Kinetics of conformational fluctuations in DNA hairpin-loops

Abstract

The kinetics of DNA hairpin-loop f luctuations has been investigated by using a combination of f luorescence energy transfer and f luorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for various salt concentrations. The rate of unzipping of the hairpin stem is essentially independent of the characteristics of the loop, whereas the rate of closing varies greatly with the loop length and sequence. The closing rate scales with the loop length, with an exponent 2.6 ؎ 0.3. The closing rate is increased at higher salt concentrations. For hairpin closing, a loop of adenosine repeats leads to smaller rates and higher activation energies than a loop with thymine repeats.DNA hairpin structures may participate in many biological functions, such as the regulation of gene expression (1), DNA recombination (2), and facilitation of mutagenic events (3). Hairpin-loops were proposed as antisense drugs with high resistance to degradation by exonucleases (4).Hairpin structures are not static: they fluctuate between different conformations. In a simplified description, all of the conformations can be divided into two main states: the open state and the closed one (Fig. 1). The closed state is characterized by a low enthalpy due to base pairing in the stem region of the hairpin. The open state has a high entropy due to the large number of configurations achievable by a single-stranded DNA chain. The closed-to-open transition requires an energy fluctuation sufficiently large to unzip all of the base pairs, whereas the opposite (closing) transition involves the collision of the two arms of a hairpin, followed by the nucleation and the propagation of a base-paired stretch. One expects the rate of closing to depend strongly on the properties of the hairpin loop, such as length and rigidity, whereas one expects the rate of opening to be relatively unaffected by these properties.Most of the previous studies of hairpins deal with the structure of the folded state or with the thermodynamics of the folding-unfolding transitions (5-7). Knowledge about the kinetics of the hairpin transitions is limited to a few isolated measurements of the characteristic rates (8, 9). The dependence of closing and opening rates on such crucial parameters as the loop size, the loop sequence, and the salt concentration have not been reported, and the activation energies of the transitions are not known.We present here measurements of the characteristic rates of closing and of opening for different lengths and sequences of the loop, done in a wide range of temperatures and salt concentrations. We find that the characteristics of the loop influence the closing rate much more strongly than the opening rate. The rate of closing decreases when the loop size increases. This dependence can be approximated by a scaling relationship with an exponent of 2.6 Ϯ 0.3. The change of poly(T) sequence in the loop to poly...

Ultrafast cooling reveals microsecond-scale biomolecular dynamics

“…This result suggests that increased ionic strength has similar stabilizing effects on the folded hairpin and the transition state, but not on the unfolded ssDNA, leading to a significant [NaCl] dependence for folding but not unfolding. The considerable increase of the HP2 folding rate with [NaCl] at 22°C and the near lack of dependence of the HP2 unfolding rate on [NaCl] at 70°C are both in agreement with what was found for a similar hairpin 17 . In addition, the rates of HP2 unfolding and folding at 100 mM NaCl and 22°C, 0.13 Â 10 3 and 1.7 Â 10 3 s À 1 (Supplementary Table 3), agree reasonably well with previously measured values for that hairpin (B0.4 Â 10 3 and 4.0 Â 10 3 s À 1 ) 17 .…”

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

“…(In an alternative MB design, different dyes rather than dye-quencher pairs can be used; excitation of the donor dye will lead to donor specific fluorescence in the absence, and FRET-induced acceptor emission in the presence of target mRNA.) MBs have several advantages such as high signal to background ratio (S/B), 5,22,23 and fast hybridization response to complementary target 24 which have been exploited for the detection of single nucleotide polymorphisms (SNPs). 20,21 However, a notable disadvantage is non-specific opening of the MB (opening of the MB in the absence of target), which gives rise to a false positive signal ( Figure 1c).…”

Direct Measurement of Sequence-Dependent Transition Path Times and Conformational Diffusion in DNA Duplex Formation

“…Here, we investigate the sequence dependence of conformational diffusion in nucleic acid duplex formation by measuring transition paths in DNA hairpins held under tension in optical tweezers. Nucleic acid hairpins provide a powerful model system for studying folding phenomena because they have been studied extensively at the single-molecule level by experiment (34)(35)(36)(37)(38)(39), theoretical and computational models have described experimental results quantitatively (38,(40)(41)(42), and hairpin folding can be manipulated predictably through sequence changes (43). We used measurements of τ tp and P TP (t) from the folding of hairpins having different stem sequences to determine whether the two canonical Watson-Crick base pairs, A:T (containing two hydrogen bonds) and G:C (containing three), give rise to sequence dependence in D. By applying Eq.…”