US-12618092-B2 - Method and apparatus for enzymatic synthesis of polynucleotides

Abstract

The invention is directed to methods and apparatus for parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a template-free polymerase that has sequence-dependent coupling efficiencies. Whenever sequences causing low efficiency coupling occur at a 3′ end of a growing chain of a polynucleotide being synthesized, one or more additional coupling cycles without de-protection steps are inserted into synthesis plans to provide additional time for completing the coupling reaction at that position of the polynucleotide.

Inventors

• Adrian Horgan
• Xavier Godron

Assignees

• DNA SCRIPT

Dates

Publication Date

20260505

Application Date

20210222

Priority Date

20200225

Claims (9)

1. 1 . A method for synthesizing with a template-free polymerase a plurality of polynucleotides each with a predetermined sequence, wherein the template-free polymerase has reduced coupling efficiency at one or more inefficiency motifs, the method comprising the steps of: (a) providing a reaction chamber for each polynucleotide of the plurality of polynucleotides, each reaction chamber having disposed therein a synthesis support with initiators attached, wherein each initiator has a free 3′-hydroxyl, and wherein each reaction chamber has an inlet and an outlet and a filter that retains the synthesis support and that is operationally associated with the outlet so that reaction solutions exiting the reaction chamber pass through the filter; (b) providing a waste manifold operationally associated with the outlets of the reaction chambers so that whenever a positive pressure differential is created between the reaction chambers and the waste manifold, reaction solutions are removed from the reaction chambers; (c) repeating for each reaction chamber, until a polynucleotide of such reaction chamber is complete, cycles of the following reaction steps: (i) contacting in a coupling solution the initiator or a deprotected elongated fragment with a 3′-protected nucleoside triphosphate and a template-free polymerase so that the initiator or the deprotected elongated fragment is elongated by the 3′-protected nucleoside triphosphate to form a 3′-protected elongated fragment, (ii) deprotecting the 3′-protected elongated fragment with a deprotection solution, and (iii) applying a pressure differential between the reaction chambers and the waste manifold to remove reaction solution(s) from the reaction chambers; wherein the kind of 3′-protected nucleoside triphosphate contacted in step (i) in a reaction chamber is determined by the predetermined sequence of the reaction chamber, and wherein, prior to each cycle, one or more short cycles of step (i) is carried out in a reaction chamber whenever an inefficiency motif is present at a 3′ end of the deprotected elongated fragment of such reaction chamber, wherein the inefficiency motif is CCA, CTA, GCA, GTA or CCT.
2. 2 . The method of claim 1 , wherein said cycles and said short cycles have the same duration.
3. 3 . The method of claim 1 , further including a step of cleaving said polynucleotides from said synthesis supports.
4. 4 . The method of claim 1 , wherein said template-free polymerase is a terminal deoxynucleotidyl transferase (TdT).
5. 5 . The method of claim 4 , wherein said TdT is a TdT variant having an amino acid sequence at least 90 percent identical to SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or an amino acid sequence at least 90 percent identical to SEQ ID NO: 16, 17, 18, 19, or 20 and having a Q4E/S/D/N substitution, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template, and (ii) is capable of incorporating a 3′-O-protected-nucleotide onto a free 3′-hydroxyl of a polynucleotide.
6. 6 . The method of claim 1 , wherein protease treatment is performed either in each cycle or periodically during the synthesis process.
7. 7 . The method of claim 1 , further comprising, after completing the synthesis of the polynucleotides, (i) cleaving the completed polynucleotides from the solid supports, and (ii) purifying the completed polynucleotides.
8. 8 . The method of claim 7 , wherein the cleaving and the purifying are performed in the reaction chambers.
9. 9 . The method of claim 7 , wherein the polynucleotides still attached to the solid supports are transferred to other reaction vessels for the cleaving and the purifying.

