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Design and construct a type of fluorescently labeled circular DNA molecules to study DNA topology and topoisomerases by fluorescence resonance energy transfer
Author(s) -
Leng Fenfei,
Gu Maxwell
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.572.2
Subject(s) - dna supercoil , topoisomerase , förster resonance energy transfer , dna , agarose gel electrophoresis , dna gyrase , topology (electrical circuits) , chemistry , fluorescence , biophysics , biology , computational biology , dna replication , biochemistry , gene , escherichia coli , physics , mathematics , quantum mechanics , combinatorics
DNA topology, an essential property of the DNA double helix, is under tight homeostatic control in vivo . Studies of DNA topology and topoisomerases are critical for understanding the mechanism underlying several infectious and genetic diseases and also for designing new anti‐cancer and anti‐microbial drugs targeting DNA topoisomerases. Typically agarose gel electrophoresis is used to study DNA supercoiling and topoisomerases. However, gel electrophoresis is time‐consuming and labor‐intensive. It is desirable to develop other assays, such as fluorescence‐based assays, for such studies. In this article, we developed a new method to construct a type of fluorescence‐labeled circular DNA molecules with high yields to study DNA topology and topoisomerases by using fluorescence resonance energy transfer (FRET). Specifically, we successfully produced relaxed and supercoiled pAB1_FL905 that carries fluorescein and dabcyl labels in the 42 bp AT sequence. Our results showed that 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 from the millions of compounds found in small molecule libraries that may target DNA topoisomerases, such as bacterial DNA gyrase and human DNA topoisomerase I and II. Support or Funding Information 1R15GM109254‐01A1 from the National Institutes of Health