Force-Dependent Fragility in RNA Hairpins

Manosas, Maria; Collin, Delphine; Ritort, Fèlix

doi:10.1103/physrevlett.96.218301

Cited by 64 publications

(105 citation statements)

References 23 publications

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“…Our results suggest that the kinetic models widely used to describe nucleic acid duplex formation (35,38,40) can now be refined, replacing the constant microscopic rates typically assumed with rates that are sequence dependent, so as to reflect the twofold difference in D between G:C and A:T base-pair formation. An interesting implication of the sequence dependence of D is that D should be position dependent along the duplex, leading to changes in the shape of the transition paths (51).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Significancementioning

confidence: 99%

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Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

Neupane

Wang

Woodside

2017

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

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The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0 to 100%, we determined both the average time to cross the transition paths, τ tp , and the distribution of individual transit times, P TP (t). We then estimated D from both τ tp and P TP (t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. τ tp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base pairs than for those with only G:C base pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0 to 100%. These results reveal that G:C base pairs form faster than A:T base pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding.folding | DNA hairpins | energy landscapes | optical tweezers S tructure formation in biological macromolecules like proteins and nucleic acids is a complex, dynamic process. Physically, it is described in the context of energy landscape theory as a diffusive search in conformational space for the minimumenergy structure (1-3). The speed at which this search takes place at the microscopic level is set by the conformational diffusion coefficient, D. Because D plays a key role in determining the kinetics of the folding, as encapsulated in the well-known expression for rates derived by Kramers (4), it is one of the fundamental physical determinants of folding phenomena: it connects the thermodynamics encoded in the energy landscape to the dynamics of conformational changes. The properties of D relate to such key issues as internal friction in the polymer chain (5-8), roughness in the energy landscape (9-12), projection of the full phase-space for conformational dynamics onto experimental reaction coordinates (13), and "speed limits" for folding transitions (14).Despite its importance, D remains challenging to determine experimentally. Several studies have found D from the reconfiguration times for unfolded proteins or polypeptides (15-19), using fluorescence probes to measure interactions between specific locations on the polymer chain. However, few stu...

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Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

Neupane

Wang

Woodside

2017

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

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“…Our results provide a benchmark for testing theoretical simulations of the pseudoknot folding transition under tension in a more detailed molecular level, such as closing of loop2 (Fig. 1B,C) by the z90°flipping of stem1 in HPp, by the reorganization of stem1 hairpin loop residues (Theimer et al 2003;Yingling and Shapiro 2005;Reipa et al 2007), and by relaxation of the stretched 39-single-strand (Hyeon and Thirumalai 2006;Manosas et al 2006). …”

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confidence: 99%

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

Chen¹,

Wen²,

Tinoco³

2007

RNA

123

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RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at ;10 and ;5 piconewtons (pN), with ensemble rate constants of ;0.1 sec À1 , by stepwise force-drop experiments. Folding studies of the isolated 59-hairpin construct suggested that the 59-hairpin within the pseudoknot forms first, followed by formation of the 39-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of ;46 pN, at an average rate of ;0.05 sec À1 . In the constant-force folding trajectories at ;10 pN and ;5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.

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“…The GRM gives quantitative agreement with the simulation results. The key results announced here provide a framework for using the measured folding landscape of nucleic acid hairpins at f Ϸ f m to obtain f-dependent folding and unfolding times and the transition state movements as f is varied (24)(25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

Force-dependent hopping rates of RNA hairpins can be estimated from accurate measurement of the folding landscapes

Hyeon

Morrison²,

Thirumalai

2008

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

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The sequence-dependent folding landscapes of nucleic acid hairpins reflect much of the complexity of biomolecular folding. Folding trajectories, generated by using single-molecule force-clamp experiments by attaching semiflexible polymers to the ends of hairpins, have been used to infer their folding landscapes. Using simulations and theory, we study the effect of the dynamics of the attached handles on the handle-free RNA free-energy profile F eq o (zm), where zm is the molecular extension of the hairpin. A molecular understanding of how proteins and RNA fold is needed to describe the functions of enzymes (1) and ribozymes (2), interactions between biomolecules, and the origins of misfolding that is linked to a number of diseases (3). The energy-landscape perspective has provided a conceptual framework for describing the mechanisms by which unfolded molecules navigate the large conformational space in search of the native state (4-6). Recently, single-molecule techniques have been used to probe features of the energy landscape of proteins and RNA that are not easily accessible in ensemble experiments (7-18). It is possible to construct the shape of the energy landscape, including the energy scales of ruggedness (19,20), by using dynamical trajectories that are generated by applying a constant force ( f ) to the ends of proteins and RNA (14,15,21,22). If the observation time is long enough for the molecule to sample the accessible conformational space, then the time average of an observable X recorded for the ␣th molecule [͗X͘ ϭ t3ϱ, and the distribution P(X) should converge to the equilibrium distribution function P eq (X). By using this strategy, laser optical tweezer (LOT) experiments have been used to obtain the sequence-dependent folding landscape of a number of RNA and DNA hairpins (8,14,15,23), by using X ϭ R m , the end-to-end distance of the hairpin that is conjugate to f, as a natural reaction coordinate. In LOT experiments, the hairpin is held between two long handles [DNA (15) or DNA/RNA hybrids (8)], whose ends are attached to polystyrene beads (Fig. 1a). The equilibrium free-energy profile ␤F eq (R m ) ϭ ϪlogP eq (R m ) (␤ ϵ 1/k B T, k B is the Boltzmann constant, and T is the absolute temperature) may be useful in describing the dynamics of the molecule, provided R m is an appropriate reaction coordinate.The dynamics of the RNA extension in the presence of f (z m ϭ z 3Ј Ϫ z 5Ј Ϸ R m , provided transverse fluctuations are small) is indirectly obtained in an LOT experiment by monitoring the distance between the attached polystyrene beads (z sys ϭ z p Ϫ z o ), one of which is optically trapped at the center of the laser focus (Fig. 1a). The goal of these experiments is to extract the folding landscape [␤F eq o (z m )] and the dynamics of the hairpin in the absence of handles by using the f-dependent trajectories z sys (t). To achieve these goals, the fluctuations in the handles should minimally perturb the dynamics of the hairpin to probe the true dynamics of a molecule of interest. However, dependi...

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Force-Dependent Fragility in RNA Hairpins

Cited by 64 publications

References 23 publications

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

Force-dependent hopping rates of RNA hairpins can be estimated from accurate measurement of the folding landscapes

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