T5 exonuclease-based method to identify DNA topoisomerase inhibitors

Patent metadata

Field	Value
Publication number	US-11268129-B1
Application number	US-202117343918-A
Country	US
Kind code	B1
Filing date	Jun 10, 2021
Priority date	Oct 16, 2020
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The present invention provides assays and methods for studying DNA topology and topoisomerases. The assays and methods utilize a circular plasmid DNA comprising one or more hairpin structures and the ability of T5 exonuclease (T5E) to digest the circular plasmid DNA in a specific configuration. The assays and methods can be used as a high throughput screening for inhibitors of, for example, DNA gyrases and DNA topoisomerases I for anticancer drug and antibiotics discovery.

First claim

We claim:

1. A method for identifying an inhibitor targeting a type II DNA topoisomerase in a sample, the method comprising adding a circular double-stranded plasmid comprising two hairpin structures upon supercoiling from a relaxed configuration, wherein the circular double-stranded plasmid does not comprise a fluorescent dye; adding the type II DNA topoisomerase; adding a T5 exonuclease (T5E); adding a DNA-staining dye; and determining the presence or absence of the inhibitor based on fluorescence in the sample.

2. The method of claim 1 , the type II DNA topoisomerase being human DNA topoisomerase II or yeast DNA topoisomerase II.

3. The method of claim 1 , the DNA topoisomerase being a DNA gyrase.

4. The method of claim 1 , the circular double-stranded plasmid being pAB1.

5. The method of claim 1 , the DNA-staining dye being Hoechest 33258, SYBR gold, ethidium bromide, EthD-1, or SYBR green.

6. The method of claim 1 , the circular double-stranded plasmid comprising about 15 ATs to about 30 ATs.

7. The method of claim 1 , the circular double-stranded plasmid comprising an adenosine-thymidine dinucleotide repeat (AT)n sequence, wherein n=21.

8. The method of claim 1 , the circular double-stranded plasmid comprising at least one DNA endonuclease recognition site.

9. The method of claim 1 , which comprises adding a circular double-stranded plasmid comprising an adenosine-thymidine dinucleotide repeat (AT)n sequence, n=21, the circular double-stranded plasmid being in a relaxed configuration; adding a DNA gyrase; adding the T5E; adding the DNA-staining dye; and determining the presence or absence of the inhibitor based on the fluorescence in the sample, wherein a higher fluorescence in the sample than a control is indicative of the presence of the inhibitor of the DNA gyrase, wherein the control comprises the circular supercoiled double-stranded plasmid in a supercoiled conformation.

10. The method of claim 1 , which comprises incubating T5E in the sample for at least 1 hour prior to adding the DNA-staining dye.

11. The method of claim 1 , the sample being a sample in a high throughput screening (HTS) sample carrier.

12. The method of claim 1 , which further comprises adding ATP in the sample.

13. The method of claim 4 , the DNA gyrase being bacterial DNA gyrase.

Assignees

• Leng Fenfei
• Deng Zifang
• The Florida International Univ Board Of Trustees

Inventors

• Leng Fenfei
• Deng Zifang

Classifications

• C12Q1/533 Primary

  involving isomerase · CPC title

• C12Q1/44

  involving esterase · CPC title

• G01N2500/20

  cell-free systems · CPC title

• G01N2500/04

  Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title