Reusable initiators for synthesizing nucleic acids

Abstract

To provide improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support.SOLUTION: Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. The invention provides improved methods for nucleic acid synthesis. Methods of the invention provide faster and longer de novo synthesis of polynucleotides.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit and priority of U.S. Patent Application No. 16 / 261,229, filed on January 29, 2019, the content of which is incorporated herein by reference in its entirety.

[0002] Field of the Invention The present invention relates to methods and apparatuses for synthesizing polynucleotides having a desired sequence without the need for a template.

Background Art

[0003] Background Most de novo nucleic acid sequencing is performed using well-established solid-phase phosphoramidite technology. The phosphoramidite technology requires sequential deprotection and synthesis of sequences constructed from phosphoramidite reagents corresponding to natural (or unnatural) nucleobases. However, phosphoramidite nucleic acid synthesis is limited in length in that nucleic acids greater than 200 base pairs (bp) in length experience high rates of breakage and side reactions. Additionally, phosphoramidite synthesis produces toxic by-products, and the disposal of this waste limits the availability of nucleic acid synthesizers and increases the cost of contract oligonucleotide production (the annual demand for oligonucleotide synthesis is predicted to cause over 300,000 gallons of hazardous chemical waste, including acetonitrile, trichloroacetic acid, toluene, tetrahydrofuran, and pyridine). See LeProust et al., Nucleic Acids Res., vol. 38(8), p.2522-2540, (2010), which is incorporated herein by reference in its entirety. Accordingly, there is a need for more efficient and cost-effective methods of oligonucleotide synthesis.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] Abstract The present invention provides an improved method for nucleic acid synthesis. The method of the present invention provides de novo synthesis of faster and longer polynucleotides. Thus, the present invention dramatically reduces the overall cost of custom nucleic acid synthesis. The method of the present invention relates to template - independent synthesis of polynucleotides by using a nucleotidyl transferase enzyme to incorporate nucleotide analogs coupled to an inhibitor by a cleavable linker. Due to the above inhibitor, the synthesis is once stopped with the addition of each new base, at which time the above linker is cleaved, separating the above inhibitor and leaving a polynucleotide essentially identical to a naturally occurring (i.e., recognized by the above enzyme as a substrate for further nucleotide incorporation) nucleotide. In particular, the present invention provides a renewable substrate for template - independent nucleic acid synthesis. De novo synthesis starts with a nucleic acid initiator bound to a solid support. In the presence of a suitable enzyme (e.g., polymerase, e.g., terminal deoxynucleotidyl transferase (TdT)), nucleotide analogs are added to the above nucleic acid initiator to create an oligonucleotide. It is preferred that the above nucleotide analog contains a removable terminating group that stops the addition by the enzyme after the addition of 1 nucleotide. The removable terminating group can be linked to the base moiety of the above nucleic acid and / or to the 3'-hydroxyl of the above nucleic acid. Deblocking of the above terminating group and / or the above 3'-blocking group creates a new active site that is a substrate for the above enzyme. Following the addition of a new nucleotide or nucleotide analog, the above oligonucleotide is extended.

[0006] In some cases, the nucleic acid initiator includes a 3' portion that is a substrate for the enzyme. The releasing agent is used to decouple the 3' portion, thereby releasing the oligonucleotide. The 3' portion, the nucleic acid initiator, and the solid support are reusable after release of the nascent oligonucleotide.

[0007] The invention further includes an apparatus that utilizes the method of the invention for the production of custom polynucleotides. The apparatus of the invention includes one or more bioreactors that provide aqueous conditions and a plurality of sources of nucleotide analogs. The bioreactor can be, for example, a reservoir, a flow cell, or a multiwell plate. The bioreactor can include a nucleic acid initiator and a solid support having a cleavable 3' portion. Starting from the solid support, the polynucleotide grows in the reactor by successively adding nucleotides through the natural activity of a nucleotidyl transferase, such as terminal deoxynucleotidyl transferase (TdT), or any other enzyme that elongates a DNA or RNA strand without the direction of a template. Upon cleavage of the linker, the native polynucleotide is released from the solid support. Once the sequence is complete, the support is cleaved and removed, or the 3' portion is contacted with a releasing agent, leaving a polynucleotide that is essentially equal to that found in nature. In some embodiments, the apparatus is designed to recycle the nucleotide analog solution by recovering the nucleotide analog solution after nucleotide addition and reusing the nucleotide analog solution for subsequent nucleotide addition. Thus, the overall cost per base is reduced compared to state-of-the-art methods, the less waste is generated. In certain embodiments, the bioreactor can include a microfluidic device and / or can use inkjet printing technology.

[0008] The terminating group may include, for example, a charged moiety or a stereochemical inhibitor. Generally, large macromolecules that prevent the nucleotidyl transferase enzyme from reaching a functional conformation are useful for inhibiting oligonucleotide synthesis. Such macromolecules include polymers, polypeptides, polypeptoids, and nanoparticles. The macromolecule should be large enough to physically block access to the active site of the nucleotidyl transferase, but not so large as to negatively alter the reaction kinetics. The macromolecule is linked to the nucleotide analog using any of a variety of linkers, as described below.

[0009] In embodiments using 3'-O-blocked nucleotide analogs, the 3'-O-blocking group is typically small and easily removable, thus allowing for use with engineered enzymes having a modified active site. For example, the 3'-O-blocking group may include an azidomethyl, amino, or allyl group.

[0010] In some embodiments, oligonucleotide synthesis may include the introduction of 1 or more 3'-exonucleases to the synthesized oligonucleotides after each nucleotide analog is added, but before the terminating group is cleaved. The terminating group blocks the 3'-exonuclease from acting on any oligonucleotide to which a nucleotide analog has been added, while oligonucleotides that did not successfully add an analog containing a terminator are removed by the 3'-exonuclease. In this manner, the present invention enables in-process quality control and may eliminate the need for post-synthesis purification.

[0011] Other aspects of the invention will be apparent to those skilled in the art upon consideration of the following drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1A

[0013]

Figure 1B

[0014]

Figure 2A

[0015]

Figure 2B

[0016]

Figure 3A

[0017]

Figure 3B

[0018]

Figure 4A

[0019]

Figure 4B

[0020]

Figure 5A

[0021]

Figure 5B

[0022]

Figure 6

[0023]

Figure 7A

[0024]

Figure 7B

[0025]

Figure 8A

[0026]

Figure 8B

[0027]

Figure 9A

[0028]

Figure 9B

[0029]

Figure 10A

[0030]

Figure 10B

[0031]

Figure 11A

[0032]

Figure 11B

[0033]

Figure 12

[0034]

Figure 13

[0035]

Figure 14

[0036]

Figure 15

[0037]

Figure 16A

[0038]

Figure 16B

[0039]

Figure 16C

[0040]

Figure 17

[0041]

Figure 18

[0042]

Figure 19

[0043]

Figure 20

[0044]

Figure 21

[0045]

Figure 22

[0046]

Figure 23

[0047]

Figure 24

[0048]

Figure 25-1

Figure 25-2

[0049]

Figure 26

[0050]

Figure 27

DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The present invention provides an improved method for synthesizing polynucleotides (e.g., DNA and RNA) using enzymes and nucleic acid analogs. Using the disclosed method, a specific polynucleotide sequence can be de novo synthesized one base at a time in an aqueous environment and without using a nucleic acid template.

[0052] The nucleotide analog may have an unmodified 3'-hydroxyl, a 3'-O-blocking group, or a blocker releasably attached to the phosphate. In any case, the blocking group is designed to leave no substantial additional molecule, i.e., to leave a "scarless" nucleotide recognized as a "natural" nucleotide by the enzyme. Thus, upon completion of the synthesis, removal of the last blocking group results in a synthesized polynucleotide that is chemically and structurally equivalent to a naturally occurring polynucleotide having the same sequence. The synthesized polynucleotide can thus be incorporated into a living system without concern that the synthesized polynucleotide will interfere with biochemical pathways or metabolism.

