DNA glycosylase/lyase and AP endonuclease substrates
A new class of nucleic acid substrates for AP endonucleases and members of the glycosylase/lyase family of enzymes is described. Representatives of each family, the enzymes Nfo and fpg, respectively, cleave nucleic acid backbones at positions in which a base has been replaced by a linker to which a variety of label moieties may be attached. The use of these synthetic substrates embedded within oligonucleotides is of utility in a number of applications.
1. A process of detecting the presence or absence of a target nucleic acid, comprising the steps of:
(a) contacting an oligonucleotide probe to a target nucleic acid, thereby forming a complex between the probe and the target nucleic acid, wherein the probe comprises a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms wherein n is 1-8;
(b) contacting the probe/nucleic acid complex with a nuclease selected from the group consisting of AP endonuclease, DNA glycosylase/lyase and DNA glycosylase, wherein the nuclease can excise the linker from the complex and/or specifically cleave the probe at the dR-O—[C]n nucleotide; and
(c) detecting whether such excision or cleavage has occurred.
2. The process of claim 1 , wherein the target nucleic acid is the product of a nucleic acid amplification reaction.
3. The process of claim 2 , wherein the amplification reaction is monitored either
(i) in real time, or
(ii) at an endpoint for the reaction.
4. The process according to claim 2 , wherein the amplification reaction is
(i) a recombinase polymerase amplification process; or
(ii) a polymerase chain reaction.
5. The process of claim 1 , wherein step (a) comprises either:
(i). hybridizing the probe to the target nucleic acid; or
(ii) contacting the probe with a recombinase to form a recombinase/probe complex and contacting the recombinase/probe complex to the nucleic acid to form the probe/target nucleic acid complex.
6. The process of claim 1 , wherein the nuclease is endonuclease IV or 8-oxoguanine DNA glycosylase.
7. The process of claim 1 , wherein n is 3-6.
8. The process of claim 1 , wherein the probe is blocked at its 3′-end to prevent polymerase extension.
9. The process of claim 1 , wherein the linker is conjugated to a detectable label.
10. The process of claim 9 , wherein the detectable label is a fluorophore and the probe is further conjugated to a quencher, or the detectable label is a quencher and the probe is further conjugated to a fluorophore.
11. The process of claim 10 , wherein the quencher or fluorophore further conjugated to the probe is conjugated at one end of the probe.
12. The process of claim 9 , wherein the detectable label is selected from the group consisting of biotin, digoxygenin, peptide, fluorophore, quencher, antibody and a quantum dot.
13. The process of claim 10 , wherein nuclease activity excises and frees the fluorophore or quencher associated with the dR-O—[C]n residue and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
14. The process of claim 10 , wherein the fluorophore and quencher are separated by 4-6 bases.
15. The process of claim 4 , wherein the recombinase polymerase amplification process comprises:
(i) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;
(ii) contacting the first and second nucleoprotein primers to a double stranded target sequence to form a first double stranded structure at a first portion of the first strand and to form a double stranded structure at a second portion of the second strand so the 3′ ends of the first nucleic acid primer and the second nucleic acid primer are oriented towards each other on a given template DNA molecule;
(iii) extending the 3′ end of the first and second nucleoprotein primers by DNA polymerases to generate first and second double stranded nucleic acids, and first and second displaced strands of nucleic acid;
and repeating (ii) and (iii) until a desired degree of amplification is reached.
16. The process of claim 4 , wherein the recombinase polymerase amplification is carried out in the presence of a crowding agent.
17. A process of cleaving an oligonucleotide in a complex with a nucleic acid, which comprises contacting the oligonucleotide/nucleic acid complex with a nuclease selected from the group consisting of AP endonuclease, DNA glycosylase/lyase and DNA glycosylase, wherein the oligonucleotide contains a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms wherein n is 1-8; and wherein the nuclease specifically cleaves the probe at the dR-O—[C]n nucleotide.
18. The process of claim 17 , wherein the nuclease is endonuclease IV or 8-oxoguanine DNA glycosylase.
19. The process of claim 17 , wherein n is 3-6.
20. The process of claim 17 , wherein the oligonucleotide is blocked at its 3′-end to prevent polymerase extension.
21. The process of claim 17 , wherein the linker is conjugated to a detectable label.
22. The process of claim 9 , wherein the detectable label is selected from the group consisting of biotin, digoxygenin, peptide, fluorophore, quencher, antibody and a quantum dot.
23. The process of claim 17 , wherein the process further comprises the step of contacting the oligonucleotide to the nucleic acid to form a oligonucleotide/nucleic acid complex, and optionally wherein:
(i) the target nucleic acid is the product of a nucleic acid amplification reaction;
(ii) contacting the oligonucleotide to the nucleic acid comprises hybridizing the oligonucleotide to the nucleic acid; or
(iii) contacting the oligonucleotide to the nucleic acid comprises contacting the oligonucleotide with a recombinase to form a recombinase/oligonucleotide complex and contacting the recombinase/oligonucleotide complex to the nucleic acid to form the oligonucleotide/nucleic acid complex.
24. The process of claim 20 , wherein the nucleic acid amplification reaction is
(i) a recombinase polymerase process; or
(ii) a polymerase chain reaction.
25. The process of claim 24 , wherein the recombinase polymerase amplification comprises process comprising the following steps:
(i) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;
(ii) contacting the first and second nucleoprotein primers to a double stranded target sequence to form a first double stranded structure at a first portion of the first strand and to
form a double stranded structure at a second portion of the second strand so the 3′ ends of the first nucleic acid primer and the second nucleic acid primer are oriented towards each other on a given template DNA molecule;
(iii) extending the 3′ end of the first and second nucleoprotein primers by DNA polymerases to generate first and second double stranded nucleic acids, and first and second displaced strands of nucleic acid;
and repeating (ii) and (iii) until a desired degree of amplification is reached.
26. The process of claim 17 , further comprising detecting cleavage of the oligonucleotide, optionally wherein detection is monitored either
(i) in real time, or
(ii) at an endpoint for the reaction.
27. The process according to claim 26 , wherein either
(i) the linker is conjugated to a fluorophore and the oligonucleotide is further conjugated to a quencher; or
(ii) the linker is conjugated to a quencher and the oligonucleotide is further conjugated to a fluorophore; and optionally wherein nuclease activity excises and frees the fluorophore or quencher associated with the dR-O—[C]n residue and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
28. The process of claim 24 , wherein the recombinase polymerase amplification is carried out in the presence of a crowding agent.