JP2016515391A - Method and apparatus for synthesizing nucleic acids - Google Patents

Abstract

The present invention provides improved methods for synthesizing polynucleotides (eg, DNA and RNA) using enzymes and specially designed nucleotide analogs. Using the methods of the invention, a specific sequence of a polynucleotide can be synthesized de novo in an aqueous environment, one base at a time, without using a nucleic acid template. Since the nucleotide analog has an unmodified 3 ′ OH (ie, as found in “natural” deoxyribose and ribose molecules), the analog has a natural polynucleotide suitable for incorporation into biological systems. Arise.

Description

  This application is filed in U.S. Patent Application No. 14 / 056,687, filed October 17, 2013, U.S. Provisional Application No. 61 / 807,327, filed Apr. 2, 2013, and October 2013. Claims priority based on US Provisional Application No. 61 / 891,162, filed on May 15, all of which are incorporated by reference in their entirety.

  The present invention relates to a method and apparatus for (de novo) synthesis of a polynucleotide having the desired sequence and without the need for a template. Thus, the present invention provides the ability to create libraries of polynucleotides of various sequences and lengths for research, genetic engineering, and gene therapy.

(background)
Genetic engineering requires tools for determining the content of genetic material as well as tools for building the desired genetic material. The tool for determining the contents of the genetic material allowed the entire human genome to be sequenced in less than $ 1,000 in about one day (Life Technologies, Press Release: Benchtop Ion Proton ^™ Sequencer, January 10, 2012). In contrast, tools for constructing the desired genetic material (eg, de novo DNA synthesis) have not improved at the same rate. As an evaluation standard, the cost of de novo low-molecular nucleic acid synthesis (per base) has decreased to 1/10 over the past 25 years, but the cost of nucleic acid sequencing (per base) has been reduced to 1 / 10,000,000. It dropped to over 000. The lack of progress in DNA synthesis now limits the speed of translational genomics (ie, the role of individual sequence variations is determined and used to develop therapeutic treatments).

  Currently, most new nucleic acid sequences are synthesized using solid phase phosphoramidite technology developed more than 30 years ago. This technique involves the sequential deprotection and synthesis of sequences composed of phosphoramidite reagents corresponding to natural (or non-natural) nucleobases. However, phosphoramidite nucleic acid synthesis is limited in length in that nucleic acids greater than 200 base pairs (bp) in length experience high breakage rates and side reaction rates. In addition, phosphoramidite synthesis produces toxic by-products, and this waste disposal limits the effectiveness of the nucleic acid synthesizer and increases the cost of contract oligo production (the usual annual synthesis of oligonucleotide synthesis). Demand is expected to be responsible for over 300,000 gallons of hazardous chemical waste, including acetonitrile, trichloroacetic acid, toluene, tetrahydrofuran, and pyridine.LeProust et al., Nucleic Acids Res., vol.38 (8), p.2522540, (2010), which is incorporated by reference herein in its entirety). Therefore, there is a need for a more efficient and cost effective method for oligonucleotide synthesis.

Life Technologies, Press Release: Benchtop Ion Proton ™ Sequencer, January 10, 2012 LeProust et al. Nucleic Acids Res. , Vol. 38 (8), p. 2522-2540, (2010)

(Summary)
The present invention provides an improved method for nucleic acid synthesis. The methods of the invention provide for faster and longer de novo synthesis of polynucleotides. Thus, the present invention dramatically reduces the overall cost of custom nucleic acid synthesis. The methods of the present invention relate to template-independent synthesis of polynucleotides by using nucleotidyl transferase enzymes to incorporate nucleotide analogs with unmodified 3 ′ hydroxyls and cleavable end groups. Due to the end groups, synthesis is paused as each new base is added, and then the end groups are cleaved, resulting in polynucleotides that are essentially identical to the naturally occurring nucleotides (ie, additional nucleotides). (Recognized by the enzyme as a substrate for incorporation).

  The invention further includes an apparatus that utilizes the methods of the invention for the production of custom polynucleotides. The apparatus of the present invention comprises one or more bioreactors that provide aqueous conditions and multiple sources of nucleotide analogs. The bioreactor can be, for example, a reservoir, a flow cell, or a multiwell plate. Starting from a solid support, the polynucleotide is a natural product of a nucleotidyl transferase (eg, terminal deoxynucleotidyl transferase (TdT), or any other enzyme that extends a DNA or RNA strand without a template indication). Through activity, grow in the reactor by successive nucleotide additions. Upon cleavage of the end group, the natural polynucleotide is exposed on the solid support. When the sequence is complete, the support is cleaved leaving a polynucleotide that is essentially equivalent to that found in nature. In some embodiments, the device is designed to reuse the nucleotide analog solution by collecting the nucleotide analog solution after nucleotide addition and reusing the solution for subsequent nucleotide addition. Thus, less waste is produced and the overall cost per base is reduced compared to state-of-the-art methods.

