Reusable initiators for synthesizing nucleic acids
Inventors
Efcavitch, J. William • Holden, Matthew T.
Assignees
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Publication Number
US-12168211-B2
Publication Date
2024-12-17
Expiration Date
Abstract
The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. 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.
Core Innovation
The invention provides a method for synthesizing oligonucleotides on a reusable solid support in a template-independent manner. A nucleic acid initiator attached to the solid support is exposed to nucleotide analogs in the presence of a polymerase to create a first oligonucleotide. The nucleic acid initiator comprises a sequence-specific cleavage element, so the resulting oligonucleotide can be released from the initiator by cleavage of that element.
After releasing the first oligonucleotide using a releasing agent that cleaves the sequence-specific cleavage element, the method includes dephosphorylating a 3′-end of the solid support-attached nucleic acid initiator. The sequence-specific cleavage element is then enzymatically renewed, enabling the initiator to be reused. In the disclosed examples, the polymerase includes terminal deoxynucleotidyl transferase (TdT) and/or modified forms, and sequence-specific cleavage elements include poly-U tract configurations.
The method also gates stepwise nucleotide incorporation using nucleotide analogs of the form NTP–cleavable linker–inhibitor, including removable terminating groups and 3′-O-blocked nucleotide analogs. The inhibitor/linker structures are described using cleavable linker chemistries such as disulfide and Staudinger cleavable linkers, with releasing agents that decouple initiator components and allow reuse.
Claims Coverage
The document provides one independent claim covering a reusable solid-support oligonucleotide synthesis workflow using a sequence-specific cleavage element that is cleaved to release product and then renewed for reuse. The claim includes multiple inventive features that collectively define initiator reuse, release by cleavage, 3′-end dephosphorylation, and enzymatic renewal; dependent claims further refine polymerase identity and sequence- or structure-specific elements of the cleavage and nucleotide analogs.
Reusable solid support-bound nucleic acid initiator with sequence-specific cleavage element
A nucleic acid initiator attached to a solid support, wherein the nucleic acid initiator comprises a sequence-specific cleavage element, is exposed to nucleotide analogs in the presence of a polymerase to create a first oligonucleotide.
Releasing agent cleaves sequence-specific cleavage element to release the first oligonucleotide
The method contacts 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 solid support-attached nucleic acid initiator
The method dephosphorylates a 3′-end of the solid support-attached nucleic acid initiator.
Enzymatically renewing the sequence-specific cleavage element
The method enzymatically renews the sequence-specific cleavage element.
Nucleotide-analog gating by cleavable terminating group
The nucleotide analogs include an unmodified 3′-OH and a cleavable terminating group that blocks subsequent nucleotidyl transferase activity until the terminating group is cleaved, yielding a nucleotide substrate for nucleotidyl transferase.
Terminal deoxynucleotidyl transferase (TdT) polymerase
The polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT.
Poly-U tract sequence-specific cleavage element
The sequence-specific cleavage element includes a poly-U tract.
Specified 5′ functional group attachment to the solid support
The nucleic acid initiator uses a specified 5′ functional group attached to a solid support, including one of specified bond/functional group attachment options.
Second sequence-specific cleavage element for releasing two oligonucleotides
The method includes adding a second sequence-specific cleavage element and then using the releasing agent to cleave both cleavage elements and release first and second oligonucleotides.
Overall, claim coverage centers on synthesizing oligonucleotides by exposing a reusable solid support-bound nucleic acid initiator with a sequence-specific cleavage element to nucleotide analogs plus a polymerase, releasing product by cleaving the cleavage element with a releasing agent, dephosphorylating the initiator 3′ end, and enzymatically renewing the cleavage element. Dependent claims refine the polymerase, define nucleotide-analog activity gating via cleavable terminating groups, define cleavage element composition, and expand structure and workflow options such as specified 5′ attachment and use of a second cleavage element to release two oligonucleotides.
Stated Advantages
Reusability of the solid support-bound nucleic acid initiator enabled by enzymatic renewal of the sequence-specific cleavage element.
Template-independent de novo synthesis of nucleic acid using reusable solid-support-bound initiators.
Documented Applications
Template-independent, aqueous de novo DNA/RNA synthesis on a reusable solid support using nucleotide analogs and a polymerase with a sequence-specific cleavage element for release and reuse.
Oligonucleotide synthesis workflows employing quality control using a 3′ exonuclease and recyclable reaction/isolation workflows [procedural detail omitted for safety].
Example synthesizer device concepts for carrying out the method, including microfluidic polynucleotide synthesis device and inkjet/piezoelectric droplet deposition concepts.
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