[0053] The processes and analogs of the invention are used for the non-template-mediated enzymatic synthesis of oligonucleotides and oligodeoxynucleotides, in particular long oligonucleotides (<5000 nt). The products can be single-stranded or partially double-stranded, depending on the initiator used. The synthesis of long oligonucleotides requires high-efficiency incorporation and high-efficiency reversible terminator removal. The initiator bound to the solid support consists of a short single-stranded DNA sequence that is either a short fragment of a user-defined sequence or a universal initiator from which the user-defined single-stranded product is removed.

[0054] In one aspect, the disclosed method synthesizes polynucleotides in a stepwise manner from nucleotide analogs using a commercially available nucleotidyl transferase enzyme (e.g., terminal deoxynucleotidyl transferase (TdT)). The nucleotide analogs are in the form: NTP-linker-inhibitor wherein NTP is a nucleotide triphosphate (i.e., dNTP or rNTP), the linker is a linker cleavable between the base and pyridine or pyrimidine, and the inhibitor is a group that prevents the enzyme from incorporating subsequent nucleotides. In each step, a new nucleotide analog is incorporated into the growing polynucleotide chain, whereupon the enzyme is blocked from adding further nucleotides by the inhibitor group. Once the enzyme has been stopped, the excess nucleotide analogs are removed from the growing chain, the inhibitor is cleaved from the NTP, and new nucleotide analogs can be introduced to add the next nucleotide to the chain. By sequentially repeating the steps, it is possible to rapidly construct a nucleotide sequence of a desired length and sequence. Advantages of using nucleotidyl transferase for polynucleotide synthesis include: 1) 3'-extension activity using a single-stranded (ss) starting primer in template-independent polymerization, 2) the ability to extend the primer in a very efficient manner that results in the addition of thousands of nucleotides, and 3) the ability to accept a wide variety of modified and substituted NTPs as efficient substrates.

[0055] Furthermore, the present invention can utilize an initiator sequence that is a substrate for nucleotidyl transferase. The initiator is attached to a solid support and serves as a recognition site for the enzyme. The initiator is preferably a universal initiator for the enzyme (e.g., a homopolymer sequence) and is recyclable on the solid support since the formed oligonucleotide is cleavable from the initiator.

[0056] The method of the present invention is well-suited for various current applications using synthetic nucleic acids, such as DNA oligonucleotides synthesized with phosphoramidites. For example, the polynucleotides synthesized using the method of the present invention are used as primers for nucleic acid amplification, as hybridization probes for the detection of specific markers, and for incorporation into plasmids for genetic manipulation. However, since the disclosed method produces synthetic nucleotide strands faster and in a longer length in an aqueous environment, the disclosed method is also suitable for high-throughput applications (e.g., screening for the expression of genetic variations in cell assays) and synthetic biology. Furthermore, the method of the present invention provides the functionality required for next-generation applications (e.g., using DNA as synthetic read / write memory or making macroscopic materials that are fully (or partially) synthesized from DNA).

[0057] The inventions and systems described herein provide for the synthesis of polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Synthetic pathways for “natural” nucleotides (e.g., DNA and RNA) have been described in the context of common nucleobases (e.g., adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U)), but it should be understood that the methods of the present invention can be applied to so-called “unnatural” nucleotides, including nucleotides that incorporate universal bases (e.g., 3-nitropyrrole 2'-deoxynucleotide and 5-nitroindole 2'-deoxynucleotide), α phosphorothiolate, phosphorothiolate nucleotide triphosphates, or purine or pyrimidine conjugates having other desired properties (e.g., fluorescence). Other examples of purine and pyrimidine bases include: pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.In some cases, it may be useful to generate nucleotide sequences having non-reactive but approximately equivalent bases, i.e., bases that do not react with other proteins (i.e., transcription enzymes) and thus allow the influence of sequence information to be decoupled from the structural effects of the bases.

[0058] Analog The present invention provides nucleotide analogs having the formula NTP-linker-inhibitor for the synthesis of polynucleotides in an aqueous environment. With respect to the analog of the form NTP-linker-inhibitor, NTP can be any nucleotide triphosphate, for example, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), uridine triphosphate (UTP), nucleotide triphosphate, deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP).

[0059] The linker can be any molecular moiety that connects the inhibitor to the NTP and can be cleaved. For example, the linker can be cleaved by adjusting the pH of the surrounding environment. The linker can also be cleaved by an enzyme that is activated at a predetermined temperature but inactivated at another temperature. In some embodiments, the linker contains a disulfide bond.

[0060] The linker can include, for example, a photocleavable, nucleophilic, or electrophilic cleavage site. Examples of photocleavable linkers (where cleavage is activated by a specific light wavelength) can include benzoin, nitroveratryl, phenacyl, pivaloyl, silyl, 2-hydroxy-cinnamyl, coumarin-4-yl-methyl, or 2-nitrobenzyl-based linkers.

[0061] Examples of nucleophilic cleavage sites include fluoride ion-cleavable silicon-oxygen bonds or esters that can be cleaved in basic solution. Examples of electrophilic linkers include trityl, tert-butyloxycarbonyl groups, acetal groups, and acid-derived cleavage sites that can include p-alkoxybenzyl esters and amides. In certain aspects, a cleavable linker can include a cysteine residue, as shown in FIG. 15.

[0062] The linker can be attached, for example, at N4 of cytosine, N3 or O4 of thymine, N2 or N3 of guanine, and N6 of adenine, or N3 or O4 of uracil. This is because attachment at carbon results in the presence of a residual scar after removal of the group that inhibits polymerase. The linker is typically at least about 10 Å in length, for example, at least about 20 Å in length, for example, at least about 25 Å in length, thus allowing the inhibitor to be present far enough from the pyrimidine or pyrimidines to allow the enzyme to bind the NTP to the polynucleotide chain via the attached sugar backbone. In some embodiments, the cleavable linker self-cyclizes in that they form a cyclic molecule that is particularly non-reactive with the growing nucleotide chain.

[0063] In certain aspects, a cleavable linker can include a variable number of methylene bridges on the inhibitor side of the NTP or disulfide bond (e.g., including 1, 2, 3, or 4 methylene bridges, as shown in FIGS. 14 and 16A-C). These methylene bridges can be used to increase the space between the NTP and the inhibitor. As noted above, the length of the cleavable linker can be selected so as not to interfere with the coupling of the NTP to the synthesized polynucleotide. In some embodiments of the invention, the distance from the charged group to the NTP plays an important role in its effectiveness in inhibiting subsequent nucleotide incorporation.

[0064] For example, in some embodiments where a charged moiety is used as an inhibitor, the charged moiety may be from about 5 to about 60 linkages away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 40 linkages away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 35 linkages away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 30 linkages away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 20 linkages away from the NTP. The number of linkages between the charged moiety and the NTP can be increased by including additional methylene bridges.

[0065] The nucleotide analog can include any moiety linked to the NTP that inhibits subsequent nucleotide coupling by the enzyme. The inhibitory group can be a charged group (e.g., a charged amino acid), or the inhibitory group can be a group that is charged depending on the ambient conditions. In some embodiments, the inhibitor can include a moiety that is negatively charged or capable of being negatively charged. For example, the inhibitor can include a chain of phosphate groups (e.g., one, two, or three phosphates), as shown in FIGS. 16A - C, where additional phosphates increase the overall anionic charge of the inhibitor. In other embodiments, the inhibitor group is positively charged or capable of being positively charged. In some other embodiments, the inhibitor is an amino acid or amino acid analog. The inhibitor can be a peptide of 2 - 20 units of amino acids or analogs, a peptide of 2 - 10 units of amino acids or analogs, a peptide of 3 - 7 units of amino acids or analogs, or a peptide of 3 - 5 units of amino acids or analogs. In some embodiments, the inhibitor includes a group selected from the group consisting of Glu, Asp, Arg, His, and Lys, and combinations thereof (e.g., Arg, Arg - Arg, Asp, Asp - Asp, Asp, Glu, Glu - Glu, Asp - Glu - Asp, Asp - Asp - Glu, or AspAspAspAsp, etc.). The peptide or group can be a combination of the same or different amino acids or analogs. In certain embodiments, the peptide inhibitor can be acetylated to stop errant bonding of the free amino group. The inhibitory group can also include a group that reacts with a residue in the active site of the enzyme, thus interfering with subsequent nucleotide coupling by the enzyme. The inhibitor can have a charged group selected from the group consisting of - COO, - NO2, - PO4, - PO3, - SO2, or - NR3 (where each R can be H or an alkyl group). In other embodiments, the inhibitor moiety does not include a - PO4 group.

