EP-4735621-A1 - NUCLEIC ACID SYNTHESIS ON REUSABLE SUPPORT

EP4735621A1EP 4735621 A1EP4735621 A1EP 4735621A1EP-4735621-A1

Abstract

The present invention relates to methods for synthesizing polynucleotides, particularly on a reusable solid surface. The present invention also relates to a kit for use with said methods, and to methods for storing information.

Inventors

HORGAN, ADRIAN
GODRON, Xavier
FOURNIER, MAXIME
BOUL, Manon

Assignees

DNA Script

Dates

Publication Date: 20260506
Application Date: 20240628

Claims (1)

CLAIMS [Claim 1] Method for synthesizing polynucleotides, comprising : (i) providing at least one anchor nucleic acid (101) immobilized to a solid surface (100); and (ii) repeating cycles of: (a) contacting the anchor nucleic acid (101) with at least one initiator nucleic acid (102) comprising a free 3’-OH end, under conditions such that the initiator nucleic acid (102) and the anchor nucleic acid (101) hybridize over at least a part of their nucleotide sequences; (b) elongating the 3’-OH end of said initiator nucleic acid (102), to produce a synthesized polynucleotide (103) bound to the initiator nucleic acid (102); (c) disrupting the hybridization of the initiator nucleic acid (102) to the anchor nucleic acid (101); and (d) optionally recovering the synthesized polynucleotide (103). [Claim 2] Method according to claim 1 , wherein step (c) comprises applying denaturing conditions to disrupt the hybridization between the initiator nucleic acid (102) and the anchor nucleic acid (101). wherein said denaturing conditions comprise the application of at least one denaturing agent, a pH change and/or heat, preferably wherein the denaturing agent is selected from formamide, an alkylsubstituted amide, urea or a urea-based denaturant, thiourea, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, and their mixtures, and preferably wherein said pH change is generated by application of an electric potential or current at an electrode in the presence of one or more redox agent. [Claim 3] Method according to claim 1 or 2, wherein said initiator nucleic acid (102) comprises at least one cleavable group (106), preferably selected from a chemically-cleavable group, a photocleavable group and an enzymatically-cleavable group, and step (c) comprises cleaving the cleavable group (106) within the initiator nucleic acid (102), thereby breaking the initiator nucleic acid into two or more nucleic acid fragments, preferably further comprising applying denaturing conditions during and/or after the cleavage step to disrupt the hybridization of the cleaved initiator nucleic acid (102) to the anchor nucleic acid (101). [Claim 4] Method according to any one of claims 1 to 3, wherein step (c) comprises contacting the anchor nucleic acid (101) with a displacement nucleic acid (108), under conditions such that said displacement nucleic acid (108) hybridizes to the anchor nucleic acid (101), thereby disrupting the hybridization of the displacement nucleic acid (108) to the anchor nucleic acid (101). [Claim 5] Method of claim 4, wherein said displacement nucleic acid (108) and said anchor nucleic acid (101) comprises complementary sequences of nucleotides, wherein the sequence complementarity between the displacement nucleic acid (108) and the anchor nucleic acid (101) is higher than the sequence complementarity between the initiator nucleic acid (102) and the anchor nucleic acid (101). [Claim 6] Method of any of the preceding claims, wherein the anchor nucleic acid (101) is a singlestranded oligonucleotide and has its 3’-end attached to the solid surface (100), or is a single-stranded oligonucleotide immobilized by hybridization to an intermediate nucleic acid attached by its 5’-end to the solid surface (100), or is a hairpin nucleic acid attached to the solid surface (100). [Claim 7] Method of any of the preceding claims, wherein at step (b) the initiator nucleic acid (102) is elongated via a template-free elongation reaction. [Claim 8] Method of claim 7, wherein said template-free elongation reaction is enzyme-driven. [Claim 9] Method of claim 8, wherein said template-free elongation reaction is performed through the activity of a template-free polymerase, preferably a terminal deoxynucleotidyl transferase. [Claim 10] Method of any of the preceding claims, comprising a further step of amplifying the synthesized polynucleotide (103), thereby producing a plurality of amplified nucleic acids, preferably wherein any reactant incompatible with the amplification is removed before amplification, and optionally further