Description

Interest in enzymatic approaches to polynucleotide synthesis has recently increased not only because of increased demand for synthetic polynucleotides in many areas, such as synthetic biology, CRISPR-Cas9 applications, and high-throughput sequencing, but also because of the limitations of chemical approaches to polynucleotide synthesis, such as upper limits on product length and the use and needed disposal of organic solvents, Jensen et al, Biochemistry, 57: 1821-1832 (2018). Enzymatic synthesis is attractive because of its specificity and efficiency and because of its use of mild aqueous reaction conditions which eliminates the need for handling hazardous wastes. Currently, most enzymatic approaches employ template-free polymerases to repeatedly add 3′-O-blocked nucleoside triphosphates to a single stranded initiator or an elongated strand attached to a support followed by deblocking until a polynucleotide of the desired sequence is obtained. Unfortunately, however, template-free polymerases often have sequence-specific inefficiencies in their coupling yields. That is, the sequence at the 3′ end of a growing strand where nucleotide coupling occurs may affect the coupling efficiency under a given set of reaction conditions and times. This makes it difficult or impossible to obtain uniform product yields in parallel synthesis operations whenever the same reaction times are used for all reaction chambers. In view of the above, parallel synthesis of polynucleotides using template-free polymerases would be advanced if methods and apparatus were available which were capable of minimizing product yield differences due to sequence-specific coupling inefficiencies. SUMMARY OF THE INVENTION The invention is directed to methods and devices, including microfluidic devices, for synthesizing in parallel a plurality of polynucleotides in separate reaction chambers. More particularly, the present invention relates to a method for synthesizing with a template-free polymerase a plurality of polynucleotides each with a predetermined sequence, wherein the template-free polymerase has reduced coupling efficiency at one or more inefficiency motifs, the method comprising the steps of: (a) providing a reaction chamber for each polynucleotide of the plurality, each reaction chamber having disposed therein a synthesis support with initiators attached, wherein each initiator has a free 3′-hydroxyl, and wherein each reaction chamber has an inlet and an outlet and a filter that retains the synthesis support and that is operationally associated with the outlet so that reaction solutions exiting the reaction chamber pass through the filter; (b) providing a waste manifold operationally associated with the outlets of the reaction chambers so that whenever a positive pressure differential is created between the reaction chambers and the waste manifold, reaction solutions are removed from the reaction chambers; (c) repeating for each reaction chamber, until a polynucleotide of such reaction chamber is complete, cycles of the following reaction steps: (i) contacting in a coupling solution the initiator or deprotected elongated fragments with a 3′-protected nucleoside triphosphate and a template-free polymerase so that initiators or deprotected elongated fragments are elongated by the 3′-protected nucleoside triphosphate to form 3′-protected elongated fragments, (ii) deprotecting the 3′-protected elongated fragments with a deprotection solution, and (iii) applying a pressure differential between the reaction chambers and the waste manifold to remove solutions from the reaction chambers; wherein the kind of 3′-protected nucleoside triphosphate contacted in step (i) in a reaction chamber is determined by the polynucleotide sequence of the reaction chamber, and wherein, prior to each cycle, one or more short cycles of step (i) is carried out in a reaction chamber whenever an inefficiency motif is present at a 3′ end of a deprotected elongated fragment of such reaction chamber. As explained more fully below, an efficiency motif is a sequence segment at the 3′ end of an elongated fragment that causes the template-free polymerase to have a reduced efficiency in coupling a nucleotide monomer to the elongated fragment. In some embodiments, template-free polymerases comprise terminal deoxynucleotidyltransferases (TdTs). In some embodiments employing TdTs disclosed herein, inefficiency motifs comprise 3-mer sequences selected from the set comprising CCA, CTA, GCA, GTA and CCT. In other embodiments, such set comprises CCA and CTA. In other embodiments, inefficiency motifs of a template-free polymerase are identified, for example, as described below, so that the method and apparatus of the invention can be implemented. The present invention also relates to an apparatus for synthesizing with a template-free polymerase a plurality of polynucleotides each with a predetermined sequence, wherein the template-free polymerase has reduced coupling efficiency at