[0066] In certain instances, a terminator or inhibitor may include a stereochemical inhibitor group. Such a stereochemical inhibitor group may enable the NTP-linker-inhibitor (i.e., nucleotide analog) to be incorporated onto the unblocked 3' OH of an oligonucleotide (such incorporation being catalyzed by a nucleotidyl transferase). The stereochemical inhibitor group may physically block the incorporation of a nucleotide or additional nucleotide analogs onto the unblocked 3' OH of the incorporated nucleotide analog. The stereochemical inhibitor may also block the action of a 3' endonuclease on the nucleotide analog and thus on an oligonucleotide into which an uncut nucleotide analog has been incorporated.

[0067] The stereochemical inhibitor may include, for example, a chemical polymer, a nanoparticle, a poly-N-substituted glycine (peptoid), or a protein. The stereochemical inhibitors of the present invention may be of various sizes including, for example, greater than 20 Å, greater than 30 Å, greater than 40 Å, greater than 50 Å, greater than 60 Å, greater than 70 Å, greater than 80 Å, greater than 90 Å, greater than 100 Å, greater than 110 Å, greater than 120 Å, greater than 130 Å, greater than 140 Å, or greater than 150 Å. In preferred embodiments, the stereochemical inhibitor may be monodisperse or substantially monodisperse. The stereochemical inhibitor may be water-soluble and conformationally constrained (i.e., in a rigid or semi-rigid form). In certain instances, the stereochemical inhibitor physically blocks access to the active site of the relevant nucleotidyl transferase enzyme due to the size or conformation of the inhibitor. In preferred embodiments, the stereochemical inhibitor may include a non-natural bio-inspired polymer (e.g., a polypeptoid or non-natural polypeptide).

[0068] In certain aspects, self-assembling polypeptoid arrays can be used as stereochemical inhibitors. Peptoid monomers are often based on N-substituted glycine backbones. Since the backbone lacks a hydrogen bond donor, the polypeptoid can still form secondary structures such as helices while being easily processed. They also provide the beneficial property of generally being chemically and thermally stable while allowing polarities and side chains similar to those of peptides. The self-assembling polypeptoid stereochemical inhibitors according to the present invention can self-assemble a single peptide helix to form microspheres having diameters in the micrometer range, including, among others, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, or 3.5 μm. In certain aspects, the stereochemical inhibitor can include a C-α-branched side chain, an N-aryl side chain, an N-1-naphthyl ethyl side chain, or a peptide having other configurations capable of forming a stable helical structure. Examples of peptide stereochemical inhibitors are shown in FIG. 18. FIG. 18 illustrates branched poly-N-methoxyethylglycine that can be used as a stereochemical inhibitor according to the present invention. In certain embodiments, the stereochemical inhibitor can include a linker group, e.g., a reactive group that can be readily linked to a cleavable linking group as described above.

[0069] In other embodiments, the stereochemical inhibitor may include a polymer (e.g., a biocompatible polymer), which may include blocks of different polymers, such that the blocks form a desired macroscopic structure (e.g., a sphere when exposed to an aqueous environment). For example, a copolymer may include hydrophilic and hydrophobic blocks, such that the polymer self-assembles into a micelle-like structure when added to water. In some embodiments, the hydrophobic block may be selected from polycaprolactone (PCL), polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), or polylactide (PLA). The hydrophilic block may include polyethylene glycol (PEG) or other polyhydric alcohols.

[0070] In other embodiments, the inhibitor may include nanoparticles of a size sufficient to block the activity of nucleotidyl transferase. Such nanoparticles may include, for example, gold, silver, silicon, cerium oxide, iron oxide, titanium dioxide, silicon nitride, boron nitride, or silica (e.g., mesoporous silica). In other embodiments, the nanoparticles may include higher-order molecular structures containing carbon (e.g., fullerenes, e.g., buckyballs and nanotubes), or semiconductors.

[0071] The stereochemical inhibitor may be uncharged or may be positively or negatively charged to provide compatibility with the nucleotide to which it is linked and with the nucleotidyl transferase enzyme. As a result, the inhibitor does not interfere with the incorporation reaction at the 5' end of the NTP analog. The stereochemical inhibitor may incorporate various amino acid residues to provide a desired conformation, charge, or attachment site.

[0072] An example of a nucleotide analog of the NTP-linker-inhibitor type is shown in FIG. 1A. The analog in FIG. 1A provides an unblocked, unmodified 3'-OH on the sugar ring and, simultaneously, through a disulfide (-S-S-) bond, the N of dCTP4 It contains an inhibitory (-Asp-Asp-) group linked to the position. The linker is constructed such that all linker atoms (including the second incorporated inhibitory moiety) can be removed, thereby enabling the nascent DNA strand to return to natural nucleotides. As shown in Figure 1B, an aqueous reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT)) can be used to cleave the -S-S- bond, resulting in the loss of the inhibitor function (deprotection). As shown in Figure 1B, a self-cyclizing linker can be incorporated, which can give rise to a cyclic oxidized tetrahydrothiophene leaving group that can be easily removed from the reagent solution upon completion of nucleotide synthesis.

[0073] Exemplary schemes for synthesizing the dCTP analog of Figure 1A are shown in Schemes 1A and 1B below:

Chemical Structure

Chemical Structure

[0074] In a manner similar to Schemes 1A and 1B, nucleotide analogs of the NTP-linker-inhibitor type can also be formed by attaching the linker-inhibitor moiety to N6 of adenine (Figure 2), N2 of guanine (Figure 3), N3 of thymine (Figure 4), or N3 of uracil (Figure 5), thereby providing analogs of "naturally occurring" dNTPs, as well as deoxyuracil nucleotides (dUTP). Although the extensive use of dUTP is unlikely, its synthesis is straightforward based on chemical properties.

[0075] Although the present invention is not limited to the chemistry of the linkage of schemes 1A and 1B, carbamate, amide, or other self-eliminating linkages can also be used. For example, nucleotides can be prepared with a Staudinger linker as shown in Scheme 2: [Chemical formula]

[0076] A deoxycytidine triphosphate (dCTP) analog (Scheme 2) prepared using a Staudinger linker to an Asp-Asp blocking group is shown in FIG. 6. As shown in FIG. 6, the above Staudinger dCTP analog undergoes cleavage under aqueous conditions by adding azide and triphenylphosphine. The Staudinger analog shown in FIG. 6 is also suitable for nucleotide extension using a nucleotidyl transferase (e.g., TdT) as described above and exemplified in FIGS. 1-5. Although not explicitly shown in the figure, one of ordinary skill in the art can use Scheme 2 with appropriate reactants to generate other nucleotide analogs having a Staudinger linker if required for complete de novo nucleotide synthesis. In a manner similar to FIG. 6, the nucleotide analog of Scheme 2 can be formed by attaching the above Staudinger moiety to N6 of adenine, N2 of guanine, N3 of thymine, or N3 of uracil, thereby providing analogs of "naturally occurring" dNTP, as well as deoxyuracil nucleotide (dUTP).

[0077] The methodology of Scheme 1A can be used to generate the corresponding ribonucleotide analogs, for example, by starting with the appropriate ribonucleotide reactants as shown in FIGS. 7-10. The ribonucleotide analogs containing the above Staudinger linker can also be prepared using Scheme 2 to form the required ribonucleotide analogs (e.g., including CTP analogs) as shown in FIG. 12. Further, all of the above ribonucleotide analogs (i.e., C, A, T, G, U) can be formed using reactions similar to Scheme 2.

