Reusable initiators for synthesizing nucleic acids
Inventors
Efcavitch, J. William • Holden, Matthew T.
Assignees
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Publication Number
US-11331643-B2
Publication Date
2022-05-17
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 document describes a template-independent, aqueous enzymatic polynucleotide synthesis approach in which a renewable nucleic acid initiator serves as a substrate. The renewable nucleic acid initiator includes an oligonucleotide with a 5′ end coupled to a solid support, and the initiator further includes a renewable, sequence-specific cleavage element that supports successive incorporation cycles.
The described synthesis is based on nucleotide analog strategies that include nucleotide analogs of the form NTP–linker–inhibitor, where a cleavable linker removes the inhibitor after each incorporation cycle to yield scarless natural nucleotides. Cleavable linker chemistries include disulfide reduction and Staudinger azide/triphenylphosphine cleavage, along with other self-eliminating or linker designs.
The document further describes a renewable initiator concept tied to solid-phase schemes and quality control, including the use of a 3′ exonuclease for quality control and reversible or 3′-blocked nucleotide analog strategies. It also discusses sequence-specific cleavage elements, including internal poly-U USER digestion, as well as solid-phase initiation and release schemes with reusable solid supports and sequence-specific cleavage elements.
Claims Coverage
The independent claim clm-00001 covers a renewable nucleic acid initiator composition for template-independent nucleic acid synthesis, anchored by a solid support and a renewable sequence-specific cleavage element. The independent claim is supported by multiple dependent claim refinements specifying coupling chemistry, irreversibility, order of introducing the cleavage element, enzymatic incorporation, and a poly-U tract cleavage element feature.
Solid support coupled renewable nucleic acid initiator
A composition comprising a solid support and renewable nucleic acid initiator comprising an oligonucleotide having a 5′ end coupled to the solid support.
Renewable sequence-specific cleavage element for template-independent synthesis
The renewable nucleic acid initiator further comprises a renewable sequence-specific cleavage element and serves as a substrate for template-independent nucleic acid synthesis.
Oligonucleotide coupling via defined 5′-end chemistries and linkage chemistries
The oligonucleotide is coupled to the solid support through a linkage formed through specified functional groups and a specified 5′ functional group on the oligonucleotide.
Irreversibly coupled oligonucleotide to solid support
An oligonucleotide is irreversibly coupled to a solid support.
Post-5′ coupling introduction of renewable cleavage element
A renewable sequence-specific cleavage element is added to an oligonucleotide after the 5′ end has been irreversibly coupled to a solid support.
Enzymatic incorporation of the renewable cleavage element
The renewable sequence-specific cleavage element is incorporated enzymatically.
Poly-U tract cleavage element
The renewable sequence-specific cleavage element includes a poly-U tract.
Overall, the claim set centers on a solid support coupled renewable nucleic acid initiator with a renewable sequence-specific cleavage element that functions as a substrate for template-independent nucleic acid synthesis, with further narrowing through defined 5′ coupling chemistries, irreversibility, adding the cleavage element after 5′ coupling, enzymatic incorporation, and a poly-U tract cleavage element.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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