  Other aspects of the invention will be apparent to those skilled in the art from consideration of the following drawings and detailed description.

FIG. 1A shows a class of deoxycytidine triphosphate (dCTP) analogs with a cleavable terminator linked at the N-4 position. FIG. 1B shows cleavage of the cleavable terminator from the dCTP analog of FIG. 1A to obtain “native” dCTP and a cyclic leaving molecule. FIG. 2A shows a class of deoxyadenosine triphosphate (dATP) analogs with a cleavable terminator linked at the N-6 position. FIG. 2B shows cleavage of the cleavable terminator from the dATP analog of FIG. 2A to obtain “native” dATP and a cyclic leaving molecule. FIG. 3A shows a class of deoxyguanosine triphosphate (dGTP) analogs with a cleavable terminator linked at the N-2 position. FIG. 3B shows cleavage of the cleavable terminator from the dGTP analog of FIG. 3A to obtain “native” dGTP and a cyclic leaving molecule. FIG. 4A shows a class of deoxythymidine triphosphate (dTTP) analogs with a cleavable terminator linked at the N-3 position. FIG. 4B shows cleavage of the cleavable terminator from the dTTP analog of FIG. 4A to obtain “natural” dTTP and cyclic leaving molecules. FIG. 5A shows a class of deoxyuridine triphosphate (dUTP) analogs with a cleavable terminator linked at the N-3 position. FIG. 5B shows cleavage of the cleavable terminator from the dUTP analog of FIG. 5A to obtain dUTP and a cyclic leaving molecule. FIG. 6 shows an exemplary deoxycytidine triphosphate (dCTP) analog with a Staudinger linker connecting the blocking Asp-Asp molecule to the N-4 position of deoxycytidine, as well as dCTP and leaving groups. FIG. 4 shows the subsequent cleavage of the Staudinger linker under aqueous conditions to obtain. FIG. 7A shows a class of cytidine triphosphate (rCTP) analogs with a cleavable terminator linked at the N-4 position. FIG. 7B shows cleavage of the cleavable terminator from the rCTP analog of FIG. 7A to obtain “native” rCTP and a cyclic leaving molecule. FIG. 8A shows a class of adenosine triphosphate (rATP) analogs with a cleavable terminator linked at the N-6 position. FIG. 8B shows cleavage of the cleavable terminator from the rATP analog of FIG. 8A to obtain “native” rATP and cyclic leaving molecules. FIG. 9A shows a class of guanosine triphosphate (rGTP) analogs with a cleavable terminator linked at the N-2 position. FIG. 9B shows cleavage of the cleavable terminator from the rGTP analog of FIG. 9A to obtain “natural” rGTP and cyclic leaving molecules. FIG. 10A shows a class of thymidine triphosphate (rTTP) analogs with a cleavable terminator linked at the N-3 position. FIG. 10B shows cleavage of the cleavable terminator from the rTTP analog of FIG. 10A to obtain “native” rTTP and a cyclic leaving molecule. FIG. 11A shows a class of uridine triphosphate (rUTP) analogs with a cleavable terminator linked at the N-3 position. FIG. 11B shows cleavage of the cleavable terminator from the rUTP analog of FIG. 11A to obtain rUTP and a cyclic leaving molecule. FIG. 12 shows an exemplary cytidine triphosphate (rCTP) analog with a Staudinger linker connecting the blocking Asp-Asp molecule to the N-4 position of cytidine, and to obtain rCTP and leaving group FIG. 4 shows subsequent cleavage of the Staudinger linker under aqueous conditions. FIG. 13 shows (a) incorporation of a cleavable terminator, a nucleotide triphosphate analog containing dN ^* TP-OH, and (b) removal of the terminal blocking group (indicated by ^* ) (hence the following dN ^* TP- FIG. 6 illustrates an exemplary terminal deoxynucleotidyl transferase (TdT) mediated polynucleotide synthesis cycle including OH (which allows OH to be incorporated), where N = A, G, C, or T.

(Detailed explanation)
The present invention provides improved methods for synthesizing polynucleotides (eg, DNA and RNA) using enzymes and nucleic acid analogs. Using the disclosed method, a specific sequence of a polynucleotide can be synthesized de novo in an aqueous environment without the use of a nucleic acid template, one base at a time. In addition, since the nucleotide analog has an unmodified 3 ′ hydroxyl (ie, as found in “natural” deoxyribose and ribose molecules), the analog may have a cleavable blocking group removed from the base. , Resulting in “natural” nucleotides. Other nucleotide analogs can also be used (for example, self-eliminating linkers or nucleotides with modified phosphate groups). In most cases, the blocking group is designed not to leave behind substantial additional molecules, i.e., after a "scarless" nucleotide that is recognized as a "natural" nucleotide by the enzyme. Designed to leave. Thus, as a result of the synthesis, upon removal of the last blocking group, the synthesized polynucleotide is chemically and structurally equivalent to a naturally occurring polynucleotide having the same sequence. The synthetic polynucleotide can thus be incorporated into a biological system without concern that the synthesized polynucleotide interferes with biochemical pathways or metabolism.