[0078] In other embodiments, 3'-O-blocked nucleotide analogs can be used with modified enzymes that can incorporate the 3'-O-blocked nucleotide analogs into oligonucleotides. Such modified enzymes allow 3'-O-blocked dNTP analogs to be used in a step-by-step manner to extend the starting nucleic acid into a user-defined sequence (see FIG. 20). Further, after each nucleotide extension step, the reactants can be recovered and returned from the solid support to the original reagent reservoir for recycling. Once the process is complete, the 3'-O-blocking group is removed, allowing the cycle to be newly initiated. At the end of n cycles of extension-recovery-deprotection-washing, the full-length single-stranded polynucleotide is cleaved from the solid support and isolated for subsequent use. Various 3'-O-blocked deoxynucleotides can be used, but the choice of a particular 3'-O-blocking group can be influenced by: 1) the smallest possible bulk to maximize substrate utilization by TdT, and 2) removal of the blocking group under the mildest and preferably aqueous conditions in the shortest period of time.

[0079] Various 3'-O-modified dNTPs and NTPs can be used with the disclosed proteins for de novo synthesis. In some embodiments, preferred removable 3'-O-blocking groups are 3'-O-amino, 3'-O-allyl or 3'-O-azidomethyl. In other embodiments, the removable 3'-O-blocking moiety is selected from the group consisting of O-phenoxyacetyl; O-methoxyacetyl; O-acetyl; O-(p-toluene)-sulfonate; O-phosphate; O-nitrate; O-[4-methoxy]-tetrahydrothiopyranyl; O-tetrahydrothiopyranyl; O-[5-methyl]-tetrahydrofuranyl; O-[2-methyl,4-methoxy]-tetrahydrothiopyranyl; O-[5-methyl]-tetrahydrothiopyranyl; and O-tetrahydrothiofuranyl (see U.S. Patent No. 8,133,669). In other embodiments, the removable blocking moiety is selected from the group consisting of esters, ethers, carbonitriles, phosphates, carbonates, carbamates, hydroxylamines, borates, nitrates, sugars, phosphoramides, phosphoramidates, phenylsulfenates, sulfates, sulfones and amino acids (see Metzker ML et al. Nuc Acids Res. 1994;22(20):4259-67, U.S. Patent Nos. 6,232,465; 7,414,116; and 7,279,563, which are all incorporated herein by reference in their entirety).

[0080] Figure 21 shows four exemplary 3'-O-blocked dNTP analogs, namely 3'-O-azidomethyl-dATP, 3'-O-azidomethyl-dCTP, 3'-O-azidomethyl-dGTP, and 3'-O-azidomethyl-dTTP. The 3'-O-blocked dNTP analogs can be purchased from specialty suppliers (e.g., Azco Biotech, Oceanside, CA). The corresponding 3'-O-blocked ribonucleotides can also be obtained commercially and may enable the production of custom RNA oligonucleotides.

[0081] Enzyme The method of the present invention uses a nucleotidyl transferase to assemble the nucleotide analogs into polynucleotides. Nucleotidyl transferases include several families of related transferases and polymerase enzymes. Some nucleotidyl transferases polymerize deoxyribonucleotides more efficiently than ribonucleotides, some nucleotidyl transferases polymerize ribonucleotides more efficiently than deoxyribonucleotides, and some nucleotidyl transferases polymerize ribonucleotides and deoxyribonucleotides at approximately the same rate.

[0082] Particularly important for the present invention, transferases with polymerase activity (e.g., terminal deoxynucleotidyl transferase (TdT)) can catalyze the addition of deoxyribonucleotides to the 3'-end of a nucleotide chain, thereby increasing the chain length in DNA nucleotides. TdT only catalyzes the addition of 1 - 2 ribonucleotides to the growing end of a DNA strand that can be useful in the construction of site-specific DNA-RNA chimeric polynucleotides. In particular, bovine thymus TdT (supplied from engineered E. coli) is suitable for use in connection with the present invention and is available from commercial sources such as Thermo Scientific (Pittsburgh, PA). The amino acid sequence corresponding to bovine TdT is listed in Table 1 as SEQ ID NO: 1.

[0083] [Table 1]

[0084] The nucleotide sequence corresponding to bovine TdT is listed in Table 2 as SEQ ID NO: 2.

[0085] [Table 2-1]

Table 2-2

[0086] Commercially available TdT is suitable for use in accordance with the methods of the present invention, while modified TdT (e.g., having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, e.g., having an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, e.g., having an amino acid sequence that is at least 99 % identical to SEQ ID NO: 1) can also be used in accordance with the methods of the present invention. Organisms expressing an appropriate nucleotide transferase can contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 2, e.g., at least 98% identical to SEQ ID NO: 2, e.g., at least 99% identical to SEQ ID NO: 2. In some cases, modified TdT results in more efficient production of polynucleotides or allows for better control of the chain length. Other modifications to TdT can change the release characteristics of the enzyme, thereby reducing the need for an aqueous reducing agent such as TCEP or DTT.

[0087] For the synthesis of RNA polynucleotides, a nucleotidyl transferase such as E. coli poly(A) polymerase can be used to catalyze the addition of ribonucleotides to the 3'-end of a ribonucleotide initiator. In other embodiments, E. coli poly(U) polymerase may be suitable for use in accordance with the methods of the present invention. Both E. coli poly(A) polymerase and E. coli poly(U) polymerase are commercially available from New England Biolabs (Ipswich, MA). These enzymes can be used with 3'-unblocked reversible terminator ribonucleotide triphosphates (rNTPs) to synthesize RNA. In certain embodiments, RNA can be synthesized using 3'-blocked, 2'-blocked, or 2'-3'-blocked rNTPs and poly(U) polymerase or poly(A) polymerase. The amino acid and nucleotide sequences of E. coli poly(A) polymerase and E. coli poly(U) polymerase are reproduced below. Modified E. coli poly(A) polymerase or E. coli poly(U) polymerase may be suitable for use in accordance with the methods of the present invention. For example, an enzyme having an amino acid sequence that is at least 95% identical to SEQ ID NO: 3, for example, an amino acid sequence that is at least 98% identical to SEQ ID NO: 3, for example, an amino acid sequence that is at least 99% identical to SEQ ID NO: 3 can be used in accordance with the methods of the present invention. An organism expressing a suitable enzyme may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 4, for example, at least 98% identical to SEQ ID NO: 4, for example, at least 99% identical to SEQ ID NO: 4. Alternatively, an enzyme having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5, for example, an amino acid sequence that is at least 98% identical to SEQ ID NO: 5, for example, an amino acid sequence that is at least 99% identical to SEQ ID NO: 5 can be used in accordance with the methods of the present invention. An organism expressing a suitable enzyme may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 6, for example, at least 98% identical to SEQ ID NO: 6, for example, at least 99% identical to SEQ ID NO: 6.

[0088]

Table 3

[0089] The nucleotide sequence corresponding to E. coli poly(A) polymerase is listed in Table 4 as SEQ ID NO: 4.

[0090]

Table 4-1

Table 4-2

[0091]

Table 5

[0092] The nucleotide sequence corresponding to E. coli poly(U) polymerase is listed in Table 6 as SEQ ID NO: 6.

[0093]

Table 6

[0094] As discussed above, the inhibitor coupled to the nucleotide analog either prevents the transferase (e.g., TdT) from being released from the polynucleotide or prevents other analogs from being incorporated into the growing chain. The charged moiety results in better inhibition, although studies suggest that the specific chemical nature of the inhibitor is not particularly important. For example, both phosphate and acidic peptides can be used to inhibit enzyme activity. See, for example, Bowers et al., Nature Methods, vol. 6, (2009) p. 593-95, and U.S. Patent No. 8,071,755, both of which are incorporated herein by reference in their entirety. In some embodiments, the inhibitor comprises a single amino acid or a dipeptide such as -(Asp)2, although the size and charge of that moiety can be adjusted as necessary based on the rates determined in the first nucleotide incorporation and second nucleotide incorporation experiments. That is, other embodiments can use more or different charged amino acids or other biocompatible charged molecules.

