Chapter 6. Nucleic acid structure and dynamics: perspectives from site-directed spin labeling

Ding, Yuan Chun; Nguyen, Phuong H.; Tangprasertchai, Narin S.; Reyes, Carolina Vazquez; Zhang, Xiaojun; Qin, Peter Z.

doi:10.1039/9781782620280-00122

Cited by 17 publications

(22 citation statements)

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“…37 Together with the results here demonstrating that inter-R5 distance measurements can distinguish different GQ folds, the R5 tool kit provides a new means for studying the conformation and conformation changes of long DNA and RNA sequences that form GQ and even multimeric GQs, as well as investigating how a ligand might impact GQ conformations. Because studying long DNA/RNA molecule remains challenging for most physicochemical tools, we believe that our joint experimental–computational protocol will provide valuable information regarding the structure–function interplay for supramolecular GQ structures, which are likely to fold from long GQ motifs in vivo .…”

Section: Discussionmentioning

confidence: 65%

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Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

Zhang

Felice

et al. 2015

Biochemistry