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

In one aspect, the disclosed method uses a commercially available nucleotidyl transferase enzyme (eg, terminal deoxynucleotidyl transferase (TdT)) to synthesize a polynucleotide from a nucleotide analog in a step-by-step manner. The nucleotide analog is of the following form:
NTP-linker-inhibitor where NTP is a nucleotide triphosphate (ie dNTP or rNTP), the linker is a cleavable linker between the base pyridine or pyrimidine, and the inhibitor is It is a group that prevents the incorporation of the nucleotide. In each step, a new nucleotide analog is incorporated into the growing polynucleotide chain, and the enzyme is then blocked by the inhibitor group from adding additional nucleotides. Once the enzyme is stopped, excess nucleotide analogs can be removed from the growing strand, the inhibitor can be cleaved from the NTP, and a new nucleotide analog adds the next nucleotide to the strand. Can be introduced. By repeating the above steps continuously, it is possible to rapidly construct a nucleotide sequence of the desired length and sequence. Advantages of using nucleotidyl transferase for polynucleotide synthesis include: 1) 3′-extension activity using a single-stranded (ss) initiation primer in template-independent polymerization, 2) The ability to extend primers in a highly efficient manner that can result in the addition of thousands of nucleotides, and 3) acceptance of a wide variety of modified and substituted NTPs as effective substrates. Furthermore, the present invention may utilize an initiator sequence that is a substrate for nucleotidyl transferase. The initiator binds to the solid support and serves as a binding site for the enzyme. The initiator is preferably a universal initiator (eg, a homopolymer sequence) of the enzyme, reusable on a solid support, and the oligonucleotide formed can be cleaved from the initiator. .

  The methods of the present invention are well suited for various applications that currently use synthetic nucleic acids, such as phosphoramidite synthetic DNA oligos. For example, the polynucleotides synthesized by the methods of the invention can be used as primers for nucleic acid amplification, as hybridization probes for detection of specific markers, and for incorporation into plasmids for genetic engineering. . However, since the disclosed methods produce longer synthetic strands of nucleotides more rapidly and in an aqueous environment, the disclosed methods can also be used in high-throughput applications (eg, screening for expression of genetic variations in cellular assays, And synthetic biology). Furthermore, the method of the present invention can be used for next generation applications (eg, using DNA as a synthetic read / write memory, or creating a macroscopic material that is fully (or partially) synthesized from DNA). Provide the required functionality.

  The present invention and the systems described herein provide for the synthesis of polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Synthetic pathways for “natural” nucleotides (eg, DNA and RNA) are common nucleobases (eg, adenine (A), guanine (G), cytosine (C), thymine (T), and uracil ( U)), while the method of the present invention can be used to produce so-called “non-natural” nucleotides (universal bases such as 3-nitropyrrole 2′-deoxynucleosides and 5-nitroindole 2′-deoxynucleosides. , Α phosphorothiolates, phosphorothioate nucleotide triphosphates, or nucleotides that incorporate purine or pyrimidine conjugates with other desirable properties (eg, fluorescence)) should be understood. Other examples of purine and pyrimidine bases include: pyrazolo [3,4-d] pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2 Amino 6-methyl and other alkyl derivatives of adenine, adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and 5 -Propynylcytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (eg 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and Other 8-substituted adeni And guanine, 5-halo (especially 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 [l, 5-a] 1,3,5triazinone, 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 produces a nucleotide sequence that is non-reactive but has a base that is nearly equivalent, i.e., a base that does not react with other proteins (i.e., transcriptases), and therefore the effect of sequence information is It may be useful to be able to be separated from the structural effects of the base.

(analog)
The present invention provides nucleotide analogs having the formula, NTP-linker-inhibitor, for synthesizing polynucleotides in an aqueous environment. With respect to analogs of the form, NTP-linker-inhibitor, the 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 tri Phosphate (dUTP).

  The linker can be any molecular moiety that links the inhibitor to the NTP and can be cleaved (eg, can be chemically cleaved, electrochemically cleaved, or cleaved enzymatically). Or can be cleaved by photolysis). 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 given temperature but inactivated at another temperature. In some embodiments, the linker comprises a disulfide bond.

  The linker may be attached, for example, at cytosine N4, thymine N3 or O4, guanine N2 or N3, and adenine N6, or uracil N3 or O4. This is because carbon binding causes the presence of the remaining wound after removal of the polymerase inhibitory group. The linker is typically on the order of at least about 10 long, such as at least about 20 long, such as at least about 25 long, thus making the inhibitor sufficiently far away from pyridine or pyrimidine, Makes it possible to link the NTP to the polynucleotide chain via a linked sugar skeleton. In some embodiments, they are self-cyclizing in that the cleavable linkers form a ring molecule that is particularly non-reactive with the growing nucleotide chain.

