 2018). In addition, phosphoramidite chemistry is not particularly amenable to the chemical modification of large numbers of components, a requirement for many current oligonucleotide therapeutics (Khvorova et al. 2017), because organic solvents and harsh conditions necessitate additional protecting groups for labile moieties that can endow the oligonucleotide with unique properties for, e.g., delivery or ligand-binding purposes, thereby complicating the synthesis scheme (Baronti et al. 2018). In vitro transcription (IVT) strategies have ameliorated some of these limitations; in particular, the generation of long RNA oligonucleotides (>120-nt), which is currently not possible with phosphoramidite chemistry (Pardi et al. 2018, Milligan et al. 1987, Sahin et al. 2014). However, IVT does not allow for site-specific labeling of oligonucleotides and requires the user to displace specific bases in addition to appropriate enzymatic catalysis using a DNA template. Unfortunately, the combination of high cost, synthetic difficulties, and the unavailability of a diverse array of nucleic acid building blocks has prevented researchers from developing innovative oligonucleotide therapeutics to combat debilitating diseases.

[0338] A potential solution to address the limitations in the aforementioned oligonucleotide synthesis and the development of oligonucleotide-based therapeutic agents is to completely avoid the use of phosphoramidite chemistry. Currently, there is particular interest in exploiting a class of polymerases called nucleotidyltransferases that catalyze the addition of monophosphonucleotides to the 3'-end of a short initiator sequence to synthesize oligonucleotides de novo (Perkel 2019, Pratt et al., 2008). Many nucleotidyltransferases do not require the use of a template sequence, and their reactions can be carried out under aqueous conditions, avoiding many of the negative effects of chemically synthesizing oligonucleotides, including nucleobase depurination, unwanted insertions or deletions, and the accumulation of irreversibly capped truncated products. Some notable nucleotidyltransferases that can synthesize oligonucleotides de novo in a template-independent manner include, but are not limited to, terminal deoxynucleotidyltransferase (TdT) (Motea et al. 2010), Cid1 poly(U) polymerase (PuP) (Munoz-Tello et al. 2012), poly(A) polymerase (PaP) (Balbo et al. 2007), poly(G) polymerase (PgP), poly(C) polymerase (PcP), CCD-adding enzyme (Cho et al. 2007), polymerase Mu (μ) (Dominguez et al. 2000), and polymerase Theta (θ) (Thomas et al. 2019). Among the aforementioned nucleotidyltransferases, only terminal deoxynucleotidyltransferase has been successfully used to demonstrate enzymatic oligonucleotide synthesis (Palluk et al. 2019). However, applications have so far only been possible in DNA data storage because terminal deoxynucleotidyltransferase is difficult to control, has a strong preference for native deoxynucleoside triphosphates, and is particularly biased for certain nucleobase and initiator combinations relative to others, properties that can be computationally calibrated post-synthesis to retrieve stored data (Ceze et al. 2019, Anavy et al. 2019, Lee et al. 2019). Therefore, it is very important to develop an enzymatic oligonucleotide synthesis platform that can (1) extend the generated sequence by one base (n+1) with reversibly blocked modified nucleoside triphosphates, (2) incorporate a range of modified nucleoside triphosphates that confer therapeutic or other value to the oligonucleotide, and (3) scale up to industrially relevant output levels.

[0339] Controlled Enzymatic Synthesis of RNA Oligonucleotides

[0340] Some methods for the enzyme-catalyzed controlled de novo synthesis of RNA oligonucleotides have been developed in the art. Engineered and wild-type polymerases with the ability to efficiently incorporate natural and modified ribonucleoside triphosphates (rNTPs) without a template sequence can be used to repeatedly add nucleotides to the 3'-OH of an initiator oligonucleotide sequence. The addition of these nucleotides can occur through single or multiple incorporation events. The biocompatible reaction conditions required for enzyme function greatly reduce the susceptibility of RNA oligonucleotides to degradation typically associated with chemical synthesis. These methods can be integrated into microfluidic or array-based formats for the cost-effective parallel synthesis of many RNA oligonucleotides. RNA oligonucleotides synthesized by this method can be produced with a low error rate and are biocompatible for downstream biotechnological applications.

[0341] DNA / RNA-directed polymerase μ, RNA-directed polymerase poly(A) polymerase (PAP), and poly(U) polymerase are three example polymerases compatible with the above RNA synthesis protocols. However, any other polymerase or enzyme with the ability to add nucleotides to the 3'-end of an initiator oligonucleotide without a template sequence can be used, such as CCA-adding enzyme. This includes possible functional mutants that show similar or increased ability for the controlled de novo synthesis of RNA.

[0342] Some possible applications of the present invention include: (1) the cost-effective and high-fidelity de novo synthesis of RNA oligonucleotides longer than 100-nt, (2) the synthesized RNA oligonucleotides can be used as a cheap and high-quality source of biomaterials, such as: synthetic transfer RNA, ribosomal RNA, self-folding RNA structures, novel ribozymes, protein-binding complexes, RNA therapeutics, CRISPR / Cas9 guide RNAs, and RNA sequencing probes (e.g., padlock probes for in situ sequencing), (3) the generation of useful PCR-amplifiable DNA oligonucleotides or gene sequences through reverse transcription conversion, and (4) enzymes for RNA synthesis such as Pol(μ) (a DNA / RNA-directed polymerase) are candidates for the controlled enzymatic synthesis of DNA oligonucleotides or gene sequences under biocompatible reaction conditions.

[0343] Using reaction conditions to impede the rate of rNTP incorporation

[0344] The synthesis of RNA oligonucleotides can be controlled by selecting reaction components that severely impede the catalytic rate of natural nucleotide incorporation and maximize the product of the desired length, such as by adding non-hydrolyzable or incompatible nucleotides ( Figure 1)。Add natural rNTPs to the 3’ end of a primer RNA or DNA oligonucleotide hetero- or homopolymer sequence of a predetermined composition and length. The initiator oligonucleotide can be less than 20-nt. The primer oligonucleotide can also include chemical modifications, such as those that are photo-labile or have electro-chemical properties, which allow for cleavage and separation from the full-length RNA oligonucleotide product after enzymatic synthesis. The number of incorporation events is proportional to the concentration ratio of natural nucleotides and non-hydrolyzable or incompatible nucleotides also present in the reaction vessel. The rate of oligonucleotide synthesis is directly affected by competitive inhibition, where low concentrations of non-hydrolyzable or incompatible nucleotides produce longer RNA oligonucleotides at a higher reaction rate, while high concentrations of non-hydrolyzable or incompatible nucleotides can produce smaller RNA oligonucleotides at a slower reaction rate. Non-hydrolyzable nucleotides include those that have modifications to the α-, β-, or γ-phosphate of the triphosphate but do not affect the binding affinity of the nucleotide to the enzyme. Incompatible nucleotides include those that have 2’- and / or 3’- moiety or nucleobase modifications that result in non-reactivity without significantly altering the binding affinity of the nucleotide to the enzyme. In one embodiment, this synthesis protocol can be carried out in a microfluidic setting where different natural nucleotides can be rapidly switched off, allowing for the incorporation of multiple bases into a surface-bound initiator oligonucleotide.

[0345] Modified rNTP incorporation reversibly blocks additional incorporation events

[0346] RNA oligonucleotide synthesis can also be controlled by incorporating modified nucleotides that transiently alter the binding affinity of the polymerase to the initiator oligonucleotide to limit the extension reaction to only one incorporation event (n+1)( Figure 2 )。Add modified rNTPs to the 3’-end of a primer oligonucleotide sequence of a predetermined composition and length. The initiator oligonucleotide can be less than 20-nt. The primer oligonucleotide can also include chemical modifications, such as photo-labile or electro-chemical modifications, which allow for cleavage and separation from the full-length RNA oligonucleotide product after enzymatic synthesis. Incorporation of a single modified nucleotide alters the binding affinity of the enzyme to the primer oligonucleotide such that the enzyme is no longer able to incorporate other nucleotides beyond (n+1). The modified nucleotide can have a non-natural chemical moiety at, for example, the 2’-, 3’-, or 2’- and 3’- positions of the nucleotide( Figure 2)。Once the modified rTNP is incorporated, a mild deprotection reaction is employed that functions optimally under biocompatible conditions to remove the modification, thereby revealing the native chemical domain. Based on the deprotection, the enzyme will regain affinity for the oligonucleotide and can incorporate other modified nucleotides ((n + 1)+1) corresponding to the next base in the sequence. The procedure of modified nucleotide incorporation, deprotection, and restoration of enzyme binding affinity is repeated until the desired RNA oligonucleotide sequence is produced. The requirement of this protocol is that the conversion from n to n + 1 is highly efficient, and thus steps are taken to ensure that the enzyme has this ability. In one embodiment, successful incorporation events can be visually monitored by selecting modified nucleotides that include a fluorophore or a reactive domain that attaches a fluorophore upon incorporation. In another embodiment, this synthesis protocol can be carried out in a microfluidic setup that can be used to wash away unused nucleotides and prepare the extended RNA oligonucleotide for the next round of extension.

[0347] Polymerases for Controlled RNA Synthesis

[0348] Family X polymerases

[0349] Polymerases from family X such as terminal deoxynucleotidyl transferase (TdT), polymerase Mu (Polμ), polymerase Beta (Polβ), and polymerase Lambda (Polλ) are candidates for controlled template-independent synthesis of RNA oligonucleotides (Fowler and Suo 2006). These highly specialized polymerases have been shown to be key drivers in critical DNA repair pathways such as non-homologous end joining (NHEJ) as well as in antibody production and T cell receptor diversity during V(D)J recombination (Moon et al. 2007, 2014; Nick McElhinny and Ramsden 2004; Bertocci et al. 2006). The involvement of family X polymerases in such biological processes is attributed to their precision in incorporating native nucleotides in a template-dependent manner while maintaining the ability to add nucleotides randomly in the primer sequence in a template-independent manner when variability is required (J.F. Ruiz et al. 2001; Domínguez et al. 2000; Motea and Berdis 2010). This unique ability is ideal for enzymatic RNA synthesis, where the natural flexibility associated with family X polymerases is substantial without the need for protein evolution protocols. It has previously been shown that TdT has the ability to incorporate native nucleotides in addition to DNA nucleotides (Roychoudhury 1972). Family X polymerases can be further engineered to be more compatible with the proposed RNA synthesis protocol.

