Determination of thermodynamics and kinetics of RNA reactions by force

Tinoco, Ignacio; Li, Pan T.X.; Bustamante, Carlos

doi:10.1017/s0033583506004446

Cited by 84 publications

(112 citation statements)

References 73 publications

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“…Single molecule force experiments have far exceeded simple proof-of-principle experiments and have been used to elucidate the folding thermodynamics and kinetics for several RNAs [101][102][103][104][105][106]. This approach has answered one of the most fundamental questions in nucleic acid structure formation, providing direct evidence for the long-held nucleation model for duplex involving formation of 2-3 base pairs prior to rapid formation of the remainder of the duplex [107].…”

Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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“…Mechanical perturbations can be used to obtain detailed sequence-dependent structural, thermodynamic, and kinetic properties of nucleic acids (17). Forces measured during unzipping of long double-stranded DNAs show clear sequence-dependent features that can largely be reproduced using calculations based on the primary sequence and assuming thermal equilibrium (18); recent measurements have been used to determine salt-dependent base pair free energies (19).…”

mentioning

confidence: 99%

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

Oberstrass

Fernandes

Bryant

2012

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

117

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B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC) n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. “Bubble” templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.

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“…In principle, other mechanical techniques, such as atomic force microscope and magnetic tweezers, can also be used to study folding of single RNA molecules. However, such applications are limited largely due to the relatively low spatial resolution 30 .…”

Section: Introductionmentioning

confidence: 99%

“…The different geometries of the hairpin and kissing complex relative to the applied force result in their different mechanical response, which can be utilized to distinguish secondary and tertiary folding 28,32 . The theoretic aspect of mechanical unfolding has been previously reviewed 8,9,30 . This work outlines the basic approaches to set up and perform a single-molecule mechanical unfolding assay.…”

Section: Introductionmentioning

confidence: 99%

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Stephenson

Wan

Tenenbaum

et al. 2014

JoVE

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A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed. Video LinkThe video component of this article can be found at

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Determination of thermodynamics and kinetics of RNA reactions by force

Cited by 84 publications

References 73 publications

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

Nanomanipulation of Single RNA Molecules by Optical Tweezers

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