title: Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle.
abstract: Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein-nucleic acid complexes.
authors.affiliation: Department of Chemistry, Wayne State University , 5101 Cass Avenue, Detroit, Michigan 48202, United States.
authors.forename: Elvin A
authors.initials: EA
authors.lastname: Alemán
authors.affiliation: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University , 100 West 18th Avenue, Columbus, Ohio 43210, United States.
authors.forename: Chamaree
authors.initials: C
authors.lastname: de Silva
authors.affiliation: Department of Chemistry, Wayne State University , 5101 Cass Avenue, Detroit, Michigan 48202, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Patrick
authors.affiliation: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University , 100 West 18th Avenue, Columbus, Ohio 43210, United States.
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
authors.affiliation: Department of Chemistry, Wayne State University , 5101 Cass Avenue, Detroit, Michigan 48202, United States ; Department of Medicine, Section of Virology and Single Molecule Imaging Group, MRC Clinical Sciences Center, Imperial College , Du Cane Road, London W12 0NN, United Kingdom ; Department of Medicine, Section of Virology and Single Molecule Imaging Group, MRC Clinical Sciences Center, Imperial College , Du Cane Road, London W12 0NN, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Rueda