Variants of family a DNA polymerase and uses thereof
Inventors
Ybert, Thomas • Champion, Elise • Vivar, Omar • Said, Ahmed
Assignees
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Publication Number
US-11390856-B2
Publication Date
2022-07-19
Expiration Date
Abstract
The present invention relates to variant of Family A polymerases able to synthesize a nucleic acid fragment without template and to incorporate a reversible modified terminator nucleotide during the nucleic acid fragment synthesis. The present invention further relates to uses thereof for enzymatic synthesis of nucleic acid molecules.
Core Innovation
The invention describes DNA polymerase theta (Pol b8) variants engineered to synthesize nucleic acids without a template. The variants are configured to incorporate reversible modified terminator nucleotides, including 33O-modified reversible terminator nucleotides, into a growing nucleic acid fragment.
A key concept is the reversible terminator modified nucleotides, including 33O-amino and 33O-modified reversible terminator nucleotide chemistries such as 33O-azidomethyl and 33O-allyl. The document provides the rationale for using reversible terminators and describes how the variants are expected to support controlled nucleic-acid synthesis by combining terminator incorporation with reversibility.
The variants are further specified by mutation motifs/positions, including residues proximal to the 33O extremity and the catalytic triad residues D2330, D2540, and E2541 with exclusions for substitutions at those catalytic triad positions. The document also outlines a use framework that includes nucleic-acid enzymatic synthesis by template-independent de novo synthesis and controlled cyclic addition/deprotection.
Claims Coverage
The partial content provides one independent claim. The claim covers a Pol b8 variant defined by sequence identity to a specified reference, a set of substitution combinations at specified amino-acid positions, and explicit exclusions at catalytic triad positions D2330, D2540, and E2541, while also requiring specific functional abilities for template-independent synthesis and incorporation of a 33O-amino reversible modified terminator nucleotide.
Template-independent nucleic acid fragment synthesis by Pol b8 variant
The DNA polymerase theta variant is able to synthesize a nucleic acid fragment without a template.
Incorporation of a 33O-amino reversible modified terminator nucleotide
The DNA polymerase theta variant is able to incorporate a 33O-amino reversible modified terminator nucleotide into a nucleic acid fragment.
Variant defined by sequence identity to SEQ ID NO:02
The DNA polymerase theta variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:02.
Specified substitution combinations at defined positions with catalytic triad exclusions
The DNA polymerase theta variant comprises at least one substitution or a combination of substitutions selected from L2336A, A2328V, Y2387F, E2335G, P2322A, and L2334M (and the enumerated combinations of these substitutions), wherein amino acid positions are numbered by reference to the amino acid sequence of SEQ ID NO:01, and amino acid positions D2330, D2540, and E2541 do not have an amino acid substitution.
Increased ability to incorporate the 33O-amino reversible modified terminator nucleotide
The variant has an increased ability to incorporate the 33O-amino reversible modified terminator nucleotide into a nucleic acid fragment as compared to a DNA polymerase comprising the sequence of SEQ ID NO:01.
Overall, independent claim clm-00001 defines a DNA polymerase theta variant by sequence identity to SEQ ID NO:02, specific allowable substitution combinations at defined positions with explicit catalytic triad non-substitution at D2330, D2540, and E2541, and required functions: template-independent nucleic acid fragment synthesis and incorporation of a 33O-amino reversible modified terminator nucleotide, with an asserted increased incorporation ability versus a DNA polymerase comprising SEQ ID NO:01.
Stated Advantages
The variant has an increased ability to incorporate the 33O-amino reversible modified terminator nucleotide into a nucleic acid fragment as compared to a DNA polymerase comprising the sequence of SEQ ID NO:01.
Documented Applications
Template-independent de novo nucleic acid synthesis and enzymatic synthesis of nucleic-acid fragments using reversible modified terminator nucleotides, including controlled cyclic addition/deprotection concepts.
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