Use of phosphorothiolate polynucleotides in ligating nucleic acids
View Patent ↗The invention provides a method of nonenzymatic ligation of a nucleic acid. The method consists of contacting a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between the polynucleotide-3′ phosphorothiolate and the acceptor polynucleotide. The invention also provides methods of molecular cloning. In one embodiment, the method consists of contacting an insert comprising a polynucleotide-3′ phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between the insert and the acceptor vector to generate a vector comprising an insert polynucleotide. The invention further provides a compound consisting of a polynucleotide-3′ phosphorothiolate and a kit containing a polynucleotide-3′ phosphorothiolate. Also provided is a method of ligating a nucleic acid using a non-sequence specific topoisomerase.
1. A method of non-enzymatic ligation of a nucleic acid, comprising contacting a first polynucleotide comprising a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide under conditions that allow nucleophilic attack by a 5′—OH group of the acceptor polynucleotide on the polynucleotide-3′ phosphorothiolate to form a phosphodiester bond between said first polynucleotide, and said acceptor polynucleotide, wherein a phosphodiester bond is formed between said first polynucleotide and said acceptor polynucleotide, whereby a ligated nucleic acid product is generated.
2. The method of claim 1 , wherein said first polynucleotide comprising a polynucleotide-3′ phosphorothiolate comprises a moiety having the formula:
wherein,
X is a nucleotide;
y is a positive integer;
R1 is a nucleotide base;
R2 is a hydrogen atom or hydroxyl; and
R3 is nitrophenyl.
3. The method of claim 1 , wherein said first polynucleotide comprising a polynucleotide-3′ phosphorothiolate further comprises a duplex polynucleotide.
4. The method of claim 1 , wherein said acceptor polynucleotide further comprises a duplex polynucleotide.
5. A method of replicating a ligated nucleic acid product, comprising:
(a) contacting a first polynucleotide comprising a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide under conditions that allow nucleophilic attach by a 5′ —OH group of the acceptor polynucleotide on the polynucleotide-3′ phosphorothiolate to form a phosphodiester bond between said first polynucleotide and said acceptor polynucleotide, wherein a phosphodiester bond is formed between said first polynucleotide and said acceptor polynucleotide to generate a ligated nucleic acid product, wherein one of said first polynucleotide or said acceptor polynucleotide comprises a vector, and
(b) transducing into a host cell said ligated nucleic acid product, wherein said polynucleotide product is replicated in said host cell.
6. A method of non-enzymatic ligation of a nucleic acid, comprising:
(a) contacting a polynucleotide-3′ phosphorothiolate precursor and an activator under conditions sufficient to react said polynucleotide-3′ phosphorothiolate precursor and said activator, wherein said polynucleotide-3′ phosphorothiolate precursor reacts with said activator to produce an intermediate polynucleotide comprising a polynucleotide-3′ phosphorothiolate, and
(b) contacting said intermediate polynucleotide with an acceptor polynucleotide under conditions that allow nucleophilic attack by a 5′—OH group of the acceptor polynucleotide on said polynucleotide-3′ phosphorothiolate to form a phosphodiester bond between said intermediate polynucleotide and said acceptor polynucleotide, wherein a phosphodiester bond is formed between said intermediate polynucleotide and said acceptor polynucleotide, whereby a ligated nucleic acid product is generated.
7. The method of claim 6 , wherein said activator is iodonitrobenzene.
8. A method of ligating a vector and an insert comprising, contacting an insert comprising a polynucleotide-3′ phosphorothiolate with an acceptor vector under conditions that allow nucleophilic attack by a 5′—OH group of the acceptor vector on the polynucleotide-3′ phosphorothiolate of the insert to form a phosphodiester bond between said insert and said acceptor vector, wherein a phosphodiester bond is formed between said insert and said acceptor vector, whereby a ligated product vector comprising said insert is generated.
9. The method of claim 8 , further comprising transforming said vector comprising said insert into a host cell.
10. The method of claim 8 , wherein said polynucleotide-3′ phosphorothiolate comprises a moiety having the formula:
wherein,
X is a nucleotide;
y is a positive integer;
R1 is a nucleotide base;
R2 is a hydrogen atom or hydroxyl; and
R3 is nitrophenyl.
11. A method of ligating a vector and an insert comprising:
(a) contacting a polynucleotide-3′ phosphorothiolate precursor and iodonitrobenzene under conditions sufficient to react said polynucleotide-3′ phosphorothiolate precursor and said iodonitrobenzene, wherein said polynucleotide-3′ phosphorothiolate precursor reacts with said iodonitrobenzene to produce a polynucleotide-3′ phosphorothiolate, and
(b) contacting an insert comprising said polynucleotide-3′ phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between said insert and said acceptor vector, wherein a phosphodiester bond is formed between said insert and said acceptor vector, whereby a ligated product vector comprising said insert is generated.
12. A method of ligating a vector and an acceptor polynucleotide comprising, contacting a vector comprising a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide, under conditions that allow nucleophilic attack by a 5′—OH group of the acceptor polynucleotide on the 3′-phosphorothiolate of the vector to form a phosphodiester bond between said vector and said acceptor polynucleotide, wherein a phosphodiester bond is formed between said vector and said acceptor polynucleotide, whereby a ligated product vector comprising said acceptor polynucleotide is generated.
13. The method of claim 1 , further comprising transforming said vector comprising said acceptor polynucleotide into a hot cell.
14. The method of claim 12 , wherein said polynucleotide-3′ phosphorothiolate comprises a moiety having the formula:
wherein,
X is a nucleotide;
y is a positive integer;
R1 is a nucleotide base;
R2 is a hydrogen atom or hydroxyl; and
R3 is nitrophenyl.
15. The method of claim 12 , wherein said vector further comprises a 3′ phosphorothiolate moiety at one or more terminal ends of said vector.
16. A method of ligating a vector and acceptor polynucleotide comprising:
(a) contacting a vector comprising a polynucleotide-3′ phosphorothiolate precursor with an activator under conditions sufficient to react said polynucleotide-3′ phosphorothiolate precursor and said activator to produce a polynucleotide-3′ phosphorothiolate, wherein said polynucleotide-3′ phosphorothiolate precursor reacts with said activator to produce a polynucleotide-3′ phosphorothiolate, and
(b) contacting said vector comprising said polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide, under conditions that allow nucleophilic attack by a 5′—OH group of the acceptor polynucleotide on the polynucleotide-3′ phosphorothiolate to form a phosphodiester bond between said vector and said acceptor polynucleotide, wherein a phosphodiester bond is formed between said vector and said acceptor polynucleotide, whereby a ligated product vector comprising said acceptor polynucleotide is generated.