Variants of family a DNA polymerase and uses thereof
Inventors
Ybert, Thomas • Champion, Elise • Vivar, Omar • Said, Ahmed
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12173333-B2
Publication Date
2024-12-24
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 relates to a Family A DNA polymerase variant engineered to synthesize nucleic acid without template. The variant is defined as an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and includes at least one amino acid modification compared to SEQ ID NO: 2. The variant comprises a substitution at a position corresponding to E2335, where positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, including an E2335G mutation.
The described variants further specify additional amino acid modification sets at defined residue positions using SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, with numbering referenced to SEQ ID NO: 1. The modification sets include catalytic-triad related constraints at positions corresponding to D2330, D2540, and E2541, including exclusions of specific substitutions at D2540 and E2541. Additional residues used in the defined mutation combinations include K2181, R2315, F2359, and A2477.
The invention also defines use of the Family A DNA polymerase variants to quantitatively incorporate reversible modified terminator nucleotides, including 3′O-modified reversible terminator nucleotides. The reversible modified terminator nucleotides are described as 3′-blocked nucleotides, and the document specifically references 3′O-amino-2′,3′-dideoxyadenosine-5′-triphosphate (ONH2) and a 3′-O-amino reversible terminator. The content further describes nucleotide incorporation reaction contexts that include an initiating fragment and solid support, and references enzymatic usage in controlled cycle contexts with downstream cleaving/deprotection steps.
The background problem addressed is the need for template-independent nucleic acid synthesis and quantitative incorporation of reversible modified terminator nucleotides. Structural analysis and an elongation assay are referenced to support the performance of the engineered polymerase variants, including an elongation assay using [32P]-labeled primers and 3′O-modified reversible terminator nucleotides with performance results referenced in FIG. 2.
Claims Coverage
Independent claim coverage includes two independent claim groups: a Family A DNA polymerase variant defined by sequence identity and E2335G, and a kit for a nucleotide incorporation reaction using a Family A DNA polymerase and nucleotides. Dependent claims add further, enumerated amino-acid substitutions at specific SEQ-ID-numbered positions and relate to nucleic acids encoding the variants.
Family A DNA polymerase variant with E2335G substitution
A variant of a DNA polymerase of family A, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, with at least one amino acid modification as compared to SEQ ID NO: 2, wherein the variant comprises a substitution at a position corresponding to E2335 and includes an E2335G mutation (positions numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1).
Kit for nucleotide incorporation reaction using Family A DNA polymerase and nucleotides
A kit for performing a nucleotide incorporation reaction that includes a DNA polymerase of family A and one or more nucleotides.
Across the provided independent claims, protection centers on a Family A DNA polymerase variant defined by sequence identity to SEQ ID NO: 2 and a specific E2335G substitution, with further dependent claim constraints at additional SEQ-ID-numbered residues, and on use of the Family A DNA polymerase in a kit for a nucleotide incorporation reaction including one or more nucleotides.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?