Reagents and methods used in deprotection of 3'-O-amino polynucleotides
Inventors
APARIN, Ilya • SOSKINE, Mikhael • Wu, Weidong • Veillet, Adeline
Assignees
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Publication Number
US-12163172-B2
Publication Date
2024-12-10
Expiration Date
Abstract
This disclosure relates to an enzymatic method of synthesizing a polynucleotide, comprising a deprotecting step which uses as a deprotecting agent a specific phosphonate compound. It also pertains to a method for deprotecting 3′-O-amino elongated fragments of a polynucleotide in an enzymatic method of synthesizing a polynucleotide, comprising contacting the elongated fragments with this phosphonate compound. This disclosure further relates to a kit for synthesizing a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of a phosphonate compound and to a specific method for preparing said phosphonate compound.
Core Innovation
A polynucleotide synthesis method is disclosed in which initiators are polynucleotides having each a free 3′-hydroxyl. The method repeatedly elongates the initiators or elongated fragments by contacting them under elongation conditions with a 3′-O–NH2 nucleoside triphosphate and a polymerase, so that a 3′-O-amino nucleoside triphosphate is incorporated to form 3′-O-amino elongated fragments.
After each elongation cycle, the elongated fragments are deprotected to form elongated fragments having free 3′-hydroxyls by contacting the elongated fragments with an effective amount of at least one phosphonate compound. The phosphonate compound is a compound of formula (I), including carbonylbisphosphonate or carbonyldiphosphonate salts, and is used as a deprotecting agent that replaces sodium nitrite.
The disclosure emphasizes improved nucleobase integrity compared with sodium nitrite, including reduced deamination and oxidation and higher purity, while maintaining polymerase activity across synthesis cycles. It also includes sequencing-by-synthesis or sequencing-by-binding, and in sequencing-by-synthesis the initiators are repeatedly elongated using a fluorescently labelled 3′-O-amino nucleotide and a polymerase, followed by washing, reading a fluorescence signal, removing the fluorescent label and the 3′-O-amino group, and converting the fragments back to free 3′-hydroxyls so that elongation and reading can continue through additional cycles.
Claims Coverage
The provided independent claim family centers on cyclic enzymatic polynucleotide synthesis using 3′-O–NH2 nucleoside triphosphates and phosphonate-compound deprotection. The inventive features are the cyclic 3′-O-amino elongation and phosphonate-based deprotection, with claim refinements covering buffered aqueous conditions, divalent metal selection, template-independent polymerase embodiments including TdT, and sequencing-by-synthesis workflows.
Phosphonate compound deprotection of 3′-O-amino elongated fragments
Deprotecting elongated fragments having 3′-O–NH2 groups to form elongated fragments having free 3′-hydroxyls by contacting the elongated fragments with an effective amount of at least one phosphonate compound, including a compound of formula (I).
Cyclic enzymatic polynucleotide elongation with 3′-O–NH2 nucleoside triphosphate
Repeating cycles of contacting initiators or elongated fragments having free 3′-hydroxyls with a 3′-O–NH2 nucleoside triphosphate and a polymerase so that the initiators or elongated fragments are elongated by incorporation of a 3′-O-amino nucleoside triphosphate to form 3′-O-amino elongated fragments, followed by deprotecting to form free 3′-hydroxyls.
Buffered aqueous effective amount conditions for phosphonate deprotection
Providing the effective amount of the phosphonate compound as a buffered aqueous solution at a pH of from about 4 to about 8, with a phosphonate concentration of about 0.1 to about 500 mM.
Divalent metal inorganic salt selection
Using a divalent metal in the reaction mixture selected from magnesium, calcium, zinc, and copper, optionally using magnesium sulfate as the inorganic salt.
Template-independent polymerase with Terminal Deoxynucleotidyl Transferase
Employing a template-independent polymerase, wherein the template-independent polymerase is Terminal Deoxynucleotidyl Transferase (TdT).
Sequencing-by-synthesis with fluorescently labelled 3′-O-amino nucleotides and phosphonate deprotection
Performing sequencing-by-synthesis with polynucleotide initiators having free 3′-hydroxyl groups, repeatedly elongating the initiators using a fluorescently labelled 3′-O-amino nucleotide and a polymerase, washing away non-incorporated nucleotides, reading fluorescence to determine an incorporated nucleotide, and removing the fluorescent label and the 3′-O-amino group using the phosphonate compound to regenerate free 3′-hydroxyls for continued cycles.
The claim coverage is directed to cyclic enzymatic polynucleotide synthesis in which 3′-O–NH2 nucleoside triphosphates are incorporated under polymerase elongation conditions and the resulting 3′-O-amino elongated fragments are repeatedly converted back to free 3′-hydroxyls using an effective amount of a phosphonate compound. Additional coverage specifies buffered aqueous pH and concentration ranges, divalent metal inorganic salt selections, template-independent polymerase embodiments including TdT, and a sequencing-by-synthesis workflow using fluorescently labelled 3′-O-amino nucleotides followed by phosphonate-based deprotection through sequencing cycles.
Stated Advantages
Improved nucleobase integrity relative to sodium nitrite, including reduced deamination and oxidation and higher purity.
Maintains polymerase activity across synthesis cycles.
Higher cleavage yield.
Documented Applications
Enzymatic DNA/RNA synthesis using the disclosed enzymatic polynucleotide synthesis and phosphonate-based deprotection framework.
Sequencing-by-synthesis using fluorescently labelled incorporation followed by phosphonate-based removal and deprotection across cycles.
Sequencing-by-binding as an application of the disclosed framework.
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