Method to detect activity of a polymerase

Patent metadata

Field	Value
Publication number	US-10883137-B2
Application number	US-201615577028-A
Country	US
Kind code	B2
Filing date	May 30, 2016
Priority date	May 29, 2015
Publication date	Jan 5, 2021
Grant date	Jan 5, 2021

Abstract

The present invention relates to methods for detection of nucleotide polymerase activity and methods of detecting compounds that modulate nucleotide polymerase activity, by detecting product formation of the nucleotide polymerase to be tested based on determination of close proximity of two labeled nucleotide probes able to bind the product of the nucleotide polymerase. It is preferred that proximity dependent energy transfer, such as forster resonance energy transfer, between said labeled nucleotide probes is determined. The invention further provides kits comprising components for carrying out the inventive methods for detection of nucleotide polymerase activity.

First claim

The invention claimed is:

1. A method for detection of nucleotide polymerase activity comprising the following steps:
a) providing a reaction mixture comprising at least said polymerase, an initiator, and nucleoside triphosphates,
b) providing conditions sufficient to allow the polymerase an assembling of nucleotides,
c) stopping polymerase assembling of nucleotides,
d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein said donor nucleotide probe is conjugated to a first non-radioactive label and said acceptor nucleotide probe is conjugated to a second non-radioactive label, wherein said labels allow determination of close proximity of said nucleotide probes,
e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and
f) determining if said donor and acceptor nucleotide probes are in close proximity wherein close proximity correlates with the activity of the nucleotide polymerase
wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and
wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).

2. The method according to claim 1 , wherein the label of the donor nucleotide probe and the label of the acceptor nucleotide probe allow energy transfer which can be determined in step f).

3. The method according to claim 1 , wherein the reaction mixture further comprises a candidate substance to test its effect on the nucleotide polymerase activity.

4. The method according to claim 3 , wherein providing a reaction mixture of step a) comprises the following:
1) contacting said polymerase with the initiator, at least one transcription factor and an appropriate co-factor of the nucleotide polymerase resulting in a first reaction mixture
2) incubating said first reaction mixture
3) adding to said first reaction mixture the candidate substance resulting in a second reaction mixture
4) incubating said second reaction mixture
5) adding to said second reaction mixture nucleoside triphosphates.

5. The method according to claim 1 , wherein the reaction mixture of step a) comprises further at least one transcription factor, replication factor or co-factor of the nucleotide polymerase.

6. The method according to claim 1 , wherein the nucleotide polymerase is selected from the group consisting of DNA dependent DNA polymerase, RNA dependent DNA polymerase, DNA dependent RNA polymerase, RNA dependent RNA polymerase, independent RNA polymerase, and independent DNA polymerase.

7. The method according to claim 1 , wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by covalent bonding or by non-covalent interaction.

8. The method according to claim 1 , wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by non-covalent interaction mediated by biotin covalently coupled to the donor nucleotide probe or respectively the acceptor nucleotide probe and streptavidin covalently coupled to the label or fluorophore.

9. The method according to claim 8 , wherein unspecific binding of the streptavidin covalently coupled to the label or fluorophore is blocked using random oligonucleotides before the streptavidin covalently coupled to the label or fluorophore is conjugated to the biotin covalently coupled to the donor nucleotide probe or the acceptor nucleotide probe.

10. The method according to claim 1 , wherein the donor nucleotide probe and the acceptor nucleotide probe are used in a ratio between 1:2 and 2:1.

11. The method according to claim 1 , wherein the method is adapted to be carried out as a homogenous high throughput screen.

12. The method according to claim 1 , wherein the method is carried out at room temperature.

13. A method for detection of nucleotide polymerase activity comprising the following steps:
a) providing a reaction mixture comprising said polymerase, an initiator, and nucleoside triphosphates,
b) providing conditions sufficient to allow the polymerase an assembling of nucleotides,
c) stopping polymerase assembling of nucleotides,
d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein the donor nucleotide probe is conjugated to a fluorophore being a donor for förster resonance energy transfer and the acceptor nucleotide probe is conjugated to a fluorophore being a fluorescence quencher or an appropriate acceptor for förster resonance energy transfer,
e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and
f) determining fluorescence wherein said fluorescence correlates with the activity of the nucleotide polymerase;
wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and
wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).

14. The method according to claim 13 , wherein the reaction mixture further comprises a candidate substance to test its effect on the nucleotide polymerase activity.

15. The method according to claim 14 , wherein providing a reaction mixture of step a) comprises the following:
1) contacting said polymerase with the initiator, at least one transcription factor and an appropriate co-factor of the nucleotide polymerase resulting in a first reaction mixture
2) incubating said first reaction mixture
3) adding to said first reaction mixture the candidate substance resulting in a second reaction mixture
4) incubating said second reaction mixture
5) adding to said second reaction mixture nucleoside triphosphates.

16. The method according to claim 13 , wherein the reaction mixture of step a) comprises further at least one transcription factor, replication factor or co-factor of the nucleotide polymerase.

17. The method according to claim 13 , wherein the nucleotide polymerase is selected from the group consisting of DNA dependent DNA polymerase, RNA dependent DNA polymerase, DNA dependent RNA polymerase, RNA dependent RNA polymerase, independent RNA polymerase, and independent DNA polymerase.

18. The method according to claim 13 , wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by covalent bonding or by non-covalent interaction.

19. The method according to claim 13 , wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by non-covalent interaction mediated by biotin covalently coupled to the donor nucleotide probe or respectively the acceptor nucleotide probe and streptavidin covalently coupled to the label or fluorophore.

20. The method according to claim 19 , wherein unspecific binding of the streptavidin covalently coupled to the label or fluorophore is blocked using random oligonucleotides before the streptavidin covalently coupled to the label or fluorophore is conjugated to the biotin covalently coupled to the donor nucleotide probe or the acceptor nucleotide probe.

21. The method according to claim 13 , wherein the donor nucleotide probe and the acceptor nucleotide probe are used in a ratio between 1:2 and 2:1.

22. The method accordin

Assignees

• Max Planck Gesellschaft
• Lead Discovery Center Gmbh

Inventors

• Bergbrede Tim
• Larsson Nils-Göran
• Gustafsson Claes
• Falkenberg-Gustafsson Maria

Classifications

• C12Q1/68

  involving nucleic acids · CPC title

• G01N2333/91245

  Nucleotidyltransferases (2.7.7) · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q1/48

  involving transferase · CPC title