  The nucleotide analog can include any moiety linked to the NTP that inhibits subsequent binding of the nucleotide by the enzyme. The inhibitory group can be a charged group (eg, a charged amino acid) or the inhibitory group can be a charged group depending on the ambient conditions. In some embodiments, the inhibitor is a negatively charged moiety or may include a moiety that can be negatively charged. In other embodiments, the inhibitor group is positively charged or may be positively charged. In some other embodiments, the inhibitor is an amino acid or amino acid analog. The inhibitor comprises 2 to 20 units of amino acid or analog peptide, 2 to 10 units of amino acid or analog peptide, 3 to 7 units of amino acid or analog peptide, 3 to 5 units of peptide It can be an amino acid or an analog peptide. In some embodiments, the inhibitor is Glu, Asp, Arg, His, and Lys, and combinations thereof (eg, Arg, Arg-Arg, Asp, Asp-Asp, Asp, Glu, Glu-Glu, Asp). -A group selected from the group consisting of Glu-Asp, Asp-Asp-Glu or AspAspAspAsp). Peptides or groups can be the same or different amino acid or analog combinations. The inhibitory group may also include a group that reacts with a residue in the active site of the enzyme and thus prevents subsequent binding of the nucleotide by the enzyme.

  An example of a nucleotide analog of the above type, NTP-linker-inhibitor is shown in FIG. 1A. The analog in FIG. 1A contains an inhibitory (-Asp-Asp-) group linked to the N4 position of dCTP via a disulfide (-SS-) bond, while being unblocked, unmodified 3'-OH is provided on the sugar ring. The linker is constructed so that all linker atoms (including the second integration inhibiting moiety) can be removed, thereby allowing the nascent DNA strand to revert to natural nucleotides. As shown in FIG. 1B, aqueous reducing agents (eg, tris (2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT) cleave the —S—S— bond resulting in loss of inhibitor function ( 1B, a cyclic oxidized tetrahydrothiophene that can incorporate a self-cyclizing linker and is easily removed from the reagent solution as a result of nucleotide synthesis, as shown in FIG. A leaving group is formed.

An exemplary scheme for synthesizing the dCTP analog of FIG. 1A is shown below:

  In a manner similar to FIG. 1, the nucleotide analog of the above type, NTP-linker-inhibitor, also has a linker-inhibitor moiety at N6 of adenine (FIG. 2), N2 of guanine (FIG. 3), and N3 of thymine. (FIG. 4) or can be formed by conjugation to N3 of uracil (FIG. 5), thereby providing “naturally occurring” dNTPs, as well as analogs of deoxyuracil nucleotides (dUTP). Although it is unlikely that there will be a wide range of uses for dUTP, its synthesis is simple based on chemistry.

Although the present invention is not limited to the linking chemistry of Scheme 1, carbamates, amides, or other self-deleted linkages can also be used. For example, nucleotides can also be prepared with Staudinger linkers as shown in Scheme 2.

  A deoxycytidine triphosphate (dCTP) analog made with a Staudinger linker (Scheme 2) to an Asp-Asp blocking group is shown in FIG. As shown in FIG. 6, the Staudinger dCTP analog is cleaved by the addition of azide and triphenylphosphine under aqueous conditions. The Staudinger analog shown in FIG. 6 is also suitable for nucleotide extension using nucleotidyl transferase (eg, TdT), as described above and illustrated in FIGS. Although not explicitly shown in the figure, those skilled in the art will be able to generate schemes with appropriate reactants to generate other nucleotide analogs with Staudinger linkers when required for complete de novo nucleotide synthesis. 2 can be used. In a manner similar to FIG. 6, the nucleotide analog of Scheme 2 attaches a Staudinger moiety to N6 of adenine, N2 of guanine, N3 of thymine, or N3 of uracil, thereby “naturally”. It can be formed by providing “present” dNTPs, as well as analogs of deoxyuracil nucleotides (dUTP).

  The methodology of Scheme 1 can be used to generate the corresponding ribonucleotide analog by starting with an appropriate ribonucleotide reactant, for example, as shown in FIGS. Ribonucleotide analogs containing the above Staudinger linkers are also made using Scheme 2 to form the required ribonucleotide analogs, including, for example, CTP analogs as shown in FIG. Can be done. Furthermore, all of the above ribonucleotide analogs, ie, C, A, T, G, U, can be formed using a reaction similar to Scheme 2.

(enzyme)
The methods of the invention use nucleotidyl transferase to construct 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 Ribonucleotides and deoxyribonucleotides are polymerized at approximately the same rate.

