Article

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: 6.72). 06/2012; 48(59):7413-5. DOI: 10.1039/c2cc31986a
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
72 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states, and the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.
    Biochemistry 02/2015; 54(10). DOI:10.1021/bi5014276 · 3.19 Impact Factor
  • [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 · 3.96 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 · 6.69 Impact Factor