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T5 exonuclease-based method to identify DNA topoisomerase inhibitors

a topoisomerase inhibitor and exonuclease technology, applied in the field of t5 exonuclease-based method to identify dna topoisomerase inhibitors, can solve the problems of inability to use high throughput screening (hts) assays, labor-intensive and time-consuming agarose gel electrophoresis, and inability to synthesis this type of fluorescently labeled dna molecules

Active Publication Date: 2022-04-21
FLORIDA INTERNATIONAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Agarose gel electrophoresis, however, is labor-intensive and time-consuming, and cannot be used as high throughput screening (HTS) assays.
However, the synthesis of this type of fluorescently labeled DNA molecules is too expensive, preventing the use of them as a regular screening agent for drug discovery.
Additionally, certain potential Topo inhibitors have fluorescence that greatly interfere with the final detection signal.

Method used

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  • T5 exonuclease-based method to identify DNA topoisomerase inhibitors
  • T5 exonuclease-based method to identify DNA topoisomerase inhibitors
  • T5 exonuclease-based method to identify DNA topoisomerase inhibitors

Examples

Experimental program
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Effect test

example 1

Property of T5 Exonuclease

[0093]FIG. 1 shows a unique property of T5 exonuclease that completely digested the sc pAB1 carrying a AT hairpin structure (FIG. 1A and lane 3 of FIG. 1B). T5 exonuclease was not able to digest rx pAB1 (lane 2 of FIG. 1B) and rx & sc pUC18. This unique property of T5 exonuclease can be used to configure HTS assays to identify Topo inhibitors.

example 2

ease-Based HTS Assay for Bacterial DNA Gyrase

[0094]FIG. 2A shows a method to screen / identify inhibitors or other molecules targeting bacterial DNA gyrase. This method can be easily configured into a high throughput format. In the absence of gyrase inhibitors, prokaryotic DNA gyrase can convert the rx DNA templates into the sc form. This conversion results in the formation of a hairpin structure in the plasmid. As a result, the sc pAB1 can be completely digested by T5 exonuclease (lane 5 of FIG. 2B). In contrast, gyrase inhibitor novobiocin completely inhibited the bacterial gyrase activities and prevented the conversion of the rx plasmid pAB1 into sc form. T5 exonuclease could not digest rx pAB1 (lane 6 of FIG. 2B).

[0095]A DNA-staining dye, such as ethidium homodimer 1 (EthD1), can differentiate these two T5 exonuclease-based reactions (FIG. 2C). In the presence of a gyrase inhibitor, the fluorescence intensity of EthD1 is significantly higher comparing with the DNA sample in the ab...

example 4

of DNA Intercalators by T5 Exonuclease-Based HTS Assay

[0104]The T5E-based HTS assay can be used to identify DNA intercalators. In the absence of the DNA intercalator, the sc pAB1 is digested by T5E, which cannot bind to the DNA dye, leading to no or litter fluorescence (FIG. 8). In the presence of the DNA intercalator, the sc pAB1 is converted into the relaxed form, which cannot be digested by T5E. As a result, the rx pAB1 can bind to the DNA dye, leading to a high fluorescence (FIG. 8).

[0105]The compounds were added in the screening plate as indicated in Table 1. The screening results are shown in Table 3. The fluorescence was measured at λem of 617 nm with λex=528 nm using a plate reader. The results indicate that the compounds in 5A (ethacridine), and 2G (echinomycin) are DNA intercalators.

TABLE 3HTS screening of 50-compound library for DNA intercalators1234567A2235752302041367207252B178286191183246249175C202153198207214207D170259218206220314E196185220213207226F173159200217222181...

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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.

Description

SEQUENCE LISTING[0001]The Sequence Listing for this application is labeled “SeqList-16Oct20_ST20”, which was created on Oct. 16, 2020, and is 8 KB. The entire content is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]DNA topoisomerases (Topos) are enzymes responsible for the relaxation of (+) and (−) supercoiled (sc) DNA and the resolution of DNA knots and catenanes during essential biological processes, such as DNA replication, transcription, recombination, and maintenance of chromosome structure. These enzymes catalyze the changes in DNA topology through creating transient DNA break. DNA topology is a tightly-regulated property of the DNA double helix that affects genomic stability and influences susceptibility to cancer and certain hereditary diseases, such as fragile X syndrome and autism.[0003]DNA Topos that control DNA topology inside cells are, thus, important targets for certain antibiotics and anticancer drugs. For example, type IIA topois...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/533C12Q1/44G01N21/64
CPCC12Q1/533C12Q1/44G01N2021/6439G01N2500/04G01N2500/20G01N21/6428
Inventor LENG, FENFEIDENG, ZIFANG
Owner FLORIDA INTERNATIONAL UNIVERSITY
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