Of particular importance to the present invention, transferases with polymerase activity (eg, terminal deoxynucleotidyl transferase (TdT)) catalyze the addition of deoxyribonucleotides to the 3 ′ end of the nucleotide strand, thereby producing DNA nucleotides. Can increase the chain length. TdT only catalyzes the addition of 1-2 ribonucleotides to the growing end of the DNA strand and may be useful in the construction of site-specific DNA-RNA chimeric polynucleotides. In particular, calf thymus TdT (obtained from engineered E. coli) is suitable for use in the present invention and is available from commercial sources such as Thermo Scientific (Pittsburgh, PA). The amino acid sequence corresponding to calf TdT is listed in Table 1 as SEQ ID NO: 1.

The nucleotide sequence corresponding to calf TdT is listed in Table 2 as SEQ ID NO: 2.

  Commercially available TdT is suitable for use in the methods of the invention, while modified TdT (eg, has an amino acid sequence that is at least 95% in common with SEQ ID NO: 1, for example, an amino acid that is at least 98% in common with SEQ ID NO: 1). Can also be used in the methods of the present invention (eg, having an amino acid sequence at least 99% in common with SEQ ID NO: 1). An organism expressing a suitable nucleotidyl transferase may comprise a nucleic acid sequence that is at least 95% in common with SEQ ID NO: 2, for example at least 98% in common with SEQ ID NO: 2, for example at least 99% in common with SEQ ID NO: 2. In some cases, modified TdT results in more efficient production of polynucleotides or allows better control of chain length. Other modifications to the TdT can alter the release characteristics of the enzyme, thereby reducing the need for a good aqueous reducing agent such as TCEP or DTT.

For the synthesis of RNA polynucleotides, E.I. A nucleotidyl transferase, such as E. coli poly (A) polymerase, can be used to catalyze the addition of ribonucleotides to the 3 ′ end of the ribonucleotide initiator. In other embodiments, E.I. E. coli poly (U) polymerase may be more suitable for use in the methods of the invention. E. E. coli poly (A) polymerase and E. coli. Both E. coli poly (U) polymerases are available from New England Biolabs (Ipswich, Mass.). E. E. coli poly (A) polymerase and E. coli. The amino acid and nucleotide sequences of E. coli poly (U) polymerase are reproduced below. Modification E. E. coli poly (A) polymerase or E. coli. E. coli poly (U) polymerase may be suitable for use in the methods of the invention. For example, an enzyme having an amino acid sequence at least 95% in common with SEQ ID NO: 3, for example, having an amino acid sequence at least 98% in common with SEQ ID NO: 3, for example having an amino acid sequence at least 99% in common with SEQ ID NO: 3, It can be used in the method of the present invention. An organism that expresses a suitable enzyme may comprise a nucleic acid sequence that is at least 95% in common with SEQ ID NO: 4, for example at least 98% in common with SEQ ID NO: 4, for example at least 99% in common with SEQ ID NO: 4. Alternatively, an enzyme having an amino acid sequence that is at least 95% common with SEQ ID NO: 5, such as having an amino acid sequence that is at least 98% common with SEQ ID NO: 5, such as having an amino acid sequence that is at least 99% common with SEQ ID NO: 5, It can be used in the method of the present invention. An organism that expresses a suitable enzyme may comprise a nucleic acid sequence that is at least 95% in common with SEQ ID NO: 6, for example at least 98% in common with SEQ ID NO: 6, for example at least 99% in common with SEQ ID NO: 6.

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

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

As discussed above, the inhibitor bound to the nucleotide analog prevents the transferase (eg, TdT) from being released from the polynucleotide, or other analogs are incorporated into the growing strand. Do not let it. Charged moieties give better inhibition, but studies suggest that the specific chemistry of the inhibitor is not particularly important. For example, both phosphate and acidic peptides can be used to inhibit enzyme activity. For example, Bowers et al. , Nature Methods, vol. 6, (2009) p. See 593-95 and US Pat. No. 8,071,755, both of which are hereby incorporated by reference in their entirety. In some embodiments, the inhibitor comprises a single amino acid or a dipeptide such as-(Asp) ₂ , but its size and the charge of its portion, if necessary, incorporate the first nucleotide and Adjustments can be made based on empirically determined rates of incorporation of the second nucleotide. That is, other embodiments may use more charged or differently charged amino acids or other biocompatible charged molecules.

  Other methods of nucleotide synthesis can be used to construct de novo oligonucleotides in a template-independent manner using nucleotidyl transferase or modified nucleotidyl transferase. In one embodiment, the polymerase / transferase enzymes can be modified such that when they encounter a modification to the phosphate of a 3 'unmodified dNTP analog, they cease nucleotide addition. This scheme requires a deblocking reagent / reaction that modifies the phosphate terminus of the nucleotide analog, releasing the nascent strand for subsequent nucleotide incorporation. Preferred embodiments of this approach use nucleotide analogs that are modified only with phosphates (α, β or γ), but the purine / pyrimidine base modifications of the nucleotides are acceptable.

