Kinetics of conformational fluctuations in DNA hairpin-loops (molecular beacons͞f luorescence correlation spectroscopy͞folding kinetics͞polymer conformation͞f luorescence energy transfer)

Grégoire Bonnet, Oleg Krichevsky, Albert Libchaber

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The kinetics of DNA hairpin-loop f luctua-tions has been investigated by using a combination of f luo-rescence energy transfer and f luorescence correlation spec-troscopy. 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 charac-terized 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 depen-dence 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 open-ing 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(A) leads to a stunning 5-fold increase in the activation energy of closing, whereas the activation energy of opening is virtually unchanged. These results illuminate some of the properties characterizing single-stranded DNA as a random polymer coil: its persistence length and the probability of end-to-end collision. We use an approach to study the kinetics of DNA tran-sitions that combines recent advances in the development of f luorescent DNA probes (molecular beacons) (10) and f luorescence correlation spectroscopy (FCS) (11, 12). Mo-lecular beacons are oligonucleotides capable of forming a hairpin loop with a f luorophore and a quencher attached to the two ends of the stem (Fig. 1). The conformational state of a beacon is directly reported by its f luorescence: in the closed state the hairpin brings together f luorophore and quencher, the molecule is not f luorescent; in the open state f luorophore and quencher are far apart, and the beacon is f luorescent. In equilibrium conditions, beacons f luctuate between the two states with the characteristic rates of opening k Ϫ and closing k ϩ . We monitor these f luctuations by accumulating the autocorrelation function of the f luorescent light (FCS technique) (13). As compared with the conven-tional way of studying DNA transformation (temperature-jump combined with UV-absorption measurement) (8, 9, 14), this approach is characterized by a high signal͞ background ratio (typically 50 for a beacon in a closed͞open configuration), low sample concentrations (Ϸ10 nM, to avoid duplex formation), and noninvasiveness: only equilib-rium f luctuations are studied.
Original languageEnglish
Pages (from-to)8602-8606
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue numberJuly
StatePublished - 1998


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