COMPOSITIONS AND METHODS FOR NUCLEIC ACID EXTENSION
The present disclosure relates to a deblocking agent and compositions thereof for cleaving an NO bond in an amineoxy group at the 3′ position of a non-extendable polynucleotide, the deblocking agent being a carbonyl phosphonate and/or sulfonate, or salt thereof. The present disclosure further relates to methods of activating and/or extending a non-extendable polynucleotide using the deblocking agent of the present disclosure. The present disclosure also relates to methods of sequencing a target nucleic acid comprising the methods of activating a non-extendable polynucleotide of the present disclosure.
1 .- 64 . (canceled)
65 . A method of extending a non-extendable polynucleotide comprising
contacting the non-extendable polynucleotide with a deblocking agent, the non-extendable polynucleotide having an amineoxy group at the 3′ position, to cleave an NO bond of the amineoxy group to obtain an extendable polynucleotide, and
extending the extendable polynucleotide in a nucleic acid polymerization,
wherein the deblocking agent is a carbonyl diphosphonate.
66 . The method of claim 65 , wherein the polymerization is a template-dependent polymerization, wherein the non-extendable polynucleotide is a primer hybridized to a template polynucleotide.
67 . The method of claim 65 , wherein the polymerization is a template-independent polymerization by a terminal deoxynucleotidyl transferase.
68 . The method of claim 65 , wherein the extending is carried out with a non-extendable polynucleotide to obtain a second non-extendable polynucleotide, wherein the non-extendable polynucleotide comprises an amineoxy group at the 3′ position of the nucleotide, and wherein the second non-extendable polynucleotide comprises an amineoxy group at the 3′ position of the second non-extendable polynucleotide.
69 . The method of claim 65 , wherein the method further comprises a plurality of cycles of contacting and extending, wherein each extending step forms a further non-extendable polynucleotide that is contacted with the deblocking agent at a later cycle.
70 . The method of claim 69 , wherein each of the plurality of cycles further comprises detecting the respective non-extendable polynucleotide obtained in each extending step.
71 . The method of claim 70 , wherein each of the plurality of cycles further comprises detecting a next cognate nucleotide in a template-based polymerization, wherein the polynucleotide is a primer of a template polynucleotide.
72 . The method of claim 71 , wherein the next cognate nucleotide is a fluorescently labeled nucleotide that is bound in a ternary complex but is not incorporated into the polynucleotide, wherein the ternary complex comprises a polymerase, the polynucleotide primer and template polynucleotide, and the fluorescently labeled nucleotide.
73 . The method of claim 72 , wherein the next cognate nucleotide is a fluorescently labeled nucleotide that is incorporated into the polynucleotide.
74 . The method of claim 65 , wherein the non-extendable polynucleotide comprises a fluorescent tag at the base of the non-extendable polynucleotide, wherein the fluorescent tag is reversibly attached to the base, and wherein the contacting further comprises cleaving an NO bond between the fluorescent the base of the non-extendable polynucleotide.
75 . The method of claim 65 , wherein extending is by a polymerase that incorporates a nucleotide having an amineoxy group at the 3′ position.
76 . The method of claim 65 , wherein the deblocking agent is comprised in a deblocking composition, the deblocking composition comprising the deblocking agent and a buffer.
77 . The method of claim 76 , wherein the deblocking composition has a pH of 4 to 7.
78 . The method of claim 77 , wherein the deblocking composition has a pH of 4.5 to 6.
79 . The method of claim 76 , wherein the buffer comprises or is acetate.
80 . The method of claim 76 , wherein the deblocking solution further comprises a solubility enhancer.
81 . The method of claim 80 , wherein the solubility enhancer is an amine.
82 . The method of claim 81 , wherein the amine is selected from substituted or unsubstituted nitrogen-containing heteroaromatic, substituted or unsubstituted arylamine, alkylamine, substituted or unsubstituted nitrogen-containing heterocycle, and combinations thereof.
83 . The method of claim 82 , wherein the amine is N,N′-dimethylethylenediamine.
84 . The method of claim 83 , wherein the solubility enhancer is present in the deblocking composition at a concentration of about 10 mM to about 50 mM.
85 . The method of claim 65 , wherein a carbonyl of the deblocking agent is an aldehyde.
86 . The method of claim 65 , wherein a carbonyl of the deblocking agent is a keytone.
87 . The method of claim 65 , wherein the deblocking agent is
or a salt thereof.
88 . A method of cyclic polynucleotide extension with a polymerase, comprising:
a) incorporating, by a polymerase, a non-extendable nucleotide at the 3′ end of a polynucleotide, wherein the non-extendable nucleotide comprises an NO bond; and
b) cleaving the NO bond, wherein cleaving is with a carbonyl phosphonate; and
c) repeating steps a) and b) in a plurality of cycles.
89 . The method of claim 88 , wherein the NO bond is of an amineoxy group.
90 . The method of claim 88 , wherein the carbonyl phosphonate is a carbonyl diphosphonate.
91 . The method of claim 88 , wherein the carbonyl diphosphonate is in the presence of an amine counterion.
92 . The method of claim 88 , wherein the polymerase remains bound to the polynucleotide across the cycles of step a) and b).
93 . The method of claim 88 , further comprising detecting a fluorescent nucleotide bound by the polymerase before or after each step a) of incorporating the non-extendible nucleotide, wherein the fluorescent nucleotide is in a stabilized ternary complex with the polymerase and the polynucleotide.
94 . The method of claim 88 , wherein the polymerization is a template-independent polymerization by a terminal deoxynucleotidyl transferase.