[0350] Polymerase Mu (Polμ)

[0351] Polμ is a family X polymerase that has been shown to efficiently incorporate deoxyribonucleoside triphosphates (dNTPs) and rNTPs into DNA, RNA, and DNA-RNA hybrid oligonucleotide substrates under optimal reaction conditions (José F. Ruiz et al. 2003; Agrawal et al. 2003). Multiple studies of the enzyme's primary structure, catalytic pocket, and various catalytic states have identified the amino acid residues associated with rNTP binding and its incorporation kinetics ((Moon et al. 2014; Jamsen et al. 2017; Moon et al. 2017). Intriguingly, wild-type Polμ has the ability to incorporate rNTPs without distorting the oligonucleotide primer or nucleotide structure and maintaining the geometry of the active site in its conformation; this phenomenon may significantly affect the ability of other family X polymerases to accommodate rNTPs in any effective capacity or rate (Moon et al. 2017). Additionally, it is noteworthy that Polμ has been shown to have a lower preference for DNA substrates compared to other family X polymerases (Moon et al. 2015).

[0352] Polμ mutagenesis

[0353] In one study, the expression of two tumor-associated human Polμ point mutants (G174S) and (R175H) produced enzymes with reduced efficiency and fidelity in NHEJ. These mutants, despite having a template sequence that guides the DNA repair process, have been shown to randomly incorporate nucleotides, resulting in a significantly altered expected error rate (Sastre-Moreno et al. 2017). Other research groups have demonstrated that removing most of Polμ, such as the N-terminal BRCT domain, which is normally associated with core factors of other DNA repair pathways, produces an active enzyme (Moon et al. 2014). These truncated variants show retention of wild-type activity and the ability to bind non-hydrolyzable nucleotides, but potentially have more physical space to incorporate modified or bulky nucleotides. However, wild-type Polμ has been identified as a predominantly template-dependent polymerase. However, a point mutation (R387K) in human wild-type Polμ produces an enzyme with significantly increased template-independent activity (Andrade et al. 2009). This point mutation (R387K) is extremely important for the feasibility of using Polμ in the de novo synthesis of RNA oligonucleotides in a template-independent manner, reaffirming the great value of its flexibility. Polμ is currently the only known polymerase in family X that shows both template-dependent and template-independent activities (Domínguez et al. 2000; Juarez et al. 2006). In addition to wild-type or mutagenized Polμ, there are other polymerases that could potentially be used for the de novo synthesis of long RNA oligonucleotides.

[0354] Poly(A) polymerase, poly(U) polymerase & other RNA polymerases

[0355] 3'-end tailing of single-stranded RNAs with ribonucleotides is important in two different contexts: (1) natural biological or biochemical processes; (2) studying these processes in vivo or in vitro (Proudfoot 2011; Strauss et al. 2012). For the latter, several research groups have used wild-type RNA polymerases such as Saccharomyces cerevisiae and Escherichia coli poly(A) polymerases (PAPs), and Schizosaccharomyces pombe Cid1 poly(U) polymerase (PUP) to directly label the 3'-ends of RNA oligonucleotides in a template-independent manner in vitro ((G. Martin and Keller 1998; Munoz-Tello, Gabus, and Thore 2012; Kwak and Wickens 2007; Winz et al. 2012). Under optimal conditions, these enzyme families, particularly PAPs, have been shown to accept modified ribonucleotides modified at the 2'- and 3'-positions of the sugar and the 8'-position of the adenosine base (Winz et al. 2012). Although the overall incorporation efficiency varies between the modified positions, nucleobases, and enzymes tested, on average 1-3 nucleotide incorporation events occur, generating RNA oligonucleotides with 3'- or internal azide functional groups attached to dyes via bioorthogonal click chemistry (Winz et al. 2012).

[0356] In addition to studying the mechanism of modified nucleotide incorporation, other research groups have examined the biochemical and structural mechanisms of substrate binding and catalysis by PAPs (Georges Martin et al. 2004; Bard et al. 2000). From these studies, including site-directed mutagenesis of many residues in the catalytic pocket and exhaustive analysis of steady-state kinetics, it is clear that there is a strong bias towards ATP incorporation compared to other nucleobases (Georges Martin et al. 2004). However, another research group determined that a single-point mutation (R215A) in bacterial PAP resulted in a complete reversal of this bias, allowing random incorporation of all nucleobases (Just et al. 2008). This result is in line with that of another template-independent RNA polymerase, namely CCA-adding enzyme (Just et al. 2008; Xiong and Steitz 2004). These studies render RNA polymerases such as PAPs highly compatible in performing Protocol II for the controlled de novo synthesis of RNA oligonucleotides. Both wild-type and mutagenized RNA polymerases appear to be flexible enough to incorporate U, A, G, or C sugar-modified nucleobases (2', 3'- or both), which can be deprotected or modified under mild conditions to restore enzyme binding affinity.

[0357] Results: Synthesis of RNA oligonucleotides

[0358] 1. Purified human polymerase μ R387K exhibits template-independent terminal transferase activity

[0359] Human polymerase μ R387K was expressed and purified as described in the Materials and Methods section. Since it was not clear under what reaction conditions polymerase μ R387K would function optimally, several reaction parameters were initially evaluated. It was found that a incubation temperature of 37 °C and the following reaction buffer conditions produced sufficient enzymatic activity in terms of dNTP incorporation: 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate. In addition, common divalent metal cofactors (Mn 2+ , Mg 2+ , Co 2+ , etc.) were supplemented in the reaction, and then the polymerase μ activity was evaluated. It was found that the combination of Mn 2+ and Mg 2+ produced the highest ssDNA generation rate of approximately 650 RFU / minute at a concentration of 0.25 mM, while 0.25 mM Co 2+ produced the worst productivity of approximately 100 RFU / minute ( Figure 3A ). Next, an attempt was made to determine whether polymerase μ R387K could incorporate rNTPs under similar reaction conditions. Denaturing gel electrophoresis showed that polymerase μ R387K could incorporate native dATP ( Figure 3B ) and could also incorporate rATP ( Figure 3C ); however, 5 mM rATP was required to produce results similar to those of 200 μM dATP. These results indicate that polymerase μ R387K exhibits terminal transferase activity like other members of the polymerase X family, functions independently of the template sequence, and can accommodate dNTPs and rNTPs. Therefore, polymerase μ R387K, which has been confirmed to be a DNA / RNA-directed polymerase, can be used for controlled enzymatic RNA synthesis.

[0360] 2. Saccharomyces cerevisiae poly(A) polymerase incorporates 2'-modified ATP nucleotides and 2'-blocked reversible terminators

[0361] The efficacy of Saccharomyces cerevisiae poly(A) polymerase (Thermo 74225Z25KU) to incorporate multiple 2'-modified nucleotides was evaluated. The modified nucleotides evaluated included 2'-F-rATP (Trilink N-1007), 2'-azido-rATP (Trilink N-1045), 2'-amino-rATP (Trilink N-1046), and 2'-O-methyl rATP (Trilink N-1015). The appropriate buffer was supplemented for the extension reaction, which included 0.5 mM Mn 2+, 200 pmol of initiator RNA oligonucleotide, 2.5 mM modified nucleotide, and 900 units of enzyme. Before analysis by denaturing gel electrophoresis, the reaction mixture was incubated at 37 °C for 60 minutes. According to gel analysis ( Figure 4A ), under these reaction conditions, Saccharomyces cerevisiae poly(A) polymerase incorporates all 2'-modified nucleotides (2'-amino-, 2'-O-methyl-, 2'-F-, and 2'-azido-rATP). These results indicate that Saccharomyces cerevisiae poly(A) polymerase is tolerant to different chemical modifications of the 2'-portion of the nucleotide sugar and is chemically compatible with 2'-reversible terminators. Under the same reaction conditions, incorporation of the reversible terminator nucleoside triphosphate 2'-O-allyl-rATP by Saccharomyces cerevisiae poly(A) polymerase was assayed. The resulting denaturing gel ( Figure 4B ) showed positive (n+1) incorporation within a range of nucleoside triphosphate concentrations (250 μM - 4000 μM) compared to a negative control reaction containing all components except enzyme and nucleotide. This indicates that the combination of Saccharomyces cerevisiae poly(A) polymerase with the reversible terminator nucleoside triphosphate bearing 2'-O-allyl can be used for the controlled enzymatic synthesis of RNA oligonucleotides.

[0362] 3. Schizosaccharomyces pombe Cid1 poly(U) polymerase incorporates natural nucleotides generally

[0363] The efficiency of incorporation of natural ribonucleotides by Schizosaccharomyces pombe Cid1 poly(U) polymerase (NEB M0337) was evaluated. For kinetic analysis, appropriate buffer, 10 pmol of labeled initiator RNA oligonucleotide (5'-Cy5-poly rU-15-mer), 1.0 mM natural nucleotide (ATP, UTP, GTP, or CTP), 1×SYBR Green II for RNA (Thermo), and 2 units of poly(U) polymerase were added to the extension reaction. The reaction was incubated at 37 °C for 30 minutes and monitored in real time. Then, 2 μL of each extension reaction mixture was analyzed using a 15% TBE-urea gel and imaged on a Typhoon FLA9500 system using EX:649 nm and EM:666 nm. It was clearly seen from the gel analysis that poly(U) polymerase has the ability to incorporate all natural ribonucleotides compared to the control; however, there was some bias in the number of extensions ( Figure 5A ). These results are consistent with previous findings (Munoz-Tello, Gabus, and Thore 2012; Lunde, Magler, and Meinhart 2012), except for rGTP and rCTP, for which poly(U) polymerase has previously been shown to have little activity. However, the results using gel electrophoresis and RNA kinetic assays confirmed that poly(U) polymerase is active towards rGTP and rCTP (Figure 5B )。

[0364] 4. Schizosaccharomyces pombe Cid1 poly(U) polymerase incorporates 2'-modified nucleotides promiscuously

[0365] The Schizosaccharomyces pombe Cid1 poly(U) polymerase is known to have the ability to incorporate all four natural ribonucleotides promiscuously. An attempt was made to determine whether this promiscuity could be extended to 2'-modified nucleotides. Using the same reaction parameters, the poly(U) polymerase was incubated with 2.5 mM of 2'-O-methyl-rATP, rUTP, rCTP, or rGTP at 37 °C for 60 min. Then, 2 μL of each extension reaction was analyzed using a 15% TBE-urea gel and imaged on a Typhoon FLA 9500 system using EX:649 nm and EM:666 nm. Gel analysis showed that the Schizosaccharomyces pombe Cid1 poly(U) polymerase incorporates 2'-modified nucleotides promiscuously and extends the primer oligonucleotide by only +1 - 2 nucleotides with very high efficiency ( Figure 6 )。As before, there is some bias in nucleotide selection; however, after 60 min of incubation, all extension products are very similar and thus negligible overall. This result is highly desirable for controlled RNA synthesis. The Schizosaccharomyces pombe Cid1 poly(U) polymerase is unique in that (1) no other currently known enzyme can incorporate modified nucleotides promiscuously and (2) the extension products are only +1 - 2 nucleotides. Like the Saccharomyces cerevisiae poly(A) polymerase, the Schizosaccharomyces pombe Cid1 poly(U) polymerase is tolerant of chemical modifications at the 2'-position of the nucleotide sugar and is chemically compatible with 2'-reversible terminators.

