Base-by-Base Dynamics in DNA Hybridization Probed by Fluorescence Correlation Spectroscopy

Abstract

Thermodynamics and dynamics of DNA hybridization/dehybridization at a terminal of a DNA duplex are investigated using steady-state fluorescence and fluorescence correlation spectroscopy (FCS). We introduce two pairs of dyes with different characteristic distances in fluorescence resonance energy transfer (FRET) to investigate the same process. The phenomenal discrepancy in the experimental observations between our two FRET pairs is incompatible with the traditional two-state model. We propose a so-called stretched exponential zipper (SEZ) model to successfully analyze the experimental data, through which the fundamental behavior of base-by-base hybridization/dehybridization is revealed. The dynamic parameters of the activation energy of single base-pair reaction derived from the two FRET pairs are consistent. The enthalpy change and entropy change of single base-pair formation are in agreement with theoretical prediction.

Dynamics of spontaneous flipping of a mismatched base in DNA duplex

“…Previous studies on dynamics of dsDNA bubbling (22), zippingunzipping (23), and DNA hairpin folding (30) imply that the relaxation time of spontaneous single-base flipping should be on the order of 10 ms. Although the conventional FCS (31) and singlemolecule imaging based on total internal reflection fluorescence microscopy (TIRFM) (32) are powerful tools for dynamic studies under equilibrium, their time windows are not suitable for relaxations around 10 ms.…”

“…In other words, the fluctuation probed by the NMR studies should be reassigned to base wobbling instead of flipping. Another well-known relaxation method, fluorescence correlation spectroscopy (FCS) coupled with fluorescence resonance energy transfer (FRET), has been applied to investigate many DNA conformational dynamics [e.g., DNA bubble breathing (22) and base zipping-unzipping (23)]. However, FRET measurement is sensitive to distance roughly from 2 to 7 nm, whereas the single-base flipping takes place at a distance shorter than 2 nm.…”

Nucleic Acid Fluorescent Probes for Biological Sensing

“…Using a FRET nucleic acid probe, the dynamics of nucleic acid itself, including hybridization/dehybridization and self-assembly, can be studied. [94][95][96][97] Majima and co-workers used a dual-labeled DNA probe to investigate the detailed conformational dynamics of i-motif shapes. 94 A donor and an acceptor are labeled at the terminal of the cytosine (C)-rich ssDNA to monitor the change of diffusion rates under different pH conditions.…”

A sandwich-type DNA electrochemical biosensor for hairpin-stem-loop structure based on multistep temperature-controlling method

“…They found that hybridization rate constants were determined by different temperatures and salt concentrations. Chen et al 27 and Chen et al 28 analyzed the origin of the specific recognition of structured DNA and investigated base thermodynamics and dynamics of DNA hybridization/ dehybridization at a terminal of a DNA duplex. They proposed that the intermediate states are existent in hairpin folding and unfolding and that the hairpin opens in a concerted manner during the hybridization.…”

“…It implies that, in order to form the duplex, the hairpin-stem-loop should be unjointed in hybridization processes and respective melting temperatures should be comparable for all reactions that were taking place, because the hybridization temperature can impact the stability of double-stranded DNA. 27,29,30 In the previous one-step hybridization experiment, we have designed shorter single-stranded probe and target molecules and increasing the incubation temperature. Nevertheless, the result showed that reducing strand length could weaken detection selectivity and increasing temperature could degrade biosensor performance.…”