[0095] Other methods of nucleotide synthesis can be used to construct de novo oligonucleotides in a template-independent manner using a nucleotidyl transferase or a modified nucleotidyl transferase. In one embodiment, the polymerase / transferase enzymes can be modified to stop nucleotide addition when they encounter a modification to the phosphate of a 3'-unmodified dNTP analog. This scheme requires a deblocking reagent / reaction that modifies the phosphate terminus of the nucleotide analog to release the nascent chain for subsequent nucleotide incorporation. A preferred embodiment of this approach uses nucleotide analogs modified at the phosphate (α, β, or γ), although modification of the purine / pyrimidine base of the nucleotide is also tolerated. only

[0096] ​In some embodiments, it may be advantageous to use a 3'-exonuclease to remove oligonucleotides that are not properly terminated with an inhibitor prior to the addition of subsequent nucleotide analogs. In particular, the inhibitor of the nucleotide analog can be selected to inhibit the activities of nucleotidyltransferase and 3'-exonuclease, such that only properly terminated oligonucleotides are constructed. Using this quality control technique, the purity of the resulting oligonucleotide sequences is improved. In some embodiments, the use of such quality control means can obviate the need for post-synthesis purification. This technique is schematically shown in FIG. 19, where the 3'-exonuclease is introduced after the washing step that removes excess nucleotide analogs and before linker cleavage. Such a cleaning step (as shown in FIG. 19) reduces the number of oligonucleotides that are of an undesirable length and / or sequence.

[0097] Another embodiment for using a template-independent polymerase / transferase enzyme is to use protein engineering or protein evolution to modify the enzyme to remain tightly bound and be inactive towards the nascent strand after each nucleotide incorporation, thus preventing any subsequent incorporations until the polymerase / transferase is released from the strand by the use of a releasing agent / condition. Such modifications are selected to allow the use of native unmodified dNTPs instead of reversible terminator dNTPs. The releasing agent can be a high salt buffer, a denaturing agent, etc. The releasing condition can be high temperature, agitation, etc. For example, mutations to the Loop1 and SD1 regions of TdT have been shown to dramatically alter the activity derived from template-independent activity to a more template-dependent activity. Specific mutations of interest include, but are not limited to, Δ3384 / 391 / 392, del loop1(386□398), L398A, D339A, F401A, and Q402K403C404□E402R403S404. Other means of achieving the goal of a tightly bound (i.e., single turnover) TdT enzyme after incorporation can include mutations to residues (including, but not limited to, K261, R432, and R454) responsible for binding the three phosphates of the initiator strand.

[0098] Another embodiment for using a template-independent polymerase / transferase enzyme is to use protein engineering or protein evolution to highly efficiently use 3-blocked reversible terminators receiveIt is to modify the above enzyme in such a way. Naturally occurring polymerase / transferase enzymes do not incorporate 3'-blocked reversible terminators due to the stereochemical constraints in the active site of the enzyme. Modifying a single or any of several amino acids in the active site of the above enzyme can enable the efficient incorporation of a 3'-blocked reversible terminator into an initiator bound to a support in a process completely analogous to that described above. After incorporation, the above 3'-reversible terminator is removed with a deblocking reagent / condition, generating a completely natural (unblemished) single-stranded molecule ready for subsequent controlled extension reactions. The above enzyme contains amino acids close to the 3'-OH of the incoming dNTP that explain the tendency of TdT to incorporate ribonucleotide triphosphates as readily as deoxyribonucleotide triphosphates; those between β1 and β2, especially R334, Loop1, and those between α13 and αal4, especially R454, although not limited to these, the amino acids seem to be targets for mutagenesis that accommodate the bulk of the 3'-reversible terminator group and enable their efficient incorporation. In certain embodiments, additional amino acid changes may be required to compensate for changes made to accommodate the 3'-reversible terminator. Another embodiment for using a template-dependent polymerase is to use a dNTP analog, either 3'-blocked or unblocked, with a plurality of primer-template pairs attached to a solid support, where the template is a nucleic acid analog that supports polymerase-mediated primer extension with any of the four bases as specified by the user.

[0099] In some embodiments, the engineered TdT is used to achieve stepwise synthesis with 3′-O-blocked nucleotide analogs. It is possible to model the active site of the TdT protein using AutoDock (Molecular Graphics Laboratory, Scripps Research Institute, La Jolla, CA). Based on this calculation, a modified TdT (having changes at Arg336 and Arg454) is estimated to have enzymatic activity against 3′-O-blocked nucleotide analogs. Gly452 and Ser453 are present in a cis-peptide bond conformation (see Delarue et al., EMBO J.. 2002; 21(3):427-39, which is incorporated herein by reference in its entirety), and the guanidium group of Arg336 is thought to assist in stabilizing this conformation. The stability provided by Arg336 may help explain why substitutions at this position have a negative impact on the reactivity of the modified TdT protein. In some cases, the instability caused by modifying position 336 can be overcome by using proline to stabilize the cis-peptide bond conformation. However, if Arg336 is substituted with, for example, alanine or glycine, the entire TGSR motif (positions 451, 452, 435, 454) may also have to be modified to compensate for this change. For example, in another embodiment, the TGSR motif can be modified to TPSR or TGPR. In another embodiment, substitution at Arg454 to accommodate the steric bulk of the 3′-O-blocking group may require further modification of the α14 region to compensate for substitution of glycine or alanine at Arg454. In other embodiments, substitution of other amino acids in the α11 region may be required to compensate for substitution at Arg336, either instead of or in addition to modification of the GSR motif.

[0100] Modifications to Arg336 and Arg454 can alter the binding interactions of 3'-O-modified dNTPs, but it may also be necessary to explore substitutions that result in improved stereochemical interactions between the 3'-O-modified dNTP and TdT. Such stereochemical modifications can also be explored computationally. Residues Gly332, Gly333, Gly452, Thr451, and Ser453 are also potential targets for substitution to accommodate the specific steric bulk of 3'-blocking groups such as 3'-O-azidomethyl or 3'-O-allyl. Residues within 1.2 nm of the 3'-OH (e.g., Glu457, Ala510, Asp509, Arg508, Lys199, Ser196, Met192, or Leu161) can also potentially interfere with the substrate utilization of 3'-O-blocked dNTPs and are thus targets for substitution in addition to, or in combination with, Arg336 and Arg454. In addition to amino acid substitutions at positions 508, 509, and 510, it may be necessary to delete amino acids to remove interference with the 3'-O-blocking group. Since these amino acids are located near the C-terminus of the protein and in a relatively unstructured region, they can be deleted either individually or together, either in place of the modifications described herein or in combination with those modifications.

[0101] Another embodiment for using a template-independent polymerase / transferase enzyme is to modify the enzyme using protein engineering or protein evolution to optimize the use of each of four different nucleotides or even different modified nucleotide analogs in an analog-specific manner. Nucleotide-specific or nucleotide analog-specific enzyme variants can be engineered to have desired biochemical properties such as reduced Km or enhanced incorporation rates that further reduce the cost of synthesizing the desired polynucleotide.

[0102] Solid-state synthesis The method of the present invention can be carried out under various reaction conditions, but the orderly construction and recovery of the desired polynucleotide mostly requires a solid support where the polynucleotide can be extended. When used with the NTPs, linkers, and inhibitor analogs discussed above, it is possible to construct specific polynucleotide sequences of DNA and RNA in an aqueous environment, for example, by using TdT or poly(A) polymerase. As shown in FIG. 13, TdT can be used to effect the stepwise construction of custom polynucleotides by extending the polynucleotide sequence in a stepwise manner. As discussed above, the inhibitor group of each NTP analog halts the enzyme upon addition of the nucleotide. After each nucleotide extension step, the reaction is washed from the solid support before removing the inhibitor by cleaving the linker, and then new reactants can be added to allow the cycle to start anew.

[0103] In certain embodiments, additional quality control steps can be incorporated, where the oligonucleotide or polynucleotide is exposed to 3'exonuclease after the nucleotide analog extension step mediated by nucleotidyl transferase and before inhibitor cleavage. 3'exonuclease degrades oligonucleotide or polynucleotide chains having an unblocked 3'OH. An uncleaved inhibitor (e.g., a stereochemical inhibitor) can physically block the degradation of chains in which an uncleaved nucleotide analog has been successfully incorporated by 3'exonuclease. Such quality control steps degrade only oligonucleotides or polynucleotides that did not successfully incorporate the desired nucleotide analog in a previous addition step, thereby eliminating any errors in the final synthesized sequence. After 3'exonuclease exposure, the enzyme can be washed away before performing the inhibitor cleavage step.