[0366] 5. Schizosaccharomyces pombe poly(U) polymerase is minimally affected by the sequence composition and secondary structure / hairpin of the primer oligonucleotide

[0367] Many terminal transferases are highly sensitive to the sequence composition of the primer oligonucleotide, where different bases at the 3'-OH terminus can greatly affect the rate of nucleotide incorporation. Therefore, in the presence of two 5'-labeled primer oligonucleotides with different compositions, the extension reactions were carried out with all four natural ribonucleotides to study whether Schizosaccharomyces pombe poly(U) polymerase is affected in this way. The reactions were carried out with 1 mM ribonucleotides and 10 pmol of the 5'-labeled primer oligonucleotide. Then 2 μL of each extension reaction was analyzed using a 15% TBE-urea gel and imaged on a Typhoon FLA 9500 system using EX: 649 nm and EM: 666 nm and EX: 495 nm and EM: 520 nm for 5'-Cy5-poly rA-15-mer. As shown by denaturing gel electrophoresis, it was found that Schizosaccharomyces pombe poly(U) polymerase was minimally affected by the primer oligonucleotide sequence composition bias( Figure 7A ). Universal ribonucleotide incorporation was observed with both primer oligonucleotides; however, the 5'-Cy5-poly rA-15-mer appeared to be slightly more efficient as there were fewer starting products present after 30 min of incubation. In addition to sequence composition, the effect of controlled enzymatic extension using primer oligonucleotides with strong secondary structures was also investigated. For this purpose, the IDT Oligoanalyzer Tool TM was used to generate several oligonucleotides to induce strong hairpin structures under the reaction conditions (considering Mg 2 + , nucleotide concentration, DNA / RNA oligonucleotide, etc.). The sequences of each oligonucleotide were similar, differing only in the position of the hairpin relative to the 3'-terminus, resulting in the following variations: 1 base at the 3'-terminus (H1), 5 bases at the 3'-terminus (H5), 10 bases at the 3'-terminus (H10), and 20 bases at the 3'-terminus. To ensure that the hairpins of the oligonucleotides were correctly formed prior to enzymatic extension, the oligonucleotides were heated to 95 °C and then slowly cooled to 15 °C at a rate of 0.1 °C / min in the appropriate enzymatic reaction buffer on a thermal cycler. After cooling, the remaining reaction components were added to the hairpin primer oligonucleotide and a 5-min extension reaction was carried out at 37 °C using reversible terminator nucleoside triphosphates 2'-O-allyl-ATP or -UTP. The extension efficiency of each hairpin primer oligonucleotide was determined using a 15% TBE-urea gel under denaturing conditions. Overall, Schizosaccharomyces pombe poly(U) polymerase was able to extend the hairpin primer oligonucleotides with strong secondary structures to (n + 1); however, there were some difficulties with the H1 oligonucleotide, where there was only 1 base after the hairpin( Figure 7B), i.e., an elongation rate of approximately 10%. Difficult secondary structures may pose risks to the synthesis protocol, but in addition to high reaction temperatures or Schizosaccharomyces pombe poly(U) polymerase mutants, other reaction components such as DMSO or betaine can be added to help solve the problem. However, these results further emphasize the unique flexibility of Schizosaccharomyces pombe poly(U) polymerase.

[0368] 6. Enhancement of Schizosaccharomyces pombe poly(U) polymerase activity by addition of inorganic pyrophosphatase

[0369] The result of high terminal transferase activity is the rapid accumulation of inorganic pyrophosphate, which is a known inhibitor of DNA and RNA-directed polymerases. To reduce the accumulation of inorganic pyrophosphate, inorganic pyrophosphatase (PPi-ase) can be used to cleave pyrophosphate into two single phosphates while the reaction is proceeding. Therefore, an attempt was made to determine whether supplementation with pyrophosphatase could enhance the terminal transferase activity of Schizosaccharomyces pombe poly(U) polymerase. The reaction mixture was incubated with 1 mM of each ribonucleotide, 10 pmol of 5’-Cy5-poly-rU-15-mer primer oligonucleotide, and 0.1 unit of yeast inorganic pyrophosphatase (New England Biolabs M2403) at 37 °C for 30 minutes. Then, 2 μL of each elongation reaction mixture was analyzed using a 15% TBE-urea gel and imaged on a Typhoon FLA 9500 system using EX:649 nm and EM:666 nm. It was found that supplementation with inorganic pyrophosphatase increased the rate of RNA synthesis by Schizosaccharomyces pombe poly(U) polymerase and increased the maximum length of the synthesized RNA ( Figures 8A - 8C ). The greatest increase was observed for the natural ribonucleotides rUTP and rATP, while rGTP and rCTP were the least affected. This observation is mainly attributed to the preference of wild-type Schizosaccharomyces pombe poly(U) polymerase for rUTP and rATP during the synthesis of long RNA chains, resulting in more accumulation of inorganic pyrophosphate. Although rGTP and rCTP can still be incorporated by Schizosaccharomyces pombe poly(U) polymerase, the total number of incorporation events is less, which leads to less accumulation of inorganic pyrophosphate. However, the addition of inorganic pyrophosphatase to the Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reaction is beneficial and includes modifications to the recommended and commercially standardized reaction conditions.

[0370] 7. Schizosaccharomyces pombe poly(U) polymerase activity can naturally incorporate base-modified ribonucleotides

[0371] The application of the method provided herein is the synthesis of bioactive molecules, such as the synthesis of transfer RNA (tRNA) or ribosomal RNA (rRNA). Generally, ribonucleotide bases containing tRNA and rRNA are subjected to natural base modifications to, for example, induce secondary structures in vivo to obtain optimal functionality. Additionally, incorporation of modified bases into RNA oligonucleotides can greatly enhance their stability and prevent unwanted nuclease digestion. Therefore, an attempt was made to determine whether Schizosaccharomyces pombe poly(U) polymerase has the ability to incorporate pseudouridine, which is one of the most common modified ribonucleotide bases in tRNA and rRNA. The reactants were incubated at 37 °C for 30 minutes with 2 mM, 1 mM, or 0.5 mM rUTP or pseudouridine (Trilink N-1019), 10 pmol of 5’-Cy5-poly-rU-15-mer primer oligonucleotide, and 0.1 unit of yeast inorganic pyrophosphatase. Then, 2 μL of each extended reactant was analyzed using a 15% TBE-urea gel and imaged on a Typhoon FLA 9500 system using EX:649 nm and EM:666 nm. It was found that Schizosaccharomyces pombe poly(U) polymerase has an inherent ability to incorporate pseudouridine, generating base-modified RNA oligonucleotides with lengths of approximately 30-45-nt ( Figure 9A ). The resulting poly-pseudouridine RNA oligonucleotides were shorter than the unmodified rUTP oligonucleotides; however, this is the first known instance indicating that Schizosaccharomyces pombe poly(U) polymerase can incorporate this nucleotide. From these results, it can be seen that Schizosaccharomyces pombe poly(U) polymerase most likely has the ability to incorporate other base-modified nucleotides, such as those with methylation or other modifications at various positions of the ribonucleotide bases. Therefore, Schizosaccharomyces pombe poly(U) polymerase was incubated with a series of base-modified nucleoside triphosphates that have modifications to all four natural bases. These include, but are not limited to, inosine 5’-triphosphate, N 1 -methyladenosine-5’-triphosphate, N 6 -methyladenosine-5’-triphosphate, N 6 -methyl-2-aminoadenosine-5’-triphosphate, 8’-azidoadenosine-5’-triphosphate, 5-methyluridine-5’-triphosphate, N 1 -methylpseudouridine-5’-triphosphate, pseudouridine-5’-triphosphate, 5-hydroxymethyluridine-5’-triphosphate, 5-methylcytidine-5’-triphosphate, 5-hydroxymethylcytidine 5’-triphosphate, N 7 -methylguanosine triphosphate, intrinsically fluorescent nucleotides such as 3’ / 2’-O-(N-methylanthraniloyl)-triphosphate, and phosphate-modified nucleotides such as α-, β-, γ-thiotriphosphates. Denaturing gel electrophoresis indicated the incorporation of each base-modified ribonucleotide detected with different numbers of incorporation events (Figure 9B ) can be synthesized with 2'-blocking groups to base-modified ribonucleotides, such as those nucleotides detected, rendering them compatible as reversible terminators. These results form the basis for the de novo synthesis of important biomolecules such as tRNA or rRNA, for which a high degree of base modification can be performed, depending on their function or RNA oligonucleotides with specific secondary structures or RNA oligonucleotides with higher resistance to degradation.

[0372] 8. RNA synthesis by Schizosaccharomyces pombe poly(U) polymerase can be controlled by competitive inhibitor nucleotides.

