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

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.

Aptamer-based electrochemical biosensor for Botulinum neurotoxin

“…Correct folding of the aptamer allows high-affinity binding between the aptamer and BoNT/A toxoid; misfolding and non-folding result in low signal-to-noise ratios (SNRs) [26]. In addition, the specific signal amplification is strongly dependent on the conformation of the aptamer before and after binding to BoNT/A.…”

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

“…1,[38][39][40][41][42] Since DNA is a polyanion, increasing the salt concentration screens the negative charge resulting in a faster hybridization rate; while increasing the temperature can sometimes decrease the rate. 43,44 On the other hand, few reports 4 exist regarding DNA hybridization in various organic solvents.…”

Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

“…Temperature affects the free energy profile of hybridization for DNA in different structures. Chen et al 34 pointed out nucleation and hairpin opening as two rate-limiting steps that influence DNA hybridization with secondary structure. At higher temperatures, hybridization is independent of secondary structures, but if the temperature is too high, it will melt the hybridization structure.…”

“…Our microfluidic approach is unique as: (a) the rapid motion of bead transfer through microfluidic channel provides minimal carry-over of non-target molecules (0.5 6 0.3% of fluid volume 34 ); (b) No flow is required for target molecules to hybridize with the capture complex, as the capture/binding of targets occurs in the reservoir (W1) with tunable size. Therefore, our method is capable of processing larger amounts of low concentration clinical samples, as well as increase sensitivity for rapid separation; (c) high efficiency separation is achieved without using any membrane or pumps, which is quite useful in resource limited settings; (d) a counter flow of clean buffer from W2 to W1 can be created by a simple fluid volume differential thus further reduce the carry over or diffusion of non-target molecules; (e) multiple microfluidic separations can be performed as needed by simply fabricating an additional microchannel from W2 to a new well 3 (W3), W3 to a new well 4 (W4), and so on, making this assay customizable for a broader scope of applications.…”