Published online 10 May 2006
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The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy
Applied Laser Physics and Laser Spectroscopy, University of Bielefeld Universitätsstrasse 25, 33615 Bielefeld, Germany
*To whom correspondence should be addressed. Tel: +49 521 106 5440; Fax: +49 521 106 2958; Email: sdoose{at}physik.uni-bielefeld.de
*Correspondence may also be addressed to Markus Sauer. Tel: +49 521 106 5450; Fax: +49 521 106 2958; Email: sauer{at}physik.uni-bielefeld.de
Received February 14, 2006. Revised March 3, 2006. Accepted March 27, 2006.
Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.
Present address: Jiho Kim, Institut Pasteur of Korea, 39-1, Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Korea
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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