title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases.
abstract: 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.
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Chemistry &Biochemistry, Florida International University, Miami, FL 33199.
authors: Maxwell
authors: M
authors: Gu
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Chemistry &Biochemistry, Florida International University, Miami, FL 33199.
authors: Andrea
authors: A
authors: Berrido
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Chemistry &Biochemistry, Florida International University, Miami, FL 33199.
authors: Walter G
authors: WG
authors: Gonzalez
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Chemistry &Biochemistry, Florida International University, Miami, FL 33199.
authors: Jaroslava
authors: J
authors: Miksovska
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199.
authors: Jeremy W
authors: JW
authors: Chambers
authors: Biomolecular Science Institute, Florida International University, Miami, FL 33199.|Department of Chemistry &Biochemistry, Florida International University, Miami, FL 33199.
authors: Fenfei
authors: F
authors: Leng
mesh_terms: D001483:Base Sequence; D027081:DNA Gyrase / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry* / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D004270:DNA, Circular; D004278:DNA, Superhelical; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase; D019793:Fluorescein