Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation

Orden, Alan Van; Jung, Jaemyeong

doi:10.1002/bip.20826

Cited by 54 publications

(59 citation statements)

References 102 publications

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“…[4,5] Although our hairpin data were consistent with a two-state model via the chi-squared metric, we also observed a systematic deviation between our data and the model. Recent studies of DNA hairpin dynamics have identified putative intermediates in the folding pathway, [36,37] which could explain this discrepancy. However, we caution that the detection of such intermediates by PDA should involve the use of novel experimental controls, and not simply an increase in the number of free parameters.…”

Section: Discussionmentioning

confidence: 99%

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Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

2010

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Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single‐molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two‐state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis‐testing models of conformational dynamics against experimental data.

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Section: Discussionmentioning

confidence: 99%

“…This could be due to more complex kinetic schemes, as identified in recent biophysical studies of DNA hairpins. [36,37] While some of these behaviors are expected to occur on timescales too short to be detected by PDA (e.g. multiple loop orientations in the open conformation), longerlived states (e.g.…”

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confidence: 99%

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

2010

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“…19 This hypothesis has been challenged by several recent studies that suggest a more complex pathway, involving intermediate states containing misfolded or partially folded configurations. [20][21][22] Additionally, there have been diverse, and sometimes contradictory, experimental observations and interpretations of hairpin formation kinetics. Fluorescence energy transfer and fluorescence correlation spectroscopy measurements 23 have reported an Arrhenius temperature dependency with a positive activation enthalpy for the hairpin closing rates.…”

Section: Introductionmentioning

confidence: 99%

The Role of Loop Stacking in the Dynamics of DNA Hairpin Formation

Mosayebi

Romano

Ouldridge³

et al. 2014

J. Phys. Chem. B

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We study the dynamics of DNA hairpin formation using oxDNA, a nucleotide-level coarse-grained model of DNA. In particular, we explore the effects of the loop stacking interactions and non-native base pairing on the hairpin closing times. We find a non-monotonic variation of the hairpin closing time with temperature, in agreement with the experimental work of Wallace et al. [Proc. Nat. Acad. Sci. USA 2001, 98, 5584-5589]. The hairpin closing process involves the formation of an initial nucleus of one or two bonds between the stems followed by a rapid zippering of the stem. At high temperatures, typically above the hairpin melting temperature, an effective negative activation enthalpy is observed because the nucleus has a lower enthalpy than the open state. By contrast, at low temperatures, the activation enthalpy becomes positive mainly due to the increasing energetic cost of bending a loop that becomes increasingly highly stacked as the temperature decreases. We show that stacking must be very strong to induce this experimentally observed behavior, and that the existence of just a few weak stacking points along the loop can substantially suppress it. Non-native base pairs are observed to have only a small effect, slightly accelerating hairpin formation.

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“…Data about the collective motion of DNA/RNA helical domains arising from structural fluctuations of the duplex (bending and twisting) are almost non-existent in the literature, especially for short duplexes. However, it can be inferred that these motions mainly cover the range from tens of nanosecond to tens of microseconds (Hogan et al 1982;Naimushin et al 2000;Orden and Jung 2008;Porschke 2007;Schwieters and Clore 2007;Shajani and Varani 2007), although motions in the range 20-400 ls have been reported (Kojima et al 2001). Together, these considerations indicate that in most cases, energy transfer captures a transient ''static'' configuration of the duplex, whereas the slow acquisition rate allows the DNA/RNA duplex and associated fluorophores to adopt all possible configurations within the experimental period used for data acquisition.…”

Section: Discussionmentioning

confidence: 99%

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1).Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation

Cited by 54 publications

References 102 publications

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

The Role of Loop Stacking in the Dynamics of DNA Hairpin Formation

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

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