[0373] As Figure 1 illustrated, by competitive inhibition, the ratio of hydrolysable to non-hydrolysable nucleotides present in the reaction directly affects the rate of RNA oligonucleotide synthesis. The inhibitor occupies the active site of the enzyme but is not incorporated, thus slowing down the RNA synthesis reaction and thereby reducing the number of binding events within a given reaction time. To demonstrate this, Schizosaccharomyces pombe poly(U) polymerase RNA synthesis reactions were incubated with increasing concentrations of the non-hydrolysable ribonucleotide uridine 5'-[(α,β)-imino]triphosphate (Jena Biosciences). It was found that higher concentrations of the non-hydrolysable ribonucleotide significantly restricted the total incorporation of hydrolysable rUTP ( Figure 10 ). The RNA synthesis reactions were incubated at 37 °C for 30 min, and then 2 μL of each elongation reaction was analysed using a 15% TBE-urea gel and imaged on a Typhoon FLA 9500 system using EX:649 nm and EM:666 nm. Concentrations of the non-hydrolysable ribonucleotide uridine-5'-[(α,β)-imino]triphosphate below 0.8 mM had little effect on controlling the RNA synthesis reaction; this may be due to the binding affinity threshold of uridine 5'-[(α,β)-imino]triphosphate to Schizosaccharomyces pombe poly(U) polymerase. Other non-hydrolysable ribonucleotides can be used in this way, which need not be uridine bases and need not be Schizosaccharomyces pombe poly(U) polymerase.

[0374] 9. RNA synthesis by Schizosaccharomyces pombe poly(U) polymerase can be controlled by 2'-O-blocked reversible terminator nucleoside triphosphates.

[0375] As Figure 2As shown in the example, RNA synthesis can be controlled by incorporating 2'-O-blocked reversible terminator nucleoside triphosphates, thereby temporarily terminating synthesis after the production of (n + 1) oligonucleotides in a single incorporation event. A mild deprotection protocol is then employed to remove the blocking groups to reconstruct the resulting oligonucleotides into substrates recognizable by the enzyme such as poly(U) polymerase. To determine whether Schizosaccharomyces pombe poly(U) polymerase has the ability to incorporate reversible terminator 2'-modified nucleoside triphosphates, 2'-O-allyl-ATP was incubated in an RNA synthesis reaction for 30 minutes under optimal reaction conditions. Analysis of the results of the RNA synthesis reaction showed that Schizosaccharomyces pombe poly(U) polymerase incorporated only a single 2'-O-allyl-ATP and extended the primer oligonucleotide by one base compared to the control reaction ( Figure 11A ). This result is particularly notable because many other modified nucleoside triphosphates (e.g., 2'-, base, etc.) produce multiple extension products, while 2'-O-allyl-ATP produces only one extension product. Further optimization of the reaction conditions and the stoichiometry of the components led to the determination that Schizosaccharomyces pombe poly(U) polymerase requires less than or equal to 0.5 minutes to completely convert the primer oligonucleotide (+0) to the extended product (+1) with a conversion rate > 99% ( Figure 11B ). Further increasing the incubation time had no apparent effect on the RNA synthesis reaction. The deblocking and further extension of the reversible terminator 2'-modified nucleoside triphosphate 2'-O-allyl-ATP were confirmed by incubating the purified (n + 1) oligonucleotide with a mixture of disodium tetrachloropalladate (Na2PdCl4) and trisodium 3,3',3''-triphenylphosphine tris(benzenesulfonate) (TPPTS) in Tris-HCl buffer at variable pH values at 50 °C for 10 minutes. The resulting deblocked oligonucleotide was then purified and extended under optimized Schizosaccharomyces pombe poly(U) polymerase reaction conditions including 1 mM reversible terminator 2'-O-allyl-ATP to obtain the (n + 2) product. Denaturing gel electrophoresis analysis showed that the purified (n + 1) oligonucleotide was efficiently converted to the (n + 2) product after optimizing the composition and pH of the deblocking buffer ( Figure 11C ). Successful deblocking of the 2'-reversible terminating group was observed in Tris-HCl buffer at pH 7.5, 6.5, 5.5, 4.5 (but not 8.5). Buffers with lower pH are known to those skilled in the art to enhance RNA stability, and a minimal buffer composition ensures access of the deblocking components (Pd & TPPTS) to the RNA oligonucleotide. A high-resolution denaturing gel showing the (n + 2) product is shown in Figure 11DShown. In addition, the stability and functionality of the 5'-fluorophore were retained throughout the deblocking process, and minimal oligonucleotide degradation was observed, indicating the biocompatibility of the chemical method. To determine whether the 2'-O-allyl reversible terminator strategy is favorable for the synthesis of longer RNA oligonucleotide fragments, the enzymatic extension and deblocking processes were repeated in bulk solution using optimized reaction conditions and 2'-O-allyl-ATP to obtain (n + 5) products. During synthesis, a small aliquot of each extension / deblocking event (also known as a synthesis cycle) was set aside for analysis. Gel electrophoresis analysis showed the successful synthesis of 25 nt fragments starting from a 20 nt initiator oligonucleotide ( Figure 11E ). Note that sample loss did occur during each purification and deblocking step, as indicated by the reduced signal on the gel. Overall, these results indicate that Schizosaccharomyces pombe poly(U) polymerase has the inherent ability to enzymatically synthesize RNA in a repetitive base-by-base manner, as Figure 2 shown. Other reversible terminator nucleoside triphosphates can also be incorporated by Schizosaccharomyces pombe poly(U) polymerase. This includes but is not limited to 2'-O-azido-methyl-NTP.

[0376] 10. Schizosaccharomyces pombe poly(U) polymerase efficiently incorporates 2'-reversible terminator nucleoside triphosphates with all four natural nucleobases

[0377] Previously, it was determined that Schizosaccharomyces pombe poly(U) polymerase has the ability to incorporate 2'-modified nucleoside triphosphates (2'-methoxy) bearing the four natural RNA nucleobases (A, U, G, C) with relatively equal efficiency ( Figure 6 ). To determine whether this is also the case for reversible terminator nucleoside triphosphates, 2'-O-allyl-ATP, -UTP, -CTP, and -GTP were synthesized. Using optimal conditions, the extension reaction was incubated in a thermal cycler with 1 mM of 2'-O-allyl-ATP, -UTP, -CTP, or -GTP reversible terminator at 37 °C for 1 minute. The reaction was then analyzed using a 15% TBE-urea denaturing gel, showing that Schizosaccharomyces pombe poly(U) polymerase efficiently incorporates the 2'-O-allyl reversible terminator in each nucleobase form, as shown by a single (n + 1) extension event ( Figure 12A)。The control reaction has all reaction components except nucleotides. To further demonstrate that this result is favorable for the synthesis of polynucleotide RNA oligonucleotides, a binary synthesis was carried out using a combination of 2'-O-allyl-ATP and -UTP. The following combinations were detected in bulk solution using optimized enzymatic extension and deblocking reaction conditions: (n+1)A, (n+1)U, (n+2)A-A, (n+2)A-U, (n+2)U-A, and (n+2)U-U. The resulting materials were analyzed using a 15% TBE-urea gel, showing positive incorporation under single (n+1) detection conditions and positive incorporation and deblocking under double (n+2) detection conditions compared to the blank reaction (all reaction components except nucleotides) that produced the (n+0) example Figure 12B )。

[0378] 11. Active Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His 6 tag can be expressed in bacteria for large-scale production and purification

[0379] By adding the amino acid "MGSSHHHHHHSSGLVPRGSH" to the N-terminus of Schizosaccharomyces pombe poly(U) polymerase to modify its basic sequence (UniProtKB - O13833). These amino acids encode an N-terminal His 6 tag with an appropriate linker. Using the protocol outlined in the Materials and Methods section, the Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His 6 tag was expressed, purified, and concentrated into a small volume. Denaturing gel electrophoresis showed that the Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His 6 tag was correctly expressed and isolated from the bacterial lysate. With an N-terminal His 6 tag, the expected molecular weight of Schizosaccharomyces pombe poly(U) polymerase is approximately 45 kDa, which corresponds to a strong band on the gel ( Figure 13A )。To determine the activity of the purified and concentrated Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His 6 tag, an extension reaction supplemented with 1 mM of the 2'-O-allyl-ATP reversible terminator nucleotide and increasing amounts of the initiator oligonucleotide was incubated with the expressed enzyme. Using the Schizosaccharomyces pombe poly(U) polymerase with an N-terminal His 6 tag, it was determined that approximately 100 pmol of the initiator oligonucleotide could be converted into the (+1) product with a conversion rate > 99% ( Figure 13B )。Reactions with a supplement greater than 100 pmol may require additional optimization to obtain a higher conversion rate. These results indicate that the N-terminal His 6The Schizosaccharomyces pombe poly(U) polymerase with a tag can be easily expressed at very low cost and can be scaled up to produce large amounts of the enzyme for RNA synthesis. In addition, the purified and concentrated N-terminal His 6 -tagged Schizosaccharomyces pombe poly(U) polymerase can convert large amounts of RNA oligonucleotide material, thereby reducing the need to repeatedly perform synthesis reactions to obtain the desired RNA sequence in high yield. No ribonuclease residues were observed in the bacterial lysate in which the Schizosaccharomyces pombe poly(U) polymerase was expressed and purified.