comprising storing the synthesized polynucleotide (103) and/or the amplified nucleic acids. [Claim 11] Method of any one of the preceding claims, comprising a quality control step, comprising controlling that a synthesized polynucleotide (103) has been synthesized, and/or controlling the sequence of synthesized polynucleotide (103), preferably wherein said quality control step comprises hybridizing a nucleic acid probe to the synthesized polynucleotide (103), wherein said nucleic acid probe comprises a detectable moiety, and detecting the nucleic acid probe hybridized to the elongated polynucleotide. [Claim 12] Method of any of the preceding claims, wherein a plurality of different anchor nucleic acids (116, 117, 118) are immobilized at a plurality of sites on the solid surface, wherein at step (a), the plurality of anchor nucleic acids (116, 117, 118)) is contacted with a plurality of different initiator nucleic acids (1 19, 120, 121), wherein each different initiator nucleic acid hybridizes to a single anchor nucleic acid of the plurality of different anchor nucleic acids 116, 117, 118) through a specific hybridization sequence. [Claim 13] Method of claim 12, wherein step (c) comprises site-specifically disrupting the hybridization of the initiator nucleic acids (119, 120, 121) to the anchor nucleic acids (116, 117, 1 18). [Claim 14] Method of any one of the preceding claims, wherein the solid surface (100) is an electrochemical device comprising a plurality of groups of electrodes, wherein each group comprises a plurality of electrodes of the same type and each group is independently addressable, preferably wherein the electrochemical device is a CMOS chip. [Claim 15] Method of claim 14, wherein step (c) comprises applying denaturing conditions to disrupt the hybridization between the initiator nucleic acid (102) and the anchor nucleic acid (101), wherein said denaturing conditions comprise the application of a pH change .wherein said pH change is generated at at least one electrode of a group of electrodes by application of an electric potential or current at said electrode in the presence of one or more redox agent. [Claim 16] Method of claim 14 or claim 15, wherein step (a) comprises the application of an electrical potential or current at at least one electrode of a group of electrodes to perform hybridization of the initiator nucleic acid (102) and the anchor nucleic acid (101). [Claim 17] Method of any of the preceding claims, wherein step (a) is carried out in a phosphate- buffered solution or in an acetate-buffered solution. [Claim 18] Method of any of the preceding claims, wherein step (c) is carried out in a phosphate- buffered solution or in an acetate-buffered solution. [Claim 19] Kit adapted for use in the method of any one of claims 1 to 18, comprising: - an anchor nucleic acid and an initiator acid wherein said anchor nucleic acid and nucleic acids comprise complementary nucleotide sequences capable of being hybridized to one another, preferably wherein said complementary sequences are respectively located at the 5’-end of said first and second nucleic acids, - at least one nucleic acid polymerase, preferably a template-free polymerase such as a terminal deoxynucleotidyl transferase; and - optionally one or more 3'-0-blocked nucleoside triphosphate. [Claim 20] Kit of claim 19, wherein said initiator nucleic acid comprises one or more cleavable group(s) such that cleavage of the cleavable group(s) breaks the initiator nucleic acid into two or more nucleic acid fragments. [Claim 21] Kit of claim 19 or 20, wherein said kit comprises at least one displacement nucleic acid (108) comprising a sequence of nucleotides complementary with the anchor nucleic acid (101), wherein the sequence complementarity between the displacement nucleic acid (108) and the anchor nucleic acid (101) is higher than the sequence complementarity between the initiator nucleic acid (102) and the anchor nucleic acid (101). [Claim 22] Kit of any one of claims 19 to 21 , wherein said kit comprises at least one denaturing agent capable of disrupting the hybridization between the anchor nucleic acid (101) and the initiator nucleic acid (102). [Claim 23] Method for storing information, comprising providing one or more items of information in the form of binary data, converting said binary data into one or more polynucleotide sequences, synthesizing polynucleotides having said polynucleotide sequences according to the method defined in claims 1 to 18, optionally amplifying said polynucleotides and storing said polynucleotides.

