Unzipping DNA with Optical Tweezers: High Sequence Sensitivity and Force Flips

Bockelmann, Ulrich; Thomen, Philippe; Essevaz-Roulet, B.; Viasnoff, Virgile; Heslot, F.

doi:10.1016/s0006-3495(02)75506-9

Cited by 275 publications

(283 citation statements)

References 33 publications

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“…These data cover the complete range of forces that can be expected for unfolding DNA. Previous work on phage DNA found that kilobase-long duplexes with Ϸ50% GC content unzip at an average force of Ϸ15 pN (23)(24)(25). Our results indicate that this asymptotic value is reached at a duplex length of only Ϸ25 bp.…”

Section: Resultssupporting

confidence: 56%

“…Especially useful is the fact that the location of the transition state along the reaction coordinate can be derived directly from the force dependence of the kinetic rates (21). The application of force also facilitates the study of molecules with a wide range of energetic stabilities under similar environmental conditions, as demonstrated by the force-induced unfolding of short duplexes (22) and hairpins (20) and very long duplexes (23)(24)(25). Nanomechanical measurements have been used to follow folding transitions in single and multiple hairpins of DNA (26) and RNA (27)(28)(29)(30), but no attempt has yet been made to investigate systematically the effects of sequence on the folding process.…”

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confidence: 99%

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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.

433

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: Resultssupporting

confidence: 56%

mentioning

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.

433

556

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show abstract

“…Previously, there have been a number of experimental and theoretical studies (9)(10)(11)(12) on the effect of sequence heterogeneity on DNA melting and unzipping transitions. Our study is along this general direction.…”

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confidence: 99%

Localization of denaturation bubbles in random DNA sequences

Hwa

Marinari²,

Sneppen

et al. 2003

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

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We study the thermodynamic and dynamic behaviors of twistinduced denaturation bubbles in a long, stretched random sequence of DNA. The small bubbles associated with weak twist are delocalized. Above a threshold torque, the bubbles of several tens of bases or larger become preferentially localized to AT-rich segments. In the localized regime, the bubbles exhibit ''aging'' and move around subdiffusively with continuously varying dynamic exponents. These properties are derived by using results of largedeviation theory together with scaling arguments and are verified by Monte Carlo simulations.L ocalized opening of double-stranded DNA is essential in a number of cellular processes such as the initiations of gene transcription and DNA replication (1). Although thermal denaturation is highly unlikely under physiological conditions, in vitro experiments show that local denaturation can be readily induced by underwinding the DNA double-helix by an amount that is physiologically reasonable (2-4). The basic physical effect is simple: An underwound double-helix suffers a reduction in binding free energy (5-7). Local openings of the double-helix (referred to as ''denaturation bubbles'') relieve the twist experienced by the remainder of the double-helix and are thus energetically favorable. The denaturation bubbles may be recruited to a specific location of the genome by a designed (e.g., AT-rich) sequence, since AT pairs bind more weakly than GC pairs (8). On the other hand, entropic effect that favors bubble delocalization is non-negligible for long sequences. Also significant is the kinetic trapping of the bubbles due to statistical agglomeration of AT-rich segments in long heterogenous sequences.To gain some quantitative understanding on the competing effects of entropy and sequence heterogeneity, we characterize in this study the thermodynamic and dynamic properties of denaturation bubbles in a long, stretched random DNA sequence with no special sequence design. Previously, there have been a number of experimental and theoretical studies (9-12) on the effect of sequence heterogeneity on DNA melting and unzipping transitions. Our study is along this general direction. The specific behaviors exhibited by the denaturation bubbles are rather complex and are typical of those observed in systems dominated by quenched disorders (13): The bubbles are localized upon increase of the applied torque beyond a certain threshold. In the localized regime, their dynamics exhibits ''aging'' (14, 15) and is subdiffusive with continuously varying exponents.Interestingly, twist-induced denaturation presents a rare physical example of the celebrated random-energy model (REM) of a disordered system (16). Consequently, detailed analysis of both the thermodynamics and dynamic properties can be made by applying the well-developed theory of disordered systems (13), together with exact results from large-deviation theory familiar in the related sequence alignment problem (17, 18). We will draw on detailed experimental knowledge of thermal denatur...

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“…For example, the energy landscape of the DNA helix was explored by unzipping dsDNA molecules. 63 Digoxigenin and biotin tags on adjacent overhangs of dsDNA were used to attach one strand to a bead and the other to a slide surface, which was then moved away from the trap. The DNA unzipping force was measured by tracking bead displacement from the beam focus.…”

Section: Optical Tweezers (Ot)mentioning

confidence: 99%

The importance of surfaces in single-molecule bioscience

2008

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The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these singlemolecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample. In this review we highlight why surface attachments are a critical aspect of many single-molecule studies and we discuss current methods for anchoring biomolecules. Finally, we provide a detailed description of a new method developed by our laboratory for anchoring and organizing hundreds of individual DNA molecules on a surface, allowing "high-throughput" studies of protein-DNA interactions at the single-molecule level.

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Unzipping DNA with Optical Tweezers: High Sequence Sensitivity and Force Flips

Cited by 275 publications

References 33 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

Localization of denaturation bubbles in random DNA sequences

The importance of surfaces in single-molecule bioscience

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