[0380] 12. Controlled RNA oligonucleotide synthesis using Schizosaccharomyces pombe poly(U) polymerase can be carried out on a solid-phase surface

[0381] Controlled synthesis of RNA oligonucleotides can be easily carried out using bulk solution; however, after the extension and deblocking steps in each synthesis cycle, the generated oligonucleotides must be purified to remove interfering components. Although efficient recovery can be achieved with modern methods, multiple purifications will ultimately result in significant loss of the sample after several synthesis cycles. Therefore, oligonucleotide synthesis on solid-phase supports such as functionalized beads, wells, slides, etc. is significantly more favorable for synthesizing longer oligonucleotide fragments and large amounts of industrially relevant materials. To evaluate the ability of Schizosaccharomyces pombe poly(U) polymerase to extend oligonucleotides anchored to the surface, an initiator oligonucleotide with a 5'-amine and an internal Cy5 dye was first used to attach a 5'-biotin-PEG-NHS linker (EZ-Link #A35389 Thermo). Analysis using a 15% TBE-urea gel determined that the efficiency of the labeling reaction was >90% (the addition of a large PEG group would cause the oligonucleotide to behave differently compared to the unlabeled oligonucleotide)( Figure 14A ). Further quality control was carried out using streptavidin-functionalized magnetic beads (Spherotech #SVM-20-10), where positive non-covalent anchoring of the initiator oligonucleotide with an internal Cy5 dye label was observed, as determined by fluorescence microscopy( Figure 14A ). Then, using the anchored initiator oligonucleotide and 2'-O-allyl reversible terminator nucleoside triphosphates, three cycles of enzymatic oligonucleotide synthesis were carried out using optimized extension and deblocking reaction conditions. The (n + 3) product was synthesized, where "A" was added in cycle 1, "C" was added in cycle 2, and "U" was added in cycle 3. After synthesis, the anchored oligonucleotides were removed from the surface of the streptavidin beads by incubating the streptavidin beads with a 90% formamide solution in water at 50 °C for 10 minutes in a thermal cycler. The collected material was purified and analyzed using a 15% TBE-urea gel, showing efficient oligonucleotide synthesis with multiple nucleobases catalyzed by Schizosaccharomyces pombe poly(U) polymerase( Figure 14B)。The main advantage of solid-phase synthesis over bulk synthesis is that the extension and deblocking reactions can be repeated to ensure that any reaction can be completed completely by purification. This can be achieved by monitoring the production of pyrophosphate during the extension reaction, measuring the fluorescence of the solid support if dye-labeled nucleoside triphosphates are used, or using a colorimetric monitor for the deblocking reaction, and then visually observing or measuring.

[0382] 13. A reusable solid support with a covalent linker can be used for the controlled enzymatic synthesis of RNA oligonucleotides mediated by Schizosaccharomyces pombe poly(U) polymerase

[0383] The main factor affecting the total cost of the controlled enzymatic synthesis of RNA oligonucleotides is the oligonucleotide initiator sequence. In previous examples of bulk synthesis, the oligonucleotide initiator sequence was consumed and generally not reusable. Additionally, it was difficult to remove the oligonucleotide initiator sequence from the final product if needed. To overcome this problem, site-specific cleavage of riboinosine (rI) and / or deoxyinosine (dI) in single-stranded RNA, DNA, or a combination thereof can be performed using endonuclease V to remove the unwanted initiator sequence from the final oligonucleotide product. Endonuclease V is highly specific for riboinosine (rI) and deoxyinosine (dI) and does not damage other bases in the oligonucleotide initiator sequence. Extending this concept to solid-phase oligonucleotide synthesis, solid-phase oligonucleotide synthesis is more favorable for the synthesis of long RNA oligonucleotides and industrial-scale synthesis compared to bulk solution synthesis, and reusable beads, wells, slides, etc. can be produced for repeated and potentially infinite use in synthetic reactions. A brief overview of this method is given in Figure 15A as follows. First, the solid support is covalently derivatized with an appropriate linker (such as a long PEG chain), which binds to an initiator oligonucleotide preferably containing riboinosine (rI) or deoxyinosine (dI) at the 3'-end. Solid-phase enzymatic RNA oligonucleotide synthesis is carried out to produce the desired product, and then endonuclease V is incubated with the complete oligonucleotide (initiator + product). This will cleave the oligonucleotide product from the solid support, and riboinosine (rI) or deoxyinosine (dI) will remain intact on the solid support for reuse in future synthetic reactions. Endonuclease V cleaves two bases downstream of riboinosine (rI) or deoxyinosine (dI), so this must be taken into account when designing the desired oligonucleotide to be synthesized. The final product is also 5'-phosphorylated, which can be easily removed using phosphatase or used for other molecular biology or sequencing applications. In some cases, the 2'-O-allyl form of this nucleobase is used, and Schizosaccharomyces pombe poly(U) polymerase can be used to introduce riboinosine (rI) into the 3'-end of the anchored initiator oligonucleotide using the 2'-O-allyl form of this nucleobase.

[0384] To determine whether this scheme operates as expected, an initiator oligonucleotide with a 5'-amine and deoxyinosine (dI) was synthesized, which would produce two equal-sized fragments upon endonuclease V digestion. The initiator oligonucleotide was anchored on the surface of amine-functionalized silica beads by introducing a bis-NHS-PEG9 linker, which would react with the 5'-amine of the oligonucleotide and the amine on the silica beads. The derivatized beads were incubated with endonuclease V (expressed and purified as described in the Materials and Methods section) at 37 °C for 1 hour. Additionally, the same digestion reaction was carried out in the bulk phase for comparison. For both the solid-phase and bulk digestion reactions, control samples were placed in reactions without endonuclease V. After incubation for 1 hour, the digestion reactions were analyzed using a 15% TBE-urea gel under denaturing conditions. The gel was stained with 1× SYBR GelStar nucleic acid dye by shaking at room temperature for 15 minutes. It was observed that endonuclease V produced digestion fragments as expected in both the bulk and solid-phase reactions ( Figure 15B ). For the bulk control reaction, full-length undigested fragments were observed. No distinguishable fragments were observed in the solid-phase control digestion reaction, suggesting that the oligonucleotide remained intact on the surface of the silica beads. This result is also important because it demonstrates that covalently bound oligonucleotides do not leech from the surface.

[0385] To confirm that the solid-phase support system functions for enzymatic synthesis and that the initiator oligonucleotide is reusable by demonstrating that the deoxyinosine nucleobase remains intact on the surface after endonuclease V digestion, the washed silica beads with digested initiator oligonucleotides were incubated with Schizosaccharomyces pombe poly(U) polymerase and native rNTPs (uncontrolled extension) as well as 2'-O-allyl-ATP reversible terminators (controlled extension) using optimized reaction conditions. For comparison, beads with newly anchored undigested initiator oligonucleotides were similarly extended. Then all the beads were washed with 10 mM Tris-HCl (pH 6.5) and incubated at 37 °C for 1 hour in the presence of endonuclease V. The efficiency of extension and cleavage from the surface was then analyzed using a 15% TBE-urea gel under denaturing conditions and stained with 1× SYBR GelStar nucleic acid dye by shaking at room temperature for 15 minutes. Gel analysis showed that under all conditions, both the reused beads and the newly derivatized beads had positive extension and cleavage ( Figure 15C)。As a final proof of feasibility, the controlled synthesis of (n+2) products was carried out using Schizosaccharomyces pombe poly(U) polymerase with a covalently attached endonuclease V-cleavable initiator oligonucleotide with 2'-O-allyl-ATP reversible terminator nucleoside triphosphates. The extension reaction was supplemented with 1 mM nucleotide and incubated at 37 °C for 15 minutes. The deblocking reaction was carried out at 50 °C for 10 minutes. The beads were washed with 10 mM Tris-HCl (pH 6.5). Endonuclease V cleavage was carried out at 37 °C for 1 hour using an appropriate buffer and then immediately electrophoresed on a denaturing gel. The control reaction was extended to (n+2), incubated in the presence of endonuclease V, but contained an anchored Cy5 initiator oligonucleotide without ribosylinosine (rI) or deoxyinosine (dI). This was used to demonstrate that the oligonucleotide did not leech during endonuclease V cleavage. After each synthesis cycle, the beads with the Cy5 initiator oligonucleotide remained visibly blue.

[0386] 14. Development of 3'-blocked reversible terminator nucleotides and their use in oligonucleotide synthesis

[0387] New nucleoside triphosphates for the enzymatic synthesis of RNA oligonucleotides and modified oligonucleotides have been developed. RNA oligonucleotides and modified oligonucleotides can be used in a variety of applications including oligonucleotide therapeutics. The nucleoside triphosphates are reversibly terminated at the 3'-position of the sugar ring with a blocking group, conferring only (n+1) extension of the resulting oligonucleotide; the extension reaction does not produce a free hydroxyl group (-OH) at the 3'-position where further extension is possible. The blocking group can be removed with a mild biocompatible deprotecting agent. This strategy complements virtual blocking at the 2'-position or at the base, where the resulting oligonucleotide is sterically blocked rather than chemically blocked (these are also referred to as "virtual terminators").

[0388] In some cases, the 3'-blocking strategy requires a compatible enzyme (such as the mutagenized poly(U) polymerase described herein) that accommodates the blocked chemical moiety. A number of chemical moieties that can be used in the 3'-blocking strategy are listed below. Some examples include but are not limited to 3'-O-allyl triphosphate (3'-O-allyl-NTP) and 3'-O-azidomethyl triphosphate (3'-O-azidomethyl-NTP).

[0389] The new 3'-reversible terminator nucleoside triphosphates can have advantages including multiple modifications that confer therapeutic or other functional value to the entire oligonucleotide. In addition to the 3'-reversible terminating group, the nucleoside triphosphates can also have a single or multiple modifications. The modifications can be introduced site-specifically into the oligonucleotide without additional protecting groups. These nucleoside triphosphates require compatible enzymes for incorporation, which may have unique sequences or a set of mutant codons for each modified nucleotide used. The modifications manifest as chemical handles, ligand-binding domains, ways of conferring nuclease resistance to the oligonucleotide, stereopure phosphorothioate oligonucleotides, ways of conferring a tendency to form the desired oligonucleotide secondary structure, or ways of conferring resistance to the formation of an undesired oligonucleotide secondary structure, etc. These modifications include:

[0390] i. Modifications to the 2'-domain of the furanose ring, which can be but are not limited to hydroxyl (-OH), hydrogen (-H), fluorine (-F), amine (-NH3), azide (-N3), mercapto (-SH), methoxy (-OCH3), methoxyethanol (-OCH2CH2OCH3), redox-active components, fluorescent or intrinsically fluorescent components, natural and unnatural amino acids, peptides, proteins, monosaccharides or oligosaccharides, functional / ligand-binding glycans, and large / gigantic groups such as polyethylene glycol (PEG).

[0391] ii. Modifications to the alpha (α) phosphate of the triphosphate, where the phosphorothioate R or S isomer is introduced site-specifically into the oligonucleotide to produce a stereopure oligonucleotide.

[0392] iii. Modifications to the beta (β) and gamma (γ) phosphates of the triphosphate: where any modification and / or both of these are beneficial to the enzymatic oligonucleotide synthesis protocol; for example, preventing or limiting undesired pyrophosphorolysis due to pyrophosphate formation.

[0393] iv. Modifications to the furanose ring, which can be but are not limited to replacing the oxygen of the ring with sulfur or introducing a bridging that restricts the ring conformation between the 2'-oxygen and 4'-carbon.

