Efficient product cleavage in template-free enzymatic synthesis of polynucleotides
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Publication Number
US-11359221-B2
Publication Date
2022-06-14
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 endonuclease V activity and initiator with a 3′-penultimate deoxyinosine.
Core Innovation
The invention provides a method of synthesizing a polynucleotide having a predetermined sequence using a template-free terminal deoxynucleotidyl transferase-like elongation scheme with an initiator and 3′-O-blocked nucleoside triphosphates. The initiator includes a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl, and each elongation cycle forms 3′-O-blocked elongated fragments by incorporation of a 3′-O-blocked nucleoside triphosphate.
The elongated fragments are then deblocked to form elongated fragments having free 3′-hydroxyls until the polynucleotide having the predetermined sequence is formed. After synthesis, the resulting polynucleotide is treated with an endonuclease V activity to cleave the polynucleotide from the initiator, and the cleavage is configured so that the polynucleotide cleaved from the initiator has a 5′-monophosphate.
The document also describes endonuclease V cleavage relying on the presence of deoxyinosine in the initiator region, and it characterizes kit components and initiator/support attachment. Selectable endonuclease V formats are discussed, including a prokaryotic endonuclease V, including E. coli, and optionally an His-tag endonuclease V for removal.
Claims Coverage
The independent claim covers a template-free TdT-like iterative elongation with one core inventive feature set and endonuclease V cleavage that yields a 5′-monophosphate. The described dependent refinements specify the endonuclease V source/format, initiator attachment to a support, and a specific 3′-terminal initiator sequence.
Penultimate deoxyinosine initiator with terminal 3′-hydroxyl
Providing an initiator having a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl.
Iterative TdT elongation with 3′-O-blocked nucleoside triphosphates and deblocking
Repeating cycles of contacting the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a terminal deoxynucleotidyl transferase to form 3′-O-blocked elongated fragments, followed by deblocking to form elongated fragments having free 3′-hydroxyls until the polynucleotide having the predetermined sequence is formed.
Endonuclease V cleavage releasing a 5′-monophosphate
Treating the polynucleotide with an endonuclease V activity to cleave the polynucleotide from the initiator such that the polynucleotide cleaved from the initiator has a 5′-monophosphate.
Prokaryotic endonuclease V
Using endonuclease V activity provided by a prokaryotic endonuclease V.
E. coli endonuclease V
Using a prokaryotic endonuclease V that is an E. coli endonuclease V.
Initiator attached to a support via its 5′ end
Performing the method such that the initiator is attached to a support via its 5′ end.
Specific 3′-terminal initiator sequence 5′-dI-dT-3′
Further defining the initiator such that the initiator includes a 3′-terminal sequence of 5′-dI-dT-3′.
The core coverage centers on iterative terminal transferase elongation with 3′-O-blocked nucleoside triphosphates and deblocking, using a 3′-penultimate deoxyinosine initiator to enable endonuclease V cleavage that yields a 5′-monophosphate, with further refinements to prokaryotic/E. coli endonuclease V, initiator attachment to a support, and a specific 3′-terminal initiator sequence.
Stated Advantages
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