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

Chen, Xudong; Zhou, Yan; Qu, Peng; Zhao, Xin

doi:10.1021/ja804628x

Cited by 51 publications

(63 citation statements)

References 37 publications

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“…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.…”

Section: Significancementioning

confidence: 97%

“…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.…”

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confidence: 99%

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Dynamics of spontaneous flipping of a mismatched base in DNA duplex

Yin

Yang

Zheng

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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111

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DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a doublestranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.fluctuation spectroscopy | integrated tempering sampling | rate constants | free-energy landscape A base in normal B-DNA spontaneously swinging out of the double helix to an extrahelical position is known as spontaneous base flipping. The dynamics of such base flipping is a fundamental issue in DNA biophysics. It is also related to how DNA repair or modification proteins search and fix the lesion bases to maintain the genome integrity or modify the DNA. Although extensive structural studies have found that many DNA base repair/modification proteins completely flip their target base out extrahelically (so-called enzymatic base flipping) (1-5), it is still under debate (6-11) whether the base flipping occurs spontaneously (9, 10, 12) or not (6-8). Accurate information on the dynamics of spontaneous base flipping is therefore of high interest and importance.However, the study of spontaneous base flipping is deemed to be difficult. The probability is extremely low for a single base to flip out of the DNA double helix in the absence of proteins. Hence only sensitive relaxation methods are able to detect such kind of fluctuation under equilibrium. As a well-known relaxation method, NMR has been applied to tackle this problem through the imino proton exchange assay (9,(13)(14)(15)(16)(17). In this assay, it is assumed that the exchange of the imino proton (in either G or T base) with the catalysts in the solution occurs only when the base flips out (13), and the extrapolated imino proton exchange rate at an infinite catalyst concentration is taken to be the base-flipping rate (14,15,17). According to these NMR studies the lifetime of the extrahelical state is on the order of microseconds, and that of the intrahelical state ranges from milliseconds to hundreds of milliseconds, depending on the stability of individual base pairs. MacKerell and coworkers as well as others have done extensive theoretical investigations and found that the target imino proton on the base already becomes accessible...

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Section: Significancementioning

confidence: 97%

mentioning

confidence: 99%

Dynamics of spontaneous flipping of a mismatched base in DNA duplex

Yin

Yang

Zheng

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

111

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“…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.…”

Section: Fluorescence Correlation Spectroscopy-based Biosensingmentioning

confidence: 99%

Nucleic Acid Fluorescent Probes for Biological Sensing

Xiao

Zhang

et al. 2012

Appl Spectrosc

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Nucleic acid fluorescent probes are playing increasingly important roles in biological sensing in recent years. In addition to the conventional functions of single-stranded DNA/RNA to hybridize with their complementary strands, affinity nucleic acids (aptamers) with specific target binding properties have also been developed, which has greatly broadened the application of nucleic acid fluorescent probes to the detection of a large variety of analytes, including small molecules, proteins, ions, and even whole cells. Another chemical property of nucleic acids is to act as substrates for various nucleic acid enzymes. This property can be utilized not only to detect those enzymes and screen their inhibitors, but also employed to develop effective signal amplification systems, which implies extensive applications. This review mainly covers the biosensing methods based on the above three types of nucleic acid fluorescent probes. The most widely used intensity-based biosensing assays are covered first, including nucleic acid probe-based signal amplification methods. Then fluorescence lifetime, fluorescence anisotropy, and fluorescence correlation spectroscopy assays are introduced, respectively. As a rapidly developing field, fluorescence imaging approaches are also briefly summarized.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Hong

Liu²,

Chen³

et al. 2012

IJN

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Abstract:A highly sensitive and selective method for amplified electrochemical detection for hairpin-stem-loop structured target sequences was developed based on the temperature regulation of DNA hybrids on a sandwich-type electrochemical DNA sensor. Multistep hybridization was applied to promote the hybridization efficiency of each section of sandwich structure. The results showed that both multistep and temperature-controlling hybridization techniques were both especially made to fabricate the sensor for the tendency of internal hybridization of target gene sequences. This strategy provides significantly enhanced hybridization efficiency and sequence specificity of electrochemical detection.

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

Cited by 51 publications

References 37 publications

Dynamics of spontaneous flipping of a mismatched base in DNA duplex

Dynamics of spontaneous flipping of a mismatched base in DNA duplex

Nucleic Acid Fluorescent Probes for Biological Sensing

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

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