Thermodynamic and Kinetic Aspects of RNA Pulling Experiments

Manosas, Maria; Ritort, Fèlix

doi:10.1529/biophysj.104.045344

Cited by 75 publications

(123 citation statements)

References 44 publications

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“…For each hairpin construct, we made a quantitative model of the folding landscape, adapting elements from previous models for dsDNA stretching (33) and unzipping (34) and RNA unfolding (27,21,35,36). This model incorporates five distinct components: the energetic contributions arising from (i) base stacking and hydrogen bonding within the folded helix, (ii) stretching of the ssDNA liberated by the hairpin unfolding, and (iii) molecular motions in the optical trap; and the effects of elastic compliance associated with (iv) the ssDNA of the unfolded state and (v) the dsDNA handles attached to the hairpin.…”

Section: Resultsmentioning

confidence: 99%

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Woodside

Behnke-Parks²,

Larizadeh³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

432

556

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Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a highresolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6 -30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.DNA hairpin ͉ energy landscape ͉ force clamp ͉ optical tweezers ͉ single molecule H airpins formed from self-complementary sequences supply a model system for studying folding and duplex formation, the most fundamental processes for generating structure in nucleic acids. Using hairpins, repeated measurements can be made on the same molecule, facilitating single-molecule studies. Furthermore, by simply altering the nucleotide sequence, physical properties such as folding energies, kinetic rates, and distances to transition states all can be changed systematically. Hairpins also play essential roles in vivo. DNA hairpins bind proteins to regulate transcription (1), and hairpin intermediates are involved in both replication and recombination (2, 3). RNA hairpins form tertiary contacts (4), bind to proteins (2), regulate transcription (5), and mediate RNA interference (6). Understanding the factors that influence hairpin folding should therefore not only elucidate principles of structure formation in nucleic acids but may also shed light on the biological roles played by these structures.Extensive calorimetric and melting studies have been carried out to generate predictive rules for the thermodynamic stability of arbitrary nucleic acid duplexes (7). The kinetic properties of duplex formation, however, remain less well understood, particularly those related to the nature of the transition state. Temperature-jump studies of annealing in short duplexes have been interpreted in terms of the nucleation of a transition state consisting of Ϸ1-3 bp, followed by zippering of the remaining stem (8). This interpretation, however, rests on the assumption that the enthalpy of activation arises ...

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Section: Resultsmentioning

confidence: 99%

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Woodside

Behnke-Parks²,

Larizadeh³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

432

556

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“…Interestingly, wiggly trajectories and reentrance have even been reported to occur in displacement driven nanoscale systems such as deformation of gold nanowires 24 and RNA unfolding. 25 A relevant quantity in hysteretic systems is dissipated energy which is given by the area enclosed within the hysteresis loops. A noteworthy feature ͑see Fig.…”

Section: Resultsmentioning

confidence: 99%

Hysteresis in a system driven by either generalized force or displacement variables: Martensitic phase transition in single-crystallineCu−Zn−Al

et al. 2007

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We report on experiments aimed at comparing the hysteretic response of a Cu-Zn-Al single crystal undergoing a martensitic transition under strain-driven and stress-driven conditions. Strain-driven experiments were performed using a conventional tensile machine while a special device was designed to perform stress-driven experiments. Significant differences in the hysteresis loops were found. The strain-driven curves show reentrant behavior ͑yield point͒ which is not observed in the stress-driven case. The dissipated energy in the stress-driven curves is larger than in the strain-driven ones. Results from recently proposed models qualitatively agree with experiments.

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“…The FDC is calculated by using a mesoscopic model that describes separately each component of the experimental setup (36): the bead in the optical trap, the handles, the released ssDNA, and the dsDNA hairpin (see SI Appendix: Fig. S1).…”

Section: Methodsmentioning

confidence: 99%

Single-molecule derivation of salt dependent base-pair free energies in DNA

Huguet

Bizarro

Forns

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

229

298

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Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol −1 precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our unique energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies, and entropies in conditions not accessible by bulk methodologies.DNA thermodynamics | DNA unzipping | nearest-neighbor model | optical tweezers T he nearest-neighbor (NN) model (1-4) for DNA thermodynamics has been successfully applied to predict the free energy of formation of secondary structures in nucleic acids. The model estimates the free-energy change to form a double helix from independent strands as a sum over all of resulting bp and adjacent-bp stacks, depending on the constituent four bases of the stack, by using 10 nearest-neighbor base-pair (NNBP) energies. These energies themselves contain contributions from stacking, hydrogen-bonding, and electrostatic interactions as well as configurational entropy loss. Accurately predicting free energies has many applications in biological science: to predict self-assembled structures in DNA origami (5, 6); achievement of high selectivity in the hybridization of synthetic DNAs (7); antigene targeting and siRNA design (8); characterization of translocating motion of enzymes that mechanically disrupt nucleic acids (9); prediction of nonnative states (e.g., RNA misfolding) (10); and DNA guided crystallization of colloids (11).Some of the most reliable estimates of the NNBP energies to date have been obtained from thermal denaturation studies of DNA oligos and polymers (2). Although early studies showed large discrepancies in the NNBP values, nowadays they are remarkably consistent among several groups. In these studies it is assumed that duplexes melt in a two-state fashion. However this assumption is not often the case and a discrepancy between the values obtained using oligomers vs. polymers remains a persistent problem that has been attributed to many factors such as the slow dissociation kinetics induced by a population of transient nondenatured intermediates that develop during thermal denaturation experiments (12). Single-molecule techniques (13) circ...

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Thermodynamic and Kinetic Aspects of RNA Pulling Experiments

Cited by 75 publications

References 44 publications

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Hysteresis in a system driven by either generalized force or displacement variables: Martensitic phase transition in single-crystallineCu−Zn−Al

Single-molecule derivation of salt dependent base-pair free energies in DNA

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