Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

0381618 - BC 2013 RIV GB eng J - Journal Article
Fessl, Tomáš - Adamec, František - Polívka, Tomáš - Foldynová-Trantírková, Silvie - Vácha, František - Trantírek, L.
Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET.
Nucleic Acids Research. Roč. 40, č. 16 (2012), s. 10. ISSN 0305-1048. E-ISSN 1362-4962
Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z60220518
Keywords : in-cell FRET * fluorescence * DNA * nucleic acid * ATTO * in vivo
Subject RIV: BO - Biophysics
Impact factor: 8.278, year: 2012

Here we explored parameters critical for characterization of NA structure using FRET in the complex cellular environment of a living E. coli cell. Measurements indicated that the uncertainty in determining the orientation factor of fluorophores, which translates into precision of inter-probe distance determination, was the main limiting factor for the quantitative structural analysis of nucleic acids in living cells. Our data suggest that despite its low precision, the in-cell FRET data could be applied to assess the consistency of structural data acquired from FRET efficiency values measured in a complex cellular environment with various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows structural investigation of NA structure in any cell type and, most importantly, in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.
Permanent Link: http://hdl.handle.net/11104/0212047