[0394] v. Modifications to the nucleobase, where the base is a natural or unnatural pyrimidine or purine, and can include but are not limited to N 1 -methyl-adenine, N 6 -methyl-adenine, 8'-azido-adenine, N,N-dimethyladenosine, aminoallyladenosine, 5'-methyluridine, pseudouridine, N 1 -methyl-pseudouridine, 5'-hydroxymethyluridine, 2'-thiouidine, 4'-thiouidine, hypoxanthine, xanthine, 5'-methylcytidine, 5'-hydroxy-methylcytidine, 6'-thioguanine, and N 7 -methylguanine.

[0395] Upon completion of synthesis, the oligonucleotide can be irreversibly capped with a terminal 3'-blocked nucleoside triphosphate, which can confer further functionality or therapeutic value. This may also require the use of compatible enzymes (e.g., mutant poly(U) polymerase), and may be the modifying groups described herein. Additionally, the 3'- and 2'-domains of the furanose ring can be irreversibly blocked with the same or different groups.

[0396] In addition to mononucleoside triphosphates, dinucleoside triphosphates, trinucleoside triphosphates, and N-nucleoside triphosphates (where N = oligonucleotide triphosphate of length N) can be used as substrates for incorporation using compatible enzyme catalysts to effectively prepare oligonucleotide ligases.

[0397] In some cases, the addition of a new nucleoside triphosphate can introduce a cleavable handle, which can be processed by chemical or biological means for post-synthesis processing and purification. For example, oligonucleotides bearing hypoxanthine (inosine) groups can be site-specifically cleaved by endonuclease V. This is particularly useful for solid-phase synthesis of oligonucleotides, where the immobilized oligonucleotide initiator can be reused indefinitely.

[0398] Enzymatic Oligonucleotide Synthesis with Wild-Type and Mutant Poly(N) Polymerases

[0399] Figures 18A - 18D Gel electrophoresis analysis showing the ability of the H336 mutant to incorporate the natural nucleotides GTP "-G" and CTP "-C". All components except the enzyme and nucleotide were added to the blank reaction. All reactions were incubated with 1 mM nucleotide, 5 pmol initiator oligonucleotide, and 1 μg enzyme at 37 °C for 30 minutes. Extension reaction analysis was performed using a 15% TBE-urea denaturing gel.

[0400] Figures 19A - 19F Gel electrophoresis analysis showing the ability of the poly(U) polymerase mutant H336R to incorporate a series of natural and analog nucleotides compared to wild-type poly(U) polymerase. Figure 19A 、 19D ATP-based nucleotide extension results for wild-type and H336R mutant are depicted respectively. Figure 19B 、 19E UTP- and ITP-based nucleotide extension results for wild-type and H336R mutant are depicted respectively. Figure 19C 、 19F CTP- and GTP-based nucleotide extension results for wild-type and H336R mutant are depicted respectively. All reactants were incubated with 1 mM nucleotide, 5 pmol initiator oligonucleotide, and 1 μg enzyme at 37 °C for 30 minutes. Extension reaction analysis was performed using a 15% TBE-urea denaturing gel.

[0401] Figure 20 Shows uncontrolled incorporation of 2'-O-methyl-ATP by various Schizosaccharomyces pombe poly(U) polymerase mutants, especially mutants at position H336; single mutants compared to wild type (WT) are shown in the figure. The blank reaction contains all components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0402] Figure 21 Shows uncontrolled incorporation of 2'-F-ATP by various Schizosaccharomyces pombe poly(U) polymerase mutants, especially mutants at position N171; single mutants compared to mutant H336R are shown in the figure. The blank reaction contains all reaction components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0403] Figure 22 Shows controlled incorporation (capping) of 3'-O-methyl-ATP by various Schizosaccharomyces pombe poly(U) polymerase mutants, especially mutants at position N171; single mutants compared to mutant H336R and wild type are shown in the figure. The upper band represents the (n + 1) product. Note: The wild type sample shows positive incorporation, but severe pyrophosphorolysis occurs. The negative reaction contains all reaction components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0404] Figure 23 Shows controlled incorporation of the reversible terminator 3'-O-allyl-ATP by various Schizosaccharomyces pombe mutants. The negative reaction contains all reaction components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0405] Figure 24 Shows controlled incorporation of the reversible terminator 3'-(O-allyl carbonate)-dATP by the poly(U) polymerase double mutant H336R-N171A. The gel image shows different input amounts of the primer oligonucleotide (2 pmol / rxn, 5 pmol / rxn, and 10 pmol / rxn) with increasing amounts of the crude purified enzyme stock solution (2 μL, 4 μL, and 6 μL). The upper band represents the (n + 1) product. The blank reaction contains all components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions.

[0406] Figure 26Shows the reaction calibration evaluation of purified poly(U) polymerase stock solution H336R with the reversible terminator 3'-O-allyl adenosine triphosphate 3'-(O-allyl)-ATP. The gel shows the (n+1) extension reaction obtained with an increasing amount of initiator oligonucleotide input. 1 mM of the reversible terminator nucleotide and 1 μL of the purified enzyme stock solution were added to the reaction. The reaction was incubated at 37 °C for 5 minutes. The upper band represents the (n+1) product. The blank reaction contained all components except the enzyme. Samples were analyzed on a 15% TBE-urea gel under denaturing conditions. This is an example of reaction scalability.

[0407] Figure 27 Shows a demonstration of the controlled enzymatic synthesis of poly(U) polymerase mutant H336R and the reversible terminator 3'-O-allyl adenosine triphosphate (3'-O-allyl-ATP) in bulk solution. Shown in the figure is the (n+5) synthesis in bulk solution. After synthesis, the reaction was analyzed on a 15% TBE-urea gel under denaturing conditions.

[0408] 3'-Reversible Terminator Structure & Synthesis

[0409] Figure 28 Shows an example structure of a 3'-reversible terminator nucleotide for enzymatic incorporation. Each example of a protecting group for the 3'-hydroxyl group. As labeled, these can be removed by redox chemistry, optics, fluoride anion, and catalysts.

[0410] Figure 29 and 30 Shows the selection of a 3'-protecting group where the furanyl ring has an oxygen. 2' can be natural ribose, deoxy, or various components that promote binding, pharmacokinetics, pharmacodynamics, general stability, and probe labeling.

[0411] Figure 31 Shows an example scheme for preparing 3'-azidomethyl ether for a triphosphate nucleotide, where 2' can be natural OH or various modifications such as -F, -OMe, -OCH2CH2CH3, or other modifications that prove beneficial to the bioactivity of the target oligomer or contribute to broader scientific impacts.

[0412] Figure 32 Shows an example scheme for preparing 3'-azidomethyl ether for a locked triphosphate nucleotide.

[0413] Figure 33 Shows an example scheme for preparing 3'-allyl ether for a triphosphate nucleotide, where 2' can be natural OH or various modifications such as -F, -OMe, -OCH2CH2CH3, or other modifications that prove beneficial to the bioactivity of the target oligomer or contribute to broader scientific impacts.

[0414] Figure 34An exemplary scheme for preparing 3'-azidomethyl ethers for locking nucleoside triphosphates is shown.

[0415] Sequence

[0416] Human polymerase Mu R387K (SEQ ID NO:1)

[0417]

[0418] Saccharomyces cerevisiae poly(A) polymerase (SEQ ID NO:2)

[0419]

[0420] Schizosaccharomyces pombe poly(U) polymerase (SEQ ID NO:3)

[0421]

[0422] Materials and Methods

[0423] Enzyme Expression and Purification

[0424] The basic sequences of wild-type or mutant enzymes were codon-optimized for E. coli expression using a custom optimization algorithm and ordered (IDT), which had 20-nt overlapping sequences to be assembled into the pET-28-c-(+)His-tag expression vector (EMD Millipore 69866-3) by Gibson assembly. Using the forward and reverse primers from IDT, PCR amplification was performed with Phusion High-Fidelity (HF) polymerase (NEB M05030). The PCR thermal cycling program was as follows: initially denatured at 98°C for 30 s, denatured at 98°C for 10 s, annealed at 68°C for 10 s, then extended at 72°C for 60 s for a total of 18 cycles, and finally extended at 72°C for 5 min. The PCR reaction products were purified and concentrated using the QIAquick PCR Purification Kit (Qiagen 28106).

[0425] Circular DNA was digested with 40 U of NDeI (NEB R0111) per 500 ng of vector at 37°C for 90 min to prepare for Inserted pET-28-c-(+) expression vector. The linear DNA was separated from the undigested material by 2% agarose gel electrophoresis and extracted by incubating the agarose containing the band corresponding to the linear DNA in buffer QG (Qiagen 19063) at 55 °C with rotation at 1000 RPM for 2 hours. The resulting mixture was cleaned and concentrated using the QIAquick PCR purification kit. The PCR-amplified insert sequence and vector sequence were combined at a ratio of 1:3 with 0.1 pmol of all materials and assembled with Gibson Assembly Master Mix (NEB E5510S) at 50 °C for 1 hour. Using the fully assembled plasmid, T7 Express chemically competent E. coli (NEB C2566I) was transformed according to the manufacturer's instructions, and positive transformants were selected on LB-kanamycin plates (50 μg / mL kanamycin).

[0426] Bacterial colonies were sequenced (Genewiz, T7-forward primer, T7-terminal - reverse primer), and those with perfect matches were grown overnight in liquid LB-kanamycin medium (50 μg / mL kanamycin), diluted 1:400 in fresh liquid LB-kanamycin, and induced with 1 mM IPTG (Sigma I6758) under conditions of approximately OD 600 = 0.8. The induced liquid culture was incubated overnight with shaking at 15 °C at 250 RPM. Then the culture was centrifuged at 3500×g for 10 minutes to pellet, and His-Tag purification was performed using the HisTalon Resin kit according to the manufacturer's instructions (Clontech 635654). Then, using a 15 mL filter column (Millipore), the eluted enzyme sample was buffer-changed to the optimal 2× protein storage buffer by centrifugation at 5000×G for 15 minutes at 4 °C under an appropriate MWCO. This process was repeated twice. In the third spin, the sample was spun for 30 minutes to concentrate the protein into a smaller volume.

