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Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization.

Nucleic acids research (2007-04-14)
Chunlai Chen, Wenjuan Wang, Zhang Wang, Fang Wei, Xin Sheng Zhao
ABSTRACT

Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 532 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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