Efficient product cleavage in template-free enzymatic synthesis of polynucleotides
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Publication Number
US-11905541-B2
Publication Date
2024-02-20
Expiration Date
Abstract
The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides that include or enable a step of efficiently cleaving the polynucleotide products from its initiator using an endonuclease V activity and initiator with a 3′-penultimate deoxyinosine.
Core Innovation
The disclosed technology synthesizes a polynucleotide having a predetermined sequence using an initiator and template-independent nucleic acid polymerization. The initiator has a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl, and elongation proceeds by repeated cycles of contacting the initiator or elongated fragments with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase. The template-independent DNA polymerase is terminal deoxynucleotidyl transferase, and elongation forms 3′-O-blocked elongated fragments, followed by deblocking to regenerate elongated fragments having free 3′-hydroxyls until the polynucleotide is formed.
After formation, the polynucleotide is treated with endonuclease V activity to cleave the polynucleotide from the initiator. The cleavage is provided by a prokaryotic endonuclease V, which cleaves an initiator-polynucleotide conjugate 3′ of a terminal nucleotide of the initiator, and the resulting cleaved polynucleotide has a 5′-monophosphate, with optional removal using a phosphatase.
A system is configured for enzymatic synthesis using an initiator attached to a support by a 5′ end, together with terminal deoxynucleotidyl transferase and a 3′-O-blocked nucleoside triphosphate for one or more nucleotides selected from deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The disclosure also describes that endonuclease V recognizes deoxyinosine and cleavage yields a polynucleotide product from the initiator, supporting template-free TdT-based polynucleotide synthesis.
Claims Coverage
The document includes two independent claims, one directed to a method and one directed to a kit. The inventive coverage centers on a deoxyinosine-containing initiator, template-free elongation using terminal deoxynucleotidyl transferase with 3′-O-blocked nucleoside triphosphates and deblocking cycles, and release of the synthesized product using prokaryotic endonuclease V.
3′-Penultimate deoxyinosine initiator with free 3′-hydroxyl
The initiator has a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl.
Template-free TdT elongation with 3′-O-blocked nucleoside triphosphates and deblocking cycles
Repeated cycles contact the initiator or elongated fragments with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase, specifically terminal deoxynucleotidyl transferase, to form 3′-O-blocked elongated fragments and then deblock them to regenerated free 3′-hydroxyl fragments until the polynucleotide is formed.
Prokaryotic endonuclease V cleavage releasing the polynucleotide from the initiator
The polynucleotide is treated with endonuclease V activity to cleave the polynucleotide from the initiator, wherein the endonuclease V activity is provided by a prokaryotic endonuclease V and cleavage occurs 3′ of a terminal nucleotide of the initiator.
Kit configuration with 5′-attached initiator, deoxyinosine, TdT, and 3′-O-blocked nucleoside triphosphate
The kit includes an initiator attached to a support by a 5′ end, having a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl; an endonuclease V capable of cleaving an initiator-polynucleotide conjugate 3′ of a terminal nucleotide of the initiator; terminal deoxynucleotidyl transferase; and a 3′-O-blocked nucleoside triphosphate for one or more of deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
The coverage focuses on combining a deoxyinosine-containing initiator with template-free TdT-based extension using 3′-O-blocked nucleoside triphosphates and deblocking cycles, followed by cleavage or release of the synthesized polynucleotide using prokaryotic endonuclease V. The kit claim defines components configured to perform the same enzymatic synthesis and release.
Stated Advantages
Improved cleavage/synthesis outcomes with Endo V/deoxyinosine-containing initiators compared with USER/deoxyuridine, as described by comparative cleavage efficiency and gel analysis.
Documented Applications
Enzymatically synthesizing a template-free polynucleotide having a predetermined sequence using a specified initiator and an endonuclease V cleavage/release step.
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