[0427] Two small aliquots were taken for determining the total protein concentration using a reducing agent-compatible MicroBCA kit (Thermo 23252), and for determining the size of the His-Tag purified protein using a 16% Tris-Gly denaturing gel (Thermo XP00165) and a 10 - 250 kDa protein ladder (Thermo 26619). After gel electrophoresis, the gel was stained with Coomassie Orange Fluorescent (Thermo C33250) with gentle stirring at room temperature for 20 minutes and observed using a GelDoc Image Station (Biorad). The remaining concentrated protein stock solution was diluted 1:2 with sterile glycerol and stored at -20 °C.

[0428] Site-directed mutagenesis of target proteins to improve RNA synthesis

[0429] Single or multiple amino acids can be mutated to improve any RNA oligonucleotide synthesis protocol, either through rational design or through high-throughput methods such as error-prone PCR mutagenesis. Using a MiniPrep kit (Qiagen 27104), plasmids carrying the target protein were harvested and purified from sequence-verified liquid bacterial cultures grown overnight at 37 °C in LB-kanamycin medium. Oligonucleotide primers were ordered from IDT and designed to amplify the protein expression plasmid by PCR while mutating the plasmid at a predetermined position, thereby generating linearized DNA. Using the reagents in the Q5 Site-Directed Mutagenesis Kit (NEB E0554S), the protein expression plasmid was PCR amplified using Q5 Hot Start High-Fidelity 2x Master Mix under the following thermal cycling conditions: initial denaturation at 98 °C for 30 s, denaturation at 98 °C for 10 s, annealing at 68 °C for 10 s, followed by extension at 72 °C for 120 s for 25 cycles, and finally extension at 72 °C for 2 min. Then 1 μL of the resulting PCR amplification reaction was treated with the enzyme reaction mixture of the kit to religate the protein expression plasmid while digesting the unsubstituted plasmid sequences remaining in the reaction mixture. After bacterial transformation and sequence verification, colonies with perfect sequence matches were used for the expression and analysis of site-directed mutant proteins as described previously. The resulting purified mutant proteins were concentrated, buffer-exchanged into a suitable 2× storage buffer as described previously, and diluted 1:2 with sterile glycerol and stored at -20 °C.

[0430] Initial activity screening with native rNTP

[0431] Expressed proteins with terminal transferase activity were screened by determining the RNA production rate based on the total RNA concentration and length / distribution produced by the protein after incubation with native rNTP. To measure the RNA production rate, a 10 μL bulk extension reaction carried out at 37 °C for 30 min was monitored on a plate reader (EX: 598 nm, EM: 522 nm), which consisted of 10 pmol of a short 5'-Cy5-labeled initiator oligonucleotide (15 - 20-nt), 100 μM rNTP, 0.25 mM divalent cation cofactor (e.g., Co 2+ 、Mg 2+ 、Mn 2+ 、Zn 2+or a combination thereof), 1× reaction buffer, 1× SYBR dye (GelStar (Lonza 50535)), Qubit ssDNA dye (Thermo Q10212) or SYBR Green II RNA gel dye (Thermo S7564), and 1 μL of purified enzyme. Signal readings were taken in triplicate (N = 3) every 1 minute. Using a custom R script, the rate of RNA production and subsequent initial enzyme activity (V o ) were determined from the slope of the best-fit curve of the plotted average RFU over time. The products were compared to a 100 nt ssDNA ladder (Simplex Biosciences) using a 15% TBE-urea denaturing gel (Thermo EC6885) according to the manufacturer's protocol to determine the length of the RNA produced in these reactions. Unless otherwise stated, ~8 μL of the initial activity screening reaction volume was loaded onto the gel and run at 185 V for 60 minutes. The gel was then stained with a solution of 1× GelStar nucleic acid dye or SYBR Green II RNA gel dye for 15 minutes with gentle agitation. The resulting gel was then imaged on a Typhoon FLA 9500 system (GE Healthcare LifeSciences) using the imaging parameters for SYBR Gold. For extension reactions performed with initiator oligonucleotides labeled with 5'-fluorophores such as FAM, Cy5, Cy3, etc., the gel was not stained and was imaged directly using appropriate parameters.

[0432] Enzyme Activity Assay - Uncontrolled Extension with Native & Analogue Nucleotides

[0433] Uncontrolled extension reactions included 5 pmol of initiator oligonucleotide, 1 mM natural or analog nucleotides, 1× poly(U) polymerase reaction buffer (10 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9, 25 °C), and 1 μg of purified enzyme. Natural and analog nucleotides were either purchased from commercial sources or custom synthesized in-house. The reaction mixture was incubated at 37 °C for 30 min and then immediately analyzed by gel electrophoresis using a 15% TBE-urea denaturing gel (Thermo EC6885) according to the manufacturer's instructions. The lengths of the oligonucleotides produced in these reactions were determined by comparing the products with a 100 nt ssDNA ladder (Simplex Biosciences). The gel was then stained for 15 min with a solution of 1× GelStar nucleic acid stain or SYBR Green II RNA gel stain with gentle agitation. The resulting gel was then imaged on a Typhoon FLA 9500 system (GE Healthcare Life Sciences) using the imaging parameters for SYBR Gold. For extension reactions performed with initiator oligonucleotides labeled with a 5'-fluorophore such as FAM, Cy5, Cy3, etc., the gel was not stained and was imaged directly using appropriate parameters.

[0434] Enzyme activity assay - Controlled extension using natural & analog reversible terminator nucleotides

[0435] Controlled extension reactions included 5 pmol of initiator oligonucleotide, 1 mM blocked reversible terminator nucleotides, 1× poly(U) polymerase reaction buffer (10 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9, 25 °C), and 1 μg of purified enzyme. The reaction mixture was incubated at 37 °C for 1 min and then immediately analyzed by gel electrophoresis using a 15% TBE-urea denaturing gel (Thermo EC6885) according to the manufacturer's instructions. The success of (N+1) events was determined by performing blank extension reactions in which no nucleotides or enzyme were added. The gel was then stained for 15 min with a solution of 1× GelStar nucleic acid stain or SYBR Green II RNA gel stain with gentle agitation. The resulting gel was then imaged on a Typhoon FLA 9500 system (GE Healthcare Life Sciences) using the imaging parameters for SYBR Gold. For extension reactions performed with initiator oligonucleotides labeled with a 5'-fluorophore (such as FAM, Cy5, Cy3, etc.), the gel was not stained and was imaged directly using appropriate parameters.

[0436] Enzyme Activity Assay - Uncontrolled or Controlled Extension Reactions on a Surface

[0437] Uncontrolled and controlled extension reactions can be carried out using surface - bound initiator oligonucleotides. The surface - bound initiator oligonucleotides are obtained from IDT with a 5'-amine C6 spacer and an internal Cy5 fluorophore. This oligonucleotide is biotinylated and PEGylated using the EZ Link NHS - PEG12 - Biotin kit (Thermo A35389) according to the manufacturer's instructions and then cleaned and concentrated using an oligonucleotide clean and concentrator spin column kit (Zymo D4060). The derived initiator oligonucleotides are bound to the surface of streptavidin - coated PCR plates (BioTez, Germany) by incubating the oligonucleotides in the wells of the plate in 2× binding and washing buffer (10 mM Tris - HCl, 2 M NaCl, 1 mM EDTA, pH 7.5, 25 °C) with gentle agitation (300 RPM) for 1 hour. The wells are aspirated and then washed once with 1× binding and washing buffer. The extension reaction mixture is prepared as described previously and incubated with the surface - bound oligonucleotides at 37 °C with shaking at 900 RPM for a predetermined time (30 minutes for the uncontrolled extension reaction and 1 minute for the controlled extension reaction). Then the wells are washed again with 1× binding and washing buffer. To remove the extended oligonucleotides from the surface, the wells are incubated with the stripping solution (95% formamide, 10 mM EDTA, pH 6.0, 25 °C) at 65 °C for 5 minutes. Then the oligonucleotides suspended in the stripping solution are cleaned and purified using an oligonucleotide spin column and eluted in 6 μL diH20. The surface extension reactants are analyzed by gel electrophoresis as described previously.

[0438] References

[0439] Agrawal, Neema, P.V.N. Dasaradhi, Asif Mohmmed, Pawan Malhotra, Raj K. Bhatnagar, and Sunil K. Mukherjee. 2003. “RNA Interference: Biology, Mechanism, and Applications.” Microbiology and Molecular Biology Reviews: MMBR 67(4):657–85.

[0440] Anavy,L.,Vaknin,I.,Atar,O.,Amit,R.&Yakhini,Z.Data storage in DNA withfewer synthesis cycles using composite DNA letters.Nat.Biotechnol.(2019).doi:10.1038 / s41587-019-0240-x

[0441] Andrade,Paula, José Raquel Juárez,Francisco de Saro,and Luis Blanco.2009.“Limited Terminal Transferase in Human DNA polymeraseμDefines the Required Balance between Accuracy and Efficiency in NHEJ.”Proceedings of the National Academy of Sciences of the United States ofAmerica 106(38):16203–8.

[0442] Balbo,P.B.&Bohm,A.Mechanism of poly(A)polymerase:structure of theenzyme-MgATP-RNAternary complex and kinetic analysis.Structure 15,1117–1131(2007).

[0443] Bard,J.,A.M.Zhelkovsky,S.Helmling,T.N.Earnest,C.L.Moore,andA.Bohm.2000.“Structure of Yeast poly(A)polymerase Alone and in Complex with3′-dATP.”Science 289(5483):1346 49.

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[0498] Equivalent solutions and ranges

[0499] Unless otherwise indicated or apparent from the context, the articles “a,” “an,” and “the” in the claims can mean one or more. Unless otherwise indicated or apparent from the context, if one, more than one, or all members of a given group are present in, applied to, or associated with a given product or process, a claim or description that includes “or” among one or more members of the group is considered to be satisfied. Embodiments of the present invention include those in which exactly one member of the group is present in, applied to, or associated with a given product or process. Embodiments of the present invention include those in which one, more than one, or all members of the group are present in, applied to, or associated with a given product or process.