[0104] The 3'-exonuclease acts by shortening or completely degrading chains that did not successfully add the desired nucleotide analog. Chains that could not be extended enzymatically in a given cycle do not have a terminal polymer-dNMP conjugate prior to the linker cleavage step. If 3'-exonuclease is introduced at this stage, the full-length chains can be protected from degradation while the "failed" chains are shortened in length or potentially completely degraded to mononucleotide phosphates. The yield of long (>500 bases) synthetic DNA depends on the highly efficient reactions that occur in each cycle and all cycles; both the enzymatic extension and deblocking / self-detachment steps must occur with similar quantitative yields. Introducing 3'-exonuclease after the enzymatic extension step but before the polymer terminator cleavage step has a positive effect on the purity of the nascent strands when the extension efficiency is low (i.e., there are chains that are not extended and thus have native, unmodified terminal nucleotides).

[0105] Conversely, the 3'-exonuclease step does not affect the quality of the synthesis even if the deblocking / removal steps are less quantitative. This is because those chains are still protected by the polymer terminator and are not extended during the next extension step. Thus, the actual improvement in synthesis quality associated with the addition of the 3'-exonuclease step can simply be determined experimentally and then evaluated if it is of value commensurate with the additional cost and cycle time.

[0106] Upon completion of n cycles of extension-removal-deblocking-washing, the final full-length single-stranded polynucleotide is completed, cleaved from the solid support, and recovered for subsequent use in applications such as DNA sequencing or PCR. Alternatively, the final full-length single-stranded polynucleotide can remain attached to the solid support for subsequent use in applications such as hybridization analysis, protein or DNA affinity capture. In other embodiments, partially double-stranded DNA can be used as an initiator to yield the synthesis of double-stranded polynucleotides.

[0107] In some embodiments, the nucleic acid initiator comprises a 3' moiety that releases the synthesized oligonucleotide when in the presence of a releasing agent. This feature is generally illustrated in FIG. 22, where the nucleic acid initiator (5'-initiator-) is shown coupled to an individual support (white circle) and a releasable 3' moiety (open star). In some embodiments, the initiator is a single-stranded oligonucleotide (e.g., dimer, trimer, tetramer, pentamer, hexamer, septamer, or octamer). Since the 3' moiety attached to the initiator is a substrate for the enzyme (e.g., TdT, e.g., modified TdT), the enzyme can add additional nucleotides or nucleotide analogs in a stepwise manner. With each addition, the length of the synthesized oligonucleotide increases. Once the oligonucleotide synthesis is complete, a releasing agent can be introduced to decouple its 3' moiety from the nucleic acid initiator. In some embodiments, the 3' moiety is a ribonucleotide (e.g., A, C, G, or U ribonucleotide). In other embodiments, the 3' moiety is abasic deoxyribose. In other embodiments, the 3' moiety is abasic ribose. In other embodiments, the 3' moiety is a non-nucleoside 5'-monophosphate. The releasing agent can comprise a basic solution or metal ions. For example, the releasing agent can be a concentrated NH4OH solution having a pH greater than 8, i.e., greater than 8.5, i.e., greater than 9.0, i.e., greater than 9.5. In some embodiments, the releasing agent is an enzyme (e.g., type II restriction nuclease). In some embodiments, the enzyme specifically interacts with the nucleic acid sequence of the initiator, lyses the synthesized oligonucleotide from the initiator, and leaves the initiator intact. In one embodiment, the initiator is a nucleic acid hexamer, and the 3' portion is a ribonucleotide (e.g., adenosine). For example, once the oligonucleotide synthesis is completed using a nucleotide containing a cleavable terminator linked at the N-4 position or a nucleotide having a 3'-O-blocked position, the oligonucleotide can be released by exposing the attached oligonucleotide to an ammonium hydroxide solution at approximately pH 8. Subsequently, the basic solution containing the synthesized oligonucleotide can be separated from the solid substrate containing the hexamer initiator. Then, the solid substrate is washed and / or neutralized to prepare the initiator and the 3' portion for the production of a new oligonucleotide. In some embodiments, the terminal ribonucleotide is regenerated prior to oligonucleotide synthesis with the use of the 3' phosphatase activity of a phosphatase or T4 polynucleotide kinase.

[0108] In some embodiments, the solid support and the nucleic acid initiator containing the 3' portion are reusable, thereby enabling the initiator coupled to the solid support to be used multiple times for the rapid synthesis of oligonucleotides. Solid supports suitable for use in accordance with the methods of the present invention can include glass and silica supports (including beads, slides, pegs, or wells). In some embodiments, the support may be attached to another structure (e.g., a polymer well plate or a pipette tip). In some embodiments, the solid support may have additional magnetic properties, thus enabling the support to be manipulated using a magnet or removed from a fixed position. In other embodiments, the solid support may be a silica-coated polymer, thereby enabling the formation of various structural shapes suitable for automated processing.

[0109] The choice of the substrate material and the chemical nature of the covalent bond between the initiator and the substrate are limited only by the ability of the construct to withstand the synthesis conditions without losing the initiator. Preferred embodiments utilize a substrate and linker of greater chemical stability than the initiator such that the overall construct stability is that of the attached oligonucleotide and is independent of the substrate. In some embodiments, the initiator can be synthesized in the 5’→3’ direction from a material presenting surface hydroxyl groups, but in preferred embodiments, the initiator is instead grafted onto the substrate so that the density and initiator quality can be accurately controlled.

[0110] The covalent bond between the initiator and the substrate can be any bond that does not compromise the stability of the construct. Preferred embodiments can utilize a coupling between an oligonucleotide containing any of a 5’-amine, 5’-hydroxyl, 5’-phosphate, 5’-sulfhydryl, or 5’-benzaldehyde group and a surface or resin on the substrate containing formyl, chloromethyl, epoxide, amine, thiol, alkene, or a terminal C-F bond.

[0111] In some embodiments, the initiator can include elements for a sequence-specific cleavage strategy (e.g., one that utilizes a restriction enzyme, uracil specific excision reagent (USER), or any of various sequence-specific nucleases). In other embodiments, these elements can instead be synthesized enzymatically or added to the initiator after the initiator has been coupled to the resin. Such scenarios can be preferred, for example, when the initiator is composed entirely of thymidine in order to minimize potential side reactions with surface functional groups during the coupling process.

[0112] Figure 23 shows a liquid phase example where a short polyuracil (U) tract is added to the 3’ end of an initiator using TdT. The resulting initiator can then be used for further elongation reactions, if desired, to generate the desired sequence. Once synthesis is complete, the newly generated sequence can be cleaved at the internal poly-U tract to separate the new sequence from the original initiator. The 30 nucleotide initiator used has the following sequence:

Chemical formula

[0113] Figure 24 shows that cleavage of the sequence containing the internal poly-U tract yields a single product of uniform length with a 5’-phosphate. The cleavage process leaves a 3’-phosphate on the initiator bound to the resin, which can be removed by treatment with T4 polynucleotide kinase, alkaline phosphatase, or other dephosphorylation processes (see Figure 25). The internal poly-U tract sequence cleaved in the example shown in Figure 24 is of the following sequence:

Chemical formula

Chemical formula

[0114] Figure 25 shows an exemplary solid-phase dephosphorylation and TdT extension process. The chart shows the absorbance of the TdT reaction mixture in solid-phase extension. As dNTPs are added to the initiator bound to the resin, they are depleted from the solution, reducing the absorbance of the supernatant. Trace A shows a negligible dNTP incorporation rate on the resin where the 3'-end of the initiator is blocked with a terminal phosphate. Trace B shows the incorporation rate after treating such an initiator with shrimp alkaline phosphatase for 15 minutes. Trace C shows the dNTP incorporation rate under the same conditions using an unblocked initiator.

[0115] After cleavage of the synthesized sequence, the resin can be used in another cycle of cleavage site installation, synthesis, product removal, and regeneration. Figure 26 provides an overview of an exemplary regeneration cycle.

