Direct Measurement of the Full, Sequence-Dependent Folding Landscape of a Nucleic Acid

Woodside, Michael T.; Anthony, Peter C.; Behnke-Parks, William M.; Larizadeh, Kevan; Herschlag, Daniel; Block, Steven M.

doi:10.1126/science.1133601

Cited by 370 publications

(546 citation statements)

References 20 publications

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“…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]. Further, it has been shown that force unfolding data can be deconstructed into a complete energy landscape [108]. It will be fascinating to see if this approach can be applied to increasingly complex RNAs.…”

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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“…23,33,34 The number of experimentally determined high resolution RNA structures 30,31,35 continues to increase, enabling us to understand the interactions that stabilize the folded states. Single molecule [36][37][38][39][40][41] and ensemble experiments [42][43][44] using a variety of biophysical methods combined with theoretical techniques 14,34 have led to a conceptual framework for predicting various mechanisms by which RNA molecules fold. In order to make further progress new computational tools are required.…”

Section: Introductionmentioning

confidence: 99%

Minimal Models for Proteins and RNA: From Folding to Function

Pincus

Cho

Hyeon

et al. 2008

Progress in Molecular Biology and Translational Science

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We present a panoramic view of the utility of coarse-grained (CG) models to study folding and functions of proteins and RNA. Drawing largely on the methods developed in our group over the last twenty years, we describe a number of key applications ranging from folding of proteins with disulfide bonds to functions of molecular machines. After presenting the theoretical basis that justifies the use of CG models, we explore the biophysical basis for the emergence of a finite number of folds from lattice models. The lattice model simulations of approach to the folded state show that non-native interactions are relevant only early in the folding process -a finding that rationalizes the success of structure-based models that emphasize native interactions. Applications of off-lattice C α and models that explicitly consider side chains (C α -SCM) to folding of β-hairpin and effects of macromolecular crowding are briefly discussed. Successful applications of a new class of off-lattice models, referred to as the Self- We also present two distinct models for RNA, namely, the Three Site Interaction (TIS) model and the SOP model, that probe forced unfolding and force quench refolding of a simple hairpin and Azoarcus ribozyme. The unfolding pathways of Azoarcus ribozyme depend on the loading rate, while constant force and constant loading rate simulations of the hairpin show that both forced-unfolding and force-quench refolding pathways are heterogeneous. The location of the transition state moves as force is varied. The predictions based on the SOP model show that force-induced unfolding pathways of the ribozyme can be dramatically changed by varying the loading rate. We conclude with a discussion of future prospects for the use of coarse-grained models in addressing problems of outstanding interest in biology.

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“…Unlike DNA or RNA hairpins, where forces on the order of 15 pN are necessary to induce mechanical unzipping (10,11), the conformations of HJs could be biased at 0.5 pN or lower. The lever arm effect makes it unlikely that a purely mechanical tool could have probed the force effect on HJ conformations because if the arms are lengthened to magnify the distance change, the force effect will occur at even lower forces.…”

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confidence: 99%

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Hohng

Zhou

Nahas

et al. 2007

Science

267

299

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Despite the recent advances in single molecule manipulation techniques, purely mechanical approaches can not detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence which allowed us to examine the effect of sub-pN forces on the nanometer scale motion of the Holliday junction (HJ) at 100 Hz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.Many biological processes are dependent on tension. In recent years, single molecule force measurements have shown directly that biochemical reactions can be influenced by applied force (1). Yet, purely mechanical tools can not detect small scale conformational changes unless strong enough and persistent force is applied. At weak forces, the flexible tether connecting the mechanical probe to the biological molecule is not stretched enough to transmit small movements. This is unfortunate because weak and transient forces are likely more prevalent in vivo but the experimental limitations confine single molecule mechanical studies to examining the effect of relatively large forces. We aimed to study the effect of weak external forces on the biomolecular conformational dynamics by combining single molecule fluorescence resonance energy transfer (smFRET) (2-4) with manipulation using optical trap (5). smFRET has high spatial resolution (≤ 5 Å) (6, 7)) and can be measured at arbitrarily low forces. Previous attempts to combine FRET and optical trap using the DNA hairpin as a model system (8, 9) did not reveal new information because the hairpin unzips at high forces (~ 15 pico Newton (pN)), a regime that had been extensively investigated using force-based techniques (10, 11). Here, we report an approach to detect nanometer-* To whom correspondence should be addressed. (tjha@uiuc.edu). † Present Address: Department of Physics and Astronomy, Seoul National University, Seoul 151-742, Korea. scale motion at sub-pN forces. We used the approach to gain insight into the reaction landscape of the Holliday junction (HJ) by gently stretching it along different directions. NIH Public AccessThe HJ is a four-stranded DNA structure that forms as an intermediate during recombination (12). To understand the mechanisms of cellular enzymes that function with the HJ, a detailed description of the static and dynamic structural properties of the HJ itself is needed. In the absence of added ions the HJ adopts an open structure, where the four helical arms point toward the corners of a square (13, 14) (Fig. 1A). In the presence ...

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Direct Measurement of the Full, Sequence-Dependent Folding Landscape of a Nucleic Acid

Cited by 370 publications

References 20 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

Minimal Models for Proteins and RNA: From Folding to Function

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

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