[0500] In addition, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same base claim. In cases where elements are presented in a list form, such as in a Markush group, each sub-combination of the elements is also disclosed, and any element can be removed from the combination. It should be understood that, generally, when the present invention or aspects of the present invention are considered to include certain elements and / or features, certain embodiments of the present invention or certain aspects of the present invention consist of or consist essentially of these elements and / or features. For the sake of simplicity, these embodiments are not specifically set forth herein.

[0501] It should also be noted that the terms “comprising” and “including” are intended to be open-ended, allowing the inclusion of additional elements or steps. In cases where a range is given, the endpoints are included. In addition, unless otherwise specified or apparent from the context and the understanding of one of ordinary skill in the art, in different embodiments of the present invention, a value expressed as a range can be assumed to be any specific value or sub-range within one-tenth of a unit of the upper limit to the lower limit of the range, unless the context clearly dictates otherwise.

[0502] This application incorporates by reference all issued patents, published patent applications, journal articles, and other publications. If any cited reference conflicts with this specification, this specification shall control. Additionally, any particular embodiment of the invention that falls within the scope of the prior art may be explicitly excluded from any one or more of the claims. Since such an embodiment is considered to be known to a person of ordinary skill in the art, it may be excluded even if not explicitly recited herein. For any reason, any particular embodiment of the invention may be excluded from any claim, whether or not related to the existence of the prior art.

[0503] Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments described herein using no more than routine experimentation. The scope of the embodiments of the invention described herein is not intended to be limited to the foregoing description, but rather as set forth in the appended claims. Those of ordinary skill in the art will understand that various changes and modifications may be made to the description of the invention without departing from the spirit or scope of the invention as defined by the appended claims.

Claims

1. A method for template-independent synthesis of RNA oligonucleotides, the method comprising: (a) Provide an initiator oligonucleotide, wherein the initiator oligonucleotide is single-stranded RNA; (b) Provide a poly(U) polymerase, wherein the poly(U) polymerase is: (i)Wild-type Schizosaccharomyces pombe of SEQ ID NO: 3 ( Schizosaccharomyces pombe ) poly(U) polymerase; (ii) A mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3 having a mutation at position H336 selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S and H336W; (iii) A mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3 having a mutation at position N171 selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H and N171K; or (iv) A mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3 having H336R and N171A mutations; and (c) Combine the initiator oligonucleotide, the poly(U) polymerase, and the 2'- and / or 3'-O-protected reversible terminator nucleotide under conditions sufficient to add the 2'- and / or 3'-O-protected reversible terminator nucleotide to the 3'-end of the initiator oligonucleotide.

2. The method according to claim 1, further comprising: (d) Repeat steps (a)-(c) until the desired RNA sequence is obtained.

3. The method according to claim 1, further comprising adding one or more natural or modified nucleotides to the 3'-end of the resulting RNA oligonucleotide until the desired RNA sequence is obtained.

4. The method according to claim 1, wherein the poly(U) polymerase is the wild-type Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO:

3.

5. The method according to claim 1, wherein the poly(U) polymerase is a mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3, having a mutation at position H336 selected from the group consisting of: H336A, H336C, H336D, H336E, H336F, H336G, H336I, H336K, H336L, H336M, H336T, H336V, H336W, H336Y, H336N, H336P, H336Q, H336R, H336S and H336W.

6. The method according to claim 1, wherein the poly(U) polymerase is a mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO:3 having an H336R mutation.

7. The method according to claim 1, wherein the poly(U) polymerase is a mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3, having a mutation at position N171 selected from the group consisting of: N171E, N171L, N171Q, N171S, N171M, N171D, N171G, N171C, N171A, N171W, N171T, N171I, N171V, N171P, N171R, N171H and N171K.

8. The method according to claim 1, wherein the poly(U) polymerase is a mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO:3 having an N171A mutation.

9. The method according to claim 1, wherein the poly(U) polymerase is a mutant Schizosaccharomyces pombe poly(U) polymerase of SEQ ID NO: 3 having H336R and N171A mutations.

10. The method according to claim 1, further comprising: (d) Deprotect the RNA oligonucleotide formed in step (c) at the protected 2'- and / or 3'-O-position of the 2'- and / or 3'-O-protected reversible terminator nucleotide.

11. The method according to claim 10, further comprising: (e) Repeat steps (a)-(d) until the desired RNA sequence is obtained.

12. The method according to any one of claims 1-11, wherein the 2'- and / or 3'-O-protected reversible terminator nucleotide is a 2'-O-protected reversible terminator nucleotide protected at the 2'-O position with an oxygen protecting group.

13. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is protected at the 2'-O position with a photo-labile protecting group.

14. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is a 2'-O-alkyl, 2'-O-silyl, 2'-O-allyl, 2'-O-azidomethyl, 2'-O-benzyl, 2'-O-coumarinyl or 2'-O-carbonate modified nucleotide.

15. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is a 2'-O-carbonate modified nucleotide selected from 2'-O-allyloxycarbonyl and 2'-O-(2-oxo-2H-chromen-4-yl)methoxycarbonyl.

16. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is 2'-O-allyl-NTP or 2'-O-azidomethyl-NTP.

17. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is 2'-O-allyl-NTP.

18. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide is 2'-O-allyl-ATP, 2'-O-allyl-UTP, 2'-O-allyl-CTP or 2'-O-allyl-GTP.

19. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide comprises a modified base moiety.

20. The method according to claim 12, wherein the 2'-O-protected reversible terminator nucleotide comprises one or more additional modifications.

21. The method according to any one of claims 1-11, wherein the 2'- and / or 3'-O-protected reversible terminator nucleotide is a 3'-O-protected reversible terminator nucleotide protected at the 3'-O position with an oxygen protecting group.

22. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is protected at the 3'-O position with a photo-labile protecting group.

23. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is a 3'-O-alkyl, 3'-O-silyl, 3'-O-allyl, 3'-O-azidomethyl, 3'-O-benzyl, 3'-O-coumarinyl or 3'-O-carbonate modified nucleotide.

24. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is a 3'-O-carbonate modified nucleotide selected from 3'-O-allyloxycarbonyl and 3'-O-(2-oxo-2H-chromen-4-yl)methoxycarbonyl.

25. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is 3'-O-allyl-NTP, 3'-O-azidomethyl-NTP, 3'-O-allyl carbonate-NTP, 3'-O-allyl carbonate-dNTP, 3'-O-azidomethyl carbonate-NTP or 3'-O-azidomethyl carbonate-dNTP.

26. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is 3'-O-allyl-NTP, 3'-O-allyl carbonate-dNTP or 3'-O-azidomethyl carbonate-dNTP.

27. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide is 3'-O-allyl-ATP, 3'-O-allyl carbonate-dATP or 3'-O-azidomethyl carbonate-dATP.

28. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide comprises a modified base moiety.

29. The method according to claim 21, wherein the 3'-O-protected reversible terminator nucleotide comprises one or more additional modifications.

30. The method according to any one of claims 1-11, wherein the 2'- and / or 3'-O-protected reversible terminator nucleotide has the following formula or a salt thereof: , wherein: Y is O or S; X is O or S; R P Each instance of R is, independently, hydrogen, an oxygen protecting group, an optionally substituted acyl group, or an amino acid; provided that at least one R P is an oxygen protecting group, an optionally substituted acyl group, or an amino acid; and "Base" is a natural or unnatural nucleobase.

31. The method according to any one of claims 1 - 11, wherein the 2'- and / or 3'-O-protected reversible terminator nucleotide is a 3'-O-protected reversible terminator nucleotide having the following formula or a salt thereof: , wherein: Y is O or S; X is O or S; R P is an oxygen protecting group, an optionally substituted acyl group or an amino acid; R is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl, optionally substituted hydroxy, optionally substituted amino or optionally substituted thiol; and "Base" is a natural or unnatural nucleobase.

32. The method according to any one of claims 1 - 11, wherein the 2'- and / or 3'-O-protected reversible terminator nucleotide is a 3'-O-protected reversible terminator nucleotide having the following formula or a salt thereof: , wherein: Y is O or S; X is O or S; R P is an oxygen protecting group, an optionally substituted acyl group or an amino acid; and "Base" is a natural or unnatural nucleobase.

33. The method according to any one of claims 1 - 11, which further comprises the following steps: (f) Using a reverse transcription initiation site, a primer, and a reverse transcriptase to perform reverse transcription on the resulting RNA oligonucleotide to produce a complementary single-stranded DNA oligonucleotide or cDNA.

34. The method according to claim 33, which further comprises the following steps: (g) Amplifying the complementary single-stranded DNA oligonucleotide or cDNA produced in step (f) with a DNA polymerase to produce double-stranded DNA.

35. The method according to any one of claims 1 - 11, wherein step (c) is carried out in the presence of a crowding agent.

36. The method according to claim 35, wherein the crowding agent is polyethylene glycol (PEG).

37. The method according to any one of claims 1 - 11, wherein step (c) is carried out in the presence of one or more additional enzymes.

38. The method according to claim 37, wherein step (c) is carried out in the presence of additional poly(N) polymerase.

39. The method according to claim 37, wherein step (c) is carried out in the presence of yeast inorganic pyrophosphatase (PPI-ase).

40. The method according to any one of claims 1-11, wherein step (c) is carried out in the presence of an RNase inhibitor.

41. The method according to any one of claims 1-11, wherein step (c) is carried out in the presence of non-hydrolyzable nucleotides.

42. The method according to any one of claims 1-11, wherein the initiator oligonucleotide is covalently linked to a solid support.

43. The method according to claim 42, wherein the initiator oligonucleotide is covalently linked to the solid support through a cleavable linker.

44. The method according to any one of claims 1-11, wherein the initiator oligonucleotide has a length of 5-20 nucleotides.

45. The method according to any one of claims 1-11, wherein the initiator oligonucleotide is poly-rU, poly-rC, poly-rG or poly-rA.

46. The method according to any one of claims 1-11, wherein the initiator oligonucleotide comprises a fluorophore or a handle for bioconjugation.

47. The method according to any one of claims 1-11, wherein the initiator oligonucleotide comprises a primer site for reverse transcription or a primer site for PCR.

48. The method according to any one of claims 1-11, wherein the initiator oligonucleotide comprises a 5' cap.

49. The method according to any one of claims 1-11, further comprising the step of isolating the resulting RNA oligonucleotide.

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