Description

NUCLEIC ACID SYNTHESIS ON REUSABLE SUPPORT FIELD OF THE INVENTION The present invention relates to methods for synthesizing polynucleotides, particularly on a reusable solid surface. The present invention also relates to a kit for use with said methods, and to methods for storing information. BACKGROUND Over the past decades, remarkable progress has been achieved in oligonucleotide synthesis, enabling numerous novel applications. Among them, oligonucleotide pool production and DNA data storage offer striking perspectives. However, there is a performance gap, today, between DNA reading and DNA writing. Hence, to make oligonucleotide pool production and DNA data storage feasible in practice, vast improvements need to be done in the synthesis of DNA, particularly in terms of cost, speed and throughput. The cost of using CMOS chips, especially, is an obstacle to the performances of high-throughput DNA synthesis. While taped-out CMOS chips can be produced in large amounts inexpensively, additional steps are required to make the chip ready for use, such as die packaging to protect the die, and connection to a PCB board. To prevent liquid damage, further packaging is also required to isolate the chip surface from the electronic interconnects. These steps add cost and reduce ease of use of the chips for oligonucleotide synthesis. The high-throughput capacity of CMOS chips could be exploited more advantageously in oligonucleotide synthesis if the cost-effectiveness and efficiency of the technology could be improved. SUMMARY OF THE INVENTION The present inventors have developed improved methods for synthesizing polynucleotides. These methods allow to use a synthesis support such as a CMOS chip, for several repeated cycles of synthesis. The reusability of the chips drastically decreases the cost of synthesis and considerably increases the synthesis workflow. By making possible to reuse a chip, the method also provides important waste reductions in the oligonucleotide process. One aspect of the invention therefore relates to a method for synthesizing polynucleotides, comprising: (i) providing at least one anchor nucleic acid immobilized to a solid surface; and (ii) repeating cycles of: (a) contacting the anchor nucleic acid with at least one initiator nucleic acid comprising a free 3’-OH end, under conditions such that the initiator nucleic acid and the anchor nucleic acid hybridize over at least a part of their nucleotide sequences; (b) elongating the 3’-OH end of said initiator nucleic acid, to produce a synthesized polynucleotide bound to the initiator nucleic acid; (c) disrupting the hybridization of the initiator nucleic acid to the anchor nucleic acid; and (d) optionally recovering the synthesized polynucleotide. In some embodiments, the initiator nucleic acid hybridizes to the anchor nucleic acid by complementary sequences of nucleotides respectively located at the 5’-end of initiator nucleic acid and anchor nucleic acid. Said complementary sequence of nucleotide within the initiator nucleic acid may comprise the sequence GCTGTTTCGCGTGACAT (SEQ ID NO:11). In preferred embodiments, only a part of the initiator nucleic acid is hybridized to the anchor nucleic acid. In other words, the initiator nucleic acid comprises a first portion, preferably at its 5’-end, which hybridizes to the anchor nucleic acid and a second portion, preferably at its 3’-end, which does not hybridize to the anchor nucleic acid. For instance, said second portion is not complementary to the anchor nucleic acid. In particular, the initiator nucleic acid comprises at least 1 , more particularly at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-hybridized nucleotides, at its end the most distal from the solid support, preferably at its 3’-terminus. In some embodiments, step (c) comprises applying denaturing conditions to disrupt the hybridization between the initiator nucleic acid and the anchor nucleic acid, wherein said denaturing conditions comprise the application of at least one denaturing agent, a pH change and/or heat. The denaturing agent may be selected from formamide, an alkyl-substituted amide, urea or a urea-based denaturant, thiourea, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol and their mixtures. Preferably, said pH change is generated by application of an electric potential or current at an electrode in the presence of one or more redox agent. In some embodiments, said initiator nucleic acid comprises at least one cleavable group, and step (c) comprises cleaving the cleavable group within the initiator nucleic acid, thereby breaking the initiator nucleic acid into two or more nucleic acid fragments. In some embodiments, said cleavable group is located within the portion of the initiator nucleic acid which hybridizes to the anchor nucleic acid. Said cleavable group may be selected from a chemically-cleavable group, a photocleavable group and an enzymatically-cleavable group. It is preferably a photocleavabl