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Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to deve...

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Autores principales: Gu, Maxwell, Berrido, Andrea, Gonzalez, Walter G., Miksovska, Jaroslava, Chambers, Jeremy W., Leng, Fenfei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087112/
https://www.ncbi.nlm.nih.gov/pubmed/27796331
http://dx.doi.org/10.1038/srep36006
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author Gu, Maxwell
Berrido, Andrea
Gonzalez, Walter G.
Miksovska, Jaroslava
Chambers, Jeremy W.
Leng, Fenfei
author_facet Gu, Maxwell
Berrido, Andrea
Gonzalez, Walter G.
Miksovska, Jaroslava
Chambers, Jeremy W.
Leng, Fenfei
author_sort Gu, Maxwell
collection PubMed
description DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases.
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spelling pubmed-50871122016-11-04 Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases Gu, Maxwell Berrido, Andrea Gonzalez, Walter G. Miksovska, Jaroslava Chambers, Jeremy W. Leng, Fenfei Sci Rep Article DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases. Nature Publishing Group 2016-10-31 /pmc/articles/PMC5087112/ /pubmed/27796331 http://dx.doi.org/10.1038/srep36006 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Gu, Maxwell
Berrido, Andrea
Gonzalez, Walter G.
Miksovska, Jaroslava
Chambers, Jeremy W.
Leng, Fenfei
Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title_full Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title_fullStr Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title_full_unstemmed Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title_short Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
title_sort fluorescently labeled circular dna molecules for dna topology and topoisomerases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087112/
https://www.ncbi.nlm.nih.gov/pubmed/27796331
http://dx.doi.org/10.1038/srep36006
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