Another embodiment for using non-template dependent polymerase / transferase enzymes uses protein engineering or protein evolution to bind tightly and remain inactive against the nascent strand after each single nucleotide incorporation. Yes, thus modifying the enzyme to prevent any subsequent incorporation until such time that the polymerase / transferase is released from the strand by use of a release reagent / condition. Such modifications are selected to allow the use of natural unmodified dNTPs instead of the reversible terminator dNTPs. The release reagent can be a high salt buffer, a denaturing agent, and the like. Release conditions can be high temperature, agitation, and the like. For example, mutations to the loop 1 and SD1 regions of TdT have been shown to dramatically change activity from template-independent activity to rather template-dependent activity. Specific mutations of interest include, but are not limited to, Δ ₃ 384/391/392, del loop 1 (386 → 398), D339A, F401A, and Q402K403C404 → E402R403S404. Other means of achieving the goal of a TdT enzyme that binds tightly after incorporation may include mutations to residues responsible for binding of the initiator chain to the three phosphates, including K261, R432, and R454, It is not limited to these.

  Another embodiment for using a non-template dependent polymerase / transferase enzyme uses protein engineering or protein evolution to modify the enzyme to accept a highly efficient 3-block reversible terminator That is. Most naturally occurring polymerase / transferase enzymes do not incorporate a 3'-blocked reversible terminator due to steric constraints in the active site of the enzyme. Modification of either a single or several aa residues in the active site of the enzyme results in a 3'-block to the initiator bound to the support in a process completely similar to that described above. A very efficient integration of the reversible terminator produced can be possible. After incorporation, the 3′-reversible terminator is removed with a deblocking reagent / condition, thus allowing a fully natural (unwound) single-stranded molecule ready for a subsequent controlled extension reaction. Generate. Several residues near the 3'-OH of the incoming dNTP explain the nature of TdT to incorporate ribonucleotide triphosphates as easily as deoxyribonucleotide triphosphates; Residues including but not limited to those in between (especially R334), loop 1, and between α13 and α14 (especially R454) are probably 3′-reversible terminator groups Is a target for mutagenesis to accommodate bulk of and to allow their efficient incorporation. Another embodiment for using a template-dependent polymerase uses either 3 ′ blocked or 3 ′ unblocked dNTP analogs and multiple primer-template pairs bound to a solid support. It is.

Another embodiment for using a non-template dependent polymerase / transferase enzyme modifies the enzyme to optimize the use of each of four different nucleotides or even different modified nucleotide analogs in an analog specific manner. Protein engineering or protein evolution can be used to do this. Nucleotide-specific or nucleotide analog-specific enzyme variants are engineered to have desirable biochemical attributes such as reduced K _m or increased addition rate that further reduces the cost of synthesis of the desired polynucleotide. Can be done.

(Solid phase synthesis)
Although the methods of the invention can be performed under a variety of reaction conditions, the ordered construction and recovery of the desired polynucleotide in most cases requires a solid support on which the polynucleotide can grow. In some embodiments, the method includes enzyme-mediated synthesis of a polynucleotide on a solid support, as illustrated in FIG. When used with the cleavable terminator nucleotide triphosphate (NTP) analogs discussed above, the specific polynucleotide sequences of DNA and RNA, for example, by using TdT or poly (A) polymerase in an aqueous environment. It is possible to build. As shown in FIG. 13, the TdT can be used to provide a step-by-step construction of custom polynucleotides by extending the polynucleotide sequence in a step-wise manner. As discussed above, the inhibitor group of each NTP analog stops the enzyme by the addition of nucleotides. After each nucleotide extension step, the reactant is washed out of the solid support before removing the inhibitor by cleaving the linker, then a new reactant is added and the cycle is started again. . As a result of n cycles of extension-removal-deblocking-washing, the final full-length single-stranded polynucleotide can be completed and cleaved from the solid support and subsequently used in applications such as DNA sequencing or PCR. To be recovered. Alternatively, the final full length single stranded polynucleotide can remain bound 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, resulting in the synthesis of a double stranded polynucleotide.

  Solid supports suitable for use in the methods of the present invention can include glass and silica supports comprising beads, slides, pegs, or wells. In some embodiments, the support can be tethered to another structure, such as a polymer well plate or a pipette tip. In some embodiments, the solid support may have additional magnetic properties, thus allowing the support to be manipulated or removed from a location using a magnet. In other embodiments, the solid support can be a silica-coated polymer, thereby allowing the formation of various structural shapes useful for automated processing.

(Synthesizer)
To take advantage of the efficiency of the disclosed method, an aqueous phase DNA synthesizer can be constructed to produce the desired polynucleotide in substantial quantities. In one embodiment, the synthesizer comprises the described NTP analog reagents, i.e., four wells of dCTP, dATP, dGTP, and dTTP, and TdT at a concentration sufficient to effect polynucleotide growth. Multiple starting sequences can be coupled to a solid support designed to be repeatedly immersed in each of the four wells using, for example, an experimental robot. The robot rinses the solid support in wash buffer during nucleotide addition, cleaves the linking group by exposing the support to a deblocking agent, and washing the solid support twice. Can then be further programmed to move the solid support to the next desired nucleotide well. With simple programming, it is possible to produce useful quantities of the desired nucleotide sequence in a matter of hours and with substantially reduced hazardous waste. Ongoing synthesis under carefully controlled conditions allows the synthesis of polynucleotides having a length of thousands of base pairs. Upon completion, the extension products are released from the solid support and they can then be used as the final nucleotide sequence.

  Highly parallel embodiments may consist of a series of initiator-solid supports on pegs in either 96-well format or 384-well format, which index the pegs in a controlled manner. It can be individually retracted or lowered to contact the liquid in the well. The synthesizer is therefore a randomly addressable peg device, four enzyme-dNTP analog reservoirs in the same format (96 or 384 spacing) as the peg device, and the same format (96 or 384) as the peg device. Additional reagent reservoirs (washing, deblocking, etc.) and transporting the peg device from one reservoir to another with user-programmable control but in a random access manner It can consist of a mechanism (eg, an experimental robot). Care must be taken to avoid contamination of each of the four enzyme-dNTP reservoirs. This is because the contents are reused throughout the synthesis process to reduce the cost of each polynucleotide synthesis.

  In alternative embodiments, the reagents (eg, nucleotide analogs, enzymes, buffers) are moved between solid supports, allowing the reagents to be reused. For example, a reservoir and pump system moves four different nucleotide analog solutions, wash buffers, and / or reducing agent solutions between one or more reactors in which the oligonucleotides are formed. obtain. The reactor and pump can be conventional or the device can be constructed using a microfluidic device. Due to the non-hydrous (aqueous) nature of this process, no special attention needs to be paid to the design of the hardware used to eliminate exposure to water. This synthesis process can only occur with precautions to control losses due to evaporation. Highly parallel embodiments can consist of an integral series of initiator-solid supports on pegs in either a 96-well format or a 384-well format that can be joined to a series of wells in the same balanced manner. Each well is actually a reaction chamber supplied by four enzyme-dNTP analog reservoirs with appropriate valves and additional reagent reservoirs (washing, deblocking, etc.). Equipment is made with fluid engineering logic to recover the enzyme-dNTP reactant in a clean manner after each extension reaction. Because they are reused throughout the synthesis process to reduce the cost of each polynucleotide synthesis. In other embodiments, the pipette tip system can be used to add and remove reagents.

  After synthesis, the released extension product can be analyzed by high resolution PAGE to determine if the initiator has extended the expected number of bases compared to the control. A portion of the recovered synthetic DNA can also be sequenced to determine whether the synthesized polynucleotide is of the expected sequence.

  Since the above synthesizer is relatively simple and does not require toxic components required for phosphoramidite synthesis, the synthesizer of the present invention is easy to use widely for research institutions, biotechnology, and hospitals. Furthermore, since the reagent can be reused / reused, the generated waste is reduced and the cost of consumables is reduced. We anticipate that the above methods and systems are useful in many applications such as DNA sequencing, PCR, and synthetic biology.

(Incorporation by reference)
References and citations to other documents (eg, patents, patent applications, patent publications, scientific journals, books, papers, web content) were made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

(Equivalent)
In addition to what is shown and described herein, various modifications of the invention and many further embodiments thereof are described in this document, including references to the scientific and patent literature cited herein. It will become apparent to those skilled in the art from the entire contents of The subject matter herein includes important information, examples and guidance that can be adapted in the various embodiments and equivalents thereof for the practice of the invention.

Claims (42)

A method for synthesizing an oligonucleotide comprising the steps of:
Exposing a nucleic acid bound to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template;
Wherein the nucleotide analog comprises an unmodified 3 ′ hydroxyl and a cleavable end group that blocks the nucleotidyl transferase but generates a nucleotide substrate for the nucleotidyl transferase upon cleavage. The method of claim 1, wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar. The method of claim 1, wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. 2. The method of claim 1, wherein the nucleotidyl transferase comprises a protein sequence that is at least about 90% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 2. The method of claim 1, wherein the nucleotidyl transferase is derived from an organism and has a nucleotide sequence that is at least about 90% identical to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. 2. The method of claim 1, wherein the cleavable end group inhibits the incorporation of a second nucleotide analog. The method of claim 1, wherein the cleavable end group comprises a charged moiety. The method of claim 7, wherein the valence charged portion is negatively charged. The method of claim 1, wherein the cleavable terminal group comprises an amino acid. The nucleotide analog has the following structure:

Has a where n = 2 or 3, -X- is, -O -, - S -, - NH-, or -CH ₂ - in which A method according to claim 1. The nucleotide analog has the following structure:

Wherein n = 2 or 3, and -X- is -O-, -S-, -NH-, or -CH2-. The nucleotide analog has the following structure:

The method of claim 1, comprising: The nucleotide analog has the following structure:

The method of claim 1, comprising: The cleavable end group is chemically cleavable, photolytically cleavable, electrochemically cleavable, or biologically cleavable. The method described in 1. The method of claim 1, wherein the nucleic acid bound to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5. The method of claim 1, wherein the nucleic acid bound to the solid support is exposed to the nucleotide analog at a temperature between about 35-39 ° C. in the presence of an aqueous solution. The method of claim 1, wherein the solid support is a bead, well, or peg. The method of claim 1, wherein the nucleic acid is single stranded. 2. The method of claim 1, wherein the cleavable end group comprises a moiety that forms a cyclic byproduct when cleaved from the nucleotide analog. Cleaving the cleavable end group to produce a natural nucleotide; and exposing the natural nucleotide to a second nucleotide analog in the presence of a transferase enzyme and in the absence of a nucleic acid template. Wherein the second nucleotide analog comprises a 3 ′ hydroxyl on the sugar ring and a cleavable end group,
The method of claim 1, further comprising: 2. The method of claim 1, further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme. A system for synthesizing oligonucleotides comprising:
A solid support to which the nucleic acid is bound;
Nucleotidyl transferase enzyme;
A first nucleotide comprising a free 3 'hydroxyl and a blocking group attached to the nucleotide by a cleavable linker;
Including
Wherein a second nucleotide can be linked to the first nucleotide via the 3 ′ position of the first nucleotide by the nucleotidyl transferase until the blocking group is removed by the cleavable linker. I ca n’t
Wherein the system does not include a nucleic acid template. 23. The system of claim 22, wherein the first and second nucleotides independently comprise a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. A method for oligonucleotide synthesis comprising the steps of:
Providing a solid support comprising a plurality of bound nucleic acids in the absence of a nucleic acid template;
The substrate is linked to a nucleotide analog having a nucleotidyl transferase and a blocking group attached via a free 3 ′ hydroxyl group and a cleavable linker, wherein only one nucleotide analog has at least one of the plurality Exposing under conditions such as adding to the member;
Cleaving the cleavable linker; and repeating the exposing step;
Including the method. 25. The method of claim 24, wherein the condition comprises an aqueous solution having a pH between about 6.5 and 8.5. 26. The method of claim 25, wherein the aqueous solution is at a temperature between about 35-39 ° C. 26. The method of claim 25, wherein the nucleotide analog comprising a free 3 'hydroxyl group and a blocking group attached through a cleavable linker is provided as a solution. 28. The method of claim 27, further comprising removing the solution from the substrate. A method for oligonucleotide synthesis comprising the steps of: binding a nucleic acid bound to a support free of a nucleic acid template:
A nucleotide analog comprising a moiety linked to said nucleotide analog by a cleavable linker and having a free 3 'hydroxyl, and a nucleotidyl transferase,
Exposure to the process,
Thereby incorporating the nucleotide analog into a nucleic acid bound to the solid support;
Washing the solid support upon incorporation of the nucleotide analog to remove unincorporated nucleotide analog;
Cleaving the cleavable linker; and repeating the exposing, washing, and cleaving steps to synthesize an oligonucleotide;
Including the method. 30. The method of claim 29, wherein the nucleotide analog comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. The nucleotide analog and the nucleotidyl transferase are present in the same solution, and the solution is substantially reused during the subsequent steps of exposing, washing, and cleaving. 30. The method of claim 29. An apparatus for synthesizing an oligonucleotide having a predetermined sequence in an aqueous environment, the apparatus comprising:
A first, second, third and fourth source of nucleotide triphosphate (NTP) reagent solution and enzyme solution in fluid communication with a solid support, wherein the first, The reagent solution in the second, third and fourth sources is selected from NTP-adenine, NTP-guanine, NTP-cytosine, NTP-thymine, and variants of any of the above. A first, second, third and fourth source. 35. The apparatus of claim 32, wherein at least a portion of the nucleotide triphosphate comprises an unmodified 3 'hydroxyl and a cleavable end group that upon cleavage generates a natural nucleotide. 35. The apparatus of claim 32, wherein the nucleotide is a deoxynucleotide. 36. The apparatus of claim 32, further comprising a wash reservoir in fluid communication with the solid support. 35. The apparatus of claim 32, further comprising a source of aqueous deblocking drug in fluid communication with the solid support. 35. The apparatus of claim 32, wherein the nucleotide triphosphate reagent is flowed to the solid support. 38. The apparatus of claim 37, wherein the reagent solution is also flushed from the solid support. 40. The apparatus of claim 38, wherein the reagent solution is substantially reused after being flushed from the solid support. 35. The apparatus of claim 32, wherein the solid support is transferred to a source of nucleotide triphosphate reagent solution / enzyme solution. 41. The apparatus of claim 40, further comprising a programmable manipulator configured to move the solid support. 35. The apparatus of claim 32, further comprising a well for holding a source of nucleotide triphosphate reagent / enzyme.

Images