Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle
Author(s):
Elvin A. Aleman, Chamaree de Silva, Eric M. Patrick, Karin Musier-Forsyth, David Rueda
Journal:
The Journal of Physical Chemistry Letters
Year:
2014
Volume:
5
Pages
777-781
DOI:
10.1021/jz4025832
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.