Panorama of DNA hairpin folding observed via diffusion-decelerated fluorescence correlation spectroscopy
Beijing National Laboratory for Molecular Sciences and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. Chemical Communications
(Impact Factor: 6.83).
06/2012; 48(59):7413-5. DOI: 10.1039/c2cc31986a
Based on a confocal microscopy platform, we extended the FCS time window by three orders of magnitude to the s timescale by attaching a polystyrene microsphere. We simultaneously monitored the relaxations of multiple intermediates involved in DNA hairpin folding, thus offering a much more detailed view of the kinetics of hairpin folding experimentally.
Available from: Jonathan B Chaires
- "Duplex DNA formation is perhaps regarded as somewhat boring because of the apparent simplicity and regularity of the canonical double helix, although classic kinetic studies of the association and unwinding of genomic DNA revealed slow, multiphasic kinetics arising from sequence complexity and rate-limiting nucleation events  . DNA kinetic studies have recently been largely focused on the folding of small duplex hairpins       , model systems that, while fundamentally interesting, are unlikely to have much functional relevance. Recent advances show that certain DNA sequence elements in the genome can fold into noncanonical forms with complex tertiary structures such as G-quadruplexes, i-motifs, or triplexes. "
[Show abstract] [Hide abstract]
ABSTRACT: Sequence analogues of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K(+)-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles, and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of "prefolded" hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1=0.1s at 25°C in 25mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intra-molecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2≈3700s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3≈750s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies (e.g. d[TTGGG(TTAGGG)3A and d[TAGGG(TTAGGG)3TT]). Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway.
Journal of Molecular Biology 01/2014; 426(8). DOI:10.1016/j.jmb.2014.01.009 · 4.33 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The amplitude of chemical relaxations in fluorescence correlation spectroscopy (FCS) is an important parameter that directly relates to not only the equilibrium constant of the relaxations but also the number of individual fluorophores that diffuse together. In this Letter we answer the question how exactly the amplitude of the relaxations in FCS changes with respect to the number of identical fluorophores on one cargo. We anchored tetramethylrhodamine molecules onto each arm of a DNA Holliday junction molecule so that the codiffusing dyes were capable of performing independent fluorescent fluctuations. We found that the amplitudes of the relaxations were inversely proportional to the number of the dyes on each cargo molecule, well agreeing with the theoretical prediction derived in this Letter. The result provides a guideline for the FCS data analysis and points out a simple way to determine the number of molecules that a cargo carries.
Journal of Physical Chemistry Letters 01/2013; 4(2):304–309. DOI:10.1021/jz301871f · 7.46 Impact Factor
Available from: Eyal Nir
[Show abstract] [Hide abstract]
ABSTRACT: The dynamics of two DNA hairpins (5'-TCGCCT-A31-AGGCGA-3' and 5'-TCGCCG-A31-CGGCGA-3') were studied using immobilization-based and diffusion-based single-molecule fluorescence techniques. The techniques enabled separated and detailed investigation of the states and of the transition reactions. Only two states, open and closed, were identified from analysis of the FRET histograms; metastable states with lifetimes longer than the technique resolution (0.3 ms) were not observed. The opening and closing reaction rates were determined directly from the FRET time trajectories and the Gibbs free energies of these states and of the transition state were calculated using the Kramer theory. The rates, which are undoubtedly of transitions between the fully closed and the fully open states, ranged from 2 to 90 s-1, were slower (~10-fold) than rates previously determined from fluorescence correlation spectroscopy. The heights of the barriers for closing were almost identical for the two hairpins. The barrier for opening the hairpin with the stronger stem was higher (4.3 kJ/mol) than that for the hairpin with the weaker stem, in a very good agreement with the difference in stability calculated by the nearest-neighbor method. The barrier for closing the hairpin decreased (~ 8 kJ/mol) and the barrier for opening increased (~ 4 kJ/mol) with increasing NaCl concentration (10-100 mM), indicating that higher ionic strength stabilizes the folded state with respect to the transition state and stabilizes the transition state relative to the unfolded state. The very good agreements in the dynamics measured for free hairpins, for hairpins anchored to origami, and for hairpins anchored to the coverslip and the very good agreement between the two single-molecule techniques demonstrate that neither the origami nor the coverslip influence the hairpin dynamics, supporting a previous demonstration that origami can serve as a platform for biophysical investigations.
The Journal of Physical Chemistry B 09/2013; 117(40). DOI:10.1021/jp4059214 · 3.30 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.