[0116] In other embodiments, the cleavage site installation can be used to homogenize the enzymatic accessibility of the initiator oligonucleotides. Each enzyme used during an enzymatic synthesis cycle has its own stereochemical footprint potentially associated with a distinct optimal loading and spacing from the surface. This can result in unexpected behavior with respect to the kinetics of stepwise addition and the yield from the enzymatic cleavage process. In some embodiments, the iterative cycles of cleavage site installation, cleavage, and regeneration can be performed prior to oligonucleotide synthesis such that the cleavage enzyme and the template-independent polymerase have access to the same population of surface oligonucleotides.

[0117] Some embodiments can install a plurality of different cleavage sites throughout the chain during synthesis. Upon digestion, a complex library of chains located between the cleavage sites can be released from the resin. Such sequences can then be further amplified and used by those skilled in the art for enzymatic assembly processes. This approach may be particularly suitable for parallel synthesis schemes to generate more diverse array fragments at relatively few different positions on the surface or to avoid the synthesis of adjacent chains that are at risk of secondary structure formation during synthesis.

[0118] In other embodiments, cleavage site installation can be used immediately after each cycle of enzymatic extension to assist in the removal of unreacted initiator sequences, similar to acetylation used in phosphoramidite-based oligonucleotide synthesis. Chains that are not extended during additional cycles act as substrates for polyU tracts, while extended initiators with chain terminators do not act as substrates. At the end of the synthesis cycle, one USER digestion is performed such that the oligonucleotide is released from the support and sequences containing failed additions are simultaneously digested to a length shorter than the full-size product. Failed sequences also resemble in that they are phosphorylated at their 3' ends and rendered non-responsive to further enzymatic extension. The full-length sequences can then undergo extension to add any element that enables selective capture, isolation, or enrichment. Such an element can be either an additional homopolymer tract (e.g., an additional polyU tract) that is isolated by a hybridization-based approach and then digested, or a biotinylated element for non-covalent capture. This approach compensates for the lack of suitable chromatography techniques for long (150 nt+ oligonucleotides), low sample amounts, or complex mixtures of sequences of various lengths.

[0119] Further embodiments may use cleavage site placement to assist in recycling highly indexed DNA-based data recording media. An example is shown in FIG. 27. In such cases, the 5'-surface immobilization sequence is terminated with a short polyA tract. Extension to generate a sufficiently long terminal 3'-polyU site enables the hairpin to be folded under appropriate conditions such that the initiator element in front of the polyA stretch can be replicated using a template-dependent polymerase. The sequence can then be extended with a new homopolymer tract to leave a free 3' end that can be used in subsequent data writing operations. Once the writing process is complete, the hairpin linker can then be digested with USER enzyme to release the data strand while leaving the template initiator, which can be regenerated by 3'-dephosphorylation, polyU addition, and re-copying of the template strand. The use of homopolymer tracts in DNA-based data recording is described in U.S. patent application Ser. No. 15 / 994,335, which is incorporated herein by reference.

[0120] Synthesizer To utilize the efficiency of the disclosed method, an aqueous phase DNA synthesizer can be configured to produce a desired polynucleotide in substantial amounts. In one embodiment, the synthesizer includes four wells of the NTP analog reagents (i.e., dCTP, dATP, dGTP, and dTTP) described, and TdT, at a concentration sufficient to effect polynucleotide growth. A plurality of starting sequences can be attached to a solid support designed to be repeatedly immersed into each of the four wells, for example, using a laboratory robot. The robot can be further programmed to rinse the solid support in a wash buffer during nucleotide addition, cleave the linker groups by exposing the support to a deblocking reagent, and wash the solid support (a second time) before moving the solid support to the well of the next desired nucleotide. Using simple programming, it is possible to produce useful amounts of the desired nucleotide sequence in approximately a few hours and with a substantial reduction in harmful waste. The synthesis proceeding under carefully controlled conditions allows for the synthesis of polynucleotides having lengths of thousands of base pairs. When complete, the elongation products are released from the solid support and, once released, they can be used as the final nucleotide sequence.

[0121] Embodiments in parallel with height can consist of a series of initiator-solid supports on pegs in either a 96-well or 384-well format that can be individually retracted or lowered. As a result, the pegs can be indexed to contact the liquid in the wells in a controlled manner. Thus, the synthesizer can consist of a randomly addressable peg device, four enzyme-dNTP analog reservoirs in the same manner as the peg device (96 or 384 spacing), additional reagent reservoirs (washing, deblocking, etc.) in the same manner as the peg device (96 or 384 spacing), and a transport mechanism (e.g., a laboratory robot) for moving the peg device from one reservoir to another in a user-programmable controlled or random access manner. Care must be taken to avoid contamination of each of the four enzyme-dNTP reservoirs. This is because their contents are reused throughout the synthesis process to reduce the cost of each polynucleotide synthesis.

[0122] In alternative embodiments, reagents (e.g., nucleotide analogs, enzymes, buffers) are moved between solid supports, enabling the reagents to be recycled. For example, a reservoir and pump system can move four different nucleotide analog solutions, wash buffers, and / or reducing agent solutions between one or more reactors in which the oligonucleotides are formed. The reactors and pumps can be conventional, or the device can be constructed using microfluidic technology. Since the nature of the process is not anhydrous (is aqueous), there is no need for special care in the design of the hardware used to eliminate exposure to water. The synthesis process can be carried out as long as precautions are taken to control losses due to evaporation. Highly parallel embodiments can consist of a monolithic series of initiator-solid supports on a peg in either a 96-well or 384-well format that can be interfaced to a series of wells in the same matching pattern. Each well can actually be a reaction chamber supplied by appropriate valves with four enzyme-dNTP analog reservoirs and additional reagent reservoirs (for washing, deblocking, etc.). Preparation can be made in fluid engineering theory to recover the enzyme-dNTP reactants in their original form after each extension reaction. This is because they are reused throughout the synthesis process to reduce the cost of each polynucleotide synthesis. In other embodiments, a system for pipetting the chip can be used to add and remove reagents.

[0123] In certain embodiments, polynucleotides can be synthesized using microfluidic devices and / or inkjet printing technology. An exemplary microfluidic polynucleotide synthesis device is shown in FIG. 17 for illustrative purposes and is not drawn to scale. Microfluidic channels 255 (including regulators 257) connect reservoirs 253 to reaction chamber 251, and outlet channels 259 (including regulators 257) can discharge waste from the reaction chamber 251. Microfluidic devices for polynucleotide synthesis can include, for example, channels 255, reservoirs 253, and / or regulators 257. Polynucleotide synthesis can occur within microfluidic reaction chamber 251. This microfluidic reaction chamber 251 can contain a number of anchored synthetic nucleotide initiators that are anchored or bound to the interior surface of the reaction chamber and can releasably bind to release NTP analogs or polynucleotide initiators. The reaction chamber 251 can include at least one inlet and one outlet channel 259 such that reagents can be added to and removed from the reaction chamber 251. The microfluidic device can include reservoirs 253 for each respective NTP analog. Each of these NTP analog reservoirs 253 can also contain an appropriate amount of TdT or any other enzyme that extends a DNA or RNA strand without template direction. Additional reservoirs 253 can contain reagents for linker / inhibitor cleavage and washing. These reservoirs 253 can be connected to the reaction chamber 251 via separate channels 255, and the flow of reagents to the reaction chamber 251 through each channel 255 can be individually regulated through the use of gates, valves, pressure regulators, or other means. From the reaction chamber 251, the flow through outlet channel 259 can be similarly regulated.

[0124] In certain cases, the reagents, in particular the NTP analog - enzyme reagents, can be recycled. The reagents can be drawn back from the reaction chamber 251 through the same channel 255 to their respective reservoirs 253. Through channel 255, the reagents can enter by inducing backflow using a gate, valve, pressure regulator, or other means. Alternatively, the reagents can be returned from the reaction chamber 251 to their respective reservoirs 253 via independent return channels. The microfluidic device can include a controller that can operate the gate, valve, pressure, or other regulator 257 described above.

[0125] An exemplary microfluidic polynucleotide synthesis reaction can include flowing a desired enzyme - NTP analog reagent into the reaction chamber 251; after a set time, removing the enzyme - NTP analog reagent from the reaction chamber 251 via the outlet channel 259 or a return channel; flowing a washing reagent into the reaction chamber 251; removing the washing reagent from the reaction chamber 251 through the outlet channel 259; flowing a de - blocking or cleavage reagent into the reaction chamber 251; removing the de - blocking or cleavage reagent from the reaction chamber 251 via the outlet channel 259 or a return channel; flowing a washing reagent into the reaction chamber 251; removing the washing reagent from the reaction chamber 251 through the outlet channel 259; flowing the enzyme - NTP analog reagent containing the next NTP in the desired sequence to be synthesized into the reaction chamber 251; and repeating until the desired polynucleotide is synthesized. After the desired polynucleotide is synthesized, it can be released from the reaction chamber anchor or substrate and collected via the outlet channel 259 or other means.

[0126] In certain aspects, reagents and compounds (including NTP analogs, TdT and / or other enzymes, and reagents for linker / inhibitor cleavage and / or washing) can be deposited into the reaction chamber using inkjet printing technology or piezoelectric drop-on-demand (DOD) inkjet printing technology. Inkjet printing technology can be used to form droplets that can be deposited through the air into the reaction chamber. The reagent droplets can have volumes on the picoliter to nanoliter scale. The droplets can be introduced using inkjet printing technology at various frequencies including 1 Hz, 10 Hz, 100 Hz, 1 kHz, 2 kHz, and 2.5 kHz. The various reagents can be stored in separate reservoirs within the inkjet printing device, which can deliver droplets of the various reagents to various separate locations (including different reaction chambers or wells within the chip). In certain embodiments, inkjet and microfluidic technologies can be combined, where certain reagents and compounds are delivered via inkjet printing technology into the reaction chamber while others are delivered via microfluidic channels or tubes. The inkjet printing device can be controlled by a computing device including at least non-transitory tangible memory coupled to a processor. The computing device can receive input from an input device (such as a touch screen, mouse, or keyboard), and be operative to control when and where the inkjet printing device deposits droplets of reagent, which reagent is deposited, and / or the amount of reagent deposited.

[0127] In certain cases, the desired polynucleotide sequence can be input into the computing device through the input device. Here the computing device can be operative to perform the necessary reactions to generate the desired polynucleotide sequence by sequentially depositing appropriate NTP analogs, enzymes, cleavage reagents, and washing reagents in the appropriate order as described above.

[0128] After synthesis, the released extension products can be analyzed by high-resolution PAGE to determine whether the initiator has been extended by the number of recognized bases compared to the control. The recovered portion of the synthesized DNA can also be sequenced to determine whether the synthesized polynucleotide is the recognized sequence.

[0129] The above synthesizer is relatively simple and does not require toxic reagents necessary for phosphoramidite synthesis. Therefore, the synthesizer of the present invention can be widely used for research institutions, biotechnology companies, and hospitals. Furthermore, the ability to reuse / recycle reagents helps reduce the generated waste and the cost of consumables. The inventors recognize that the above methods and systems are useful in many applications such as DNA sequencing, PCR, and synthetic biology.

[0130] Incorporation by reference References and citations to other documents (e.g., patents, patent applications, patent publications, magazines, books, papers, web content) are made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0131] Equivalents Various modifications of the present invention and many of its further embodiments will become apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent literature cited herein, in addition to those shown and described herein. The subject matter herein includes important information, examples, and guidance that can be adapted for the practice of the present invention in various embodiments of the present invention and their equivalents. The present invention provides, for example, the following items. (Item 1) A composition comprising a solid support and an oligonucleotide, wherein the 5'-end of the oligonucleotide is coupled to the solid support, and the oligonucleotide comprises a sequence-specific cleavage element. (Item 2) The composition according to item 1, wherein the oligonucleotide is coupled to the solid support containing formyl, chloromethyl, epoxide, amine, thiol, alkene, or a terminal C-F bond and contains a 5'-amine, 5'-hydroxyl, 5'-phosphate, 5'-sulfhydryl, or 5'-benzaldehyde group. (Item 3) The composition according to item 1, wherein the oligonucleotide is irreversibly coupled to the solid support. (Item 4) The composition according to item 1, wherein the sequence-specific cleavage element is incorporated into the oligonucleotide after the 5'-end is irreversibly coupled to the solid support. (Item 5) The composition according to item 4, wherein the sequence-specific cleavage element is incorporated by an enzyme. (Item 6) The composition according to item 1, wherein the sequence-specific cleavage element comprises a polyU tract. (Item 7) The composition according to item 1, wherein the oligonucleotide coupled to the support contains an index element so as to act as a template during oligonucleotide strand synthesis from the 3'-end of the oligonucleotide coupled to the support. (Item 8) A method for synthesizing an oligonucleotide on a reusable solid support, the method comprising: exposing a nucleic acid initiator attached to a solid support to nucleotide analogs in the presence of a polymerase to produce a first oligonucleotide, wherein the nucleic acid initiator comprises a sequence-specific cleavage element; contacting the nucleic acid initiator with a releasing agent to cleave the sequence-specific cleavage element and release the first oligonucleotide from the nucleic acid initiator; dephosphorylating the 3'-end of the nucleic acid initiator attached to the solid support; and regenerating the sequence-specific cleavage element by an enzyme. A method comprising the above steps. (Item 9) The method according to item 8, wherein the nucleic acid initiator is attached to the solid support containing formyl, chloromethyl, epoxide, amine, thiol, alkene, or a 5'-amine, 5'-hydroxyl, 5'-phosphate, 5'-sulfhydryl, or 5'-benzaldehyde group containing a terminal C-F bond. (Item 10) The method according to item 8, wherein the nucleic acid initiator is irreversibly attached to the solid support. (Item 11) The method according to item 8, wherein the sequence-specific cleavage element is incorporated into the nucleic acid initiator after the 5'-end of the nucleic acid initiator is irreversibly coupled to the solid support. (Item 12) The method according to item 11, wherein the sequence-specific cleavage element is incorporated by an enzyme. (Item 13) The method according to item 8, wherein the sequence-specific cleavage element comprises a poly U tract. (Item 14) The nucleic acid initiator comprises an index element, and the method comprises the step of synthesizing the first oligonucleotide under suitable conditions to allow hairpin formation by extending the 3'-end of the nucleic acid initiator with a complementary nucleotide analog to the 3'-sequence of the nucleic acid initiator; the step of extending the first oligonucleotide after hairpin formation using the index element as a template, and further comprises the method according to item 8. (Item 15) The method according to item 14, wherein the 3'-sequence of the nucleic acid initiator is complementary to the sequence-specific cleavage element. (Item 16) The step of incorporating sufficient spacer nucleotides into the first oligonucleotide to create a single-stranded 3'-end following the hairpin, and further comprises the method according to item 14. (Item 17) Before contacting the nucleic acid initiator with the releasing agent, incorporating a second sequence-specific cleavage element at the 3'-end of the first oligonucleotide to create a second nucleic acid initiator and exposing the second nucleic acid initiator attached to the first oligonucleotide to nucleotide analogs in the presence of a polymerase to produce a second oligonucleotide, further comprising the steps of: wherein the step of contacting the nucleic acid initiator and the second nucleic acid initiator with the releasing agent cleaves the sequence-specific cleavage element and the second sequence-specific cleavage element, and releases the first oligonucleotide and the second oligonucleotide from the nucleic acid initiator and the second nucleic acid initiator. The method according to item 8. (Item 18) The method according to item 8, wherein the polymerase is a nucleotidyl transferase or a modified nucleotidyl transferase. (Item 19) The method according to item 8, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT. (Item 20) The method according to item 8, wherein the nucleotide analog has an unmodified 3'-OH and a cleavable terminating group, the cleavable terminating group blocks subsequent nucleotidyl transferase activity, but upon cleavage of the terminating group, a nucleotide substrate for nucleotidyl transferase is generated.

Claims

【Claim 1】 The invention described in the specification.

Images