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of each family, the enzymes Nfo and fpg, respectively, cleave nucleic acid backbones at positions in which a base has been replaced by a linker to which a variety of label moieties may be attached.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. An oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising:
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 said oligonucleotide contains a fluorophore-quencher pair separated by 10 nucleotides or less,
wherein the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8.
2. The probe of claim 1 , wherein n is 3-6.
3. The probe of claim 2 , wherein n is 6.
4. The probe of claim 1 , wherein the fluorophore and quencher are separated by 4-6 bases.
5. The probe of claim 1 , wherein the fluorophore or quencher not conjugated to the dR-O—[C]n nucleotide is conjugated at one end of the probe.
6. The probe of claim 5 , wherein the conjugated end of the probe is the 5′-end.
7. A kit, comprising
(i) an oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising 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 the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8; and
(ii) 8 oxoguanine DNA glycosylase (fpg).
8. The kit of claim 7 , wherein n is 3-6.
9. The kit of claim 7 , wherein n is 6.
10. A reaction mixture comprising
an oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising 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 the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8; and
8-oxoguanine DNA glycosylase (fpg).
11. The reaction mixture of claim 10 wherein n is 3-6.
12. The reaction mixture of claim 11 , wherein n is 6.
13. The reaction mixture of claim 10 , wherein the reaction mixture is lyophilized.
14. The reaction mixture of claim 10 , further comprising a container.
15. The reaction mixture according to claim 14 , wherein the container is a tube.
16. The reaction mixture of claim 10 , further comprising a target nucleic acid that contains a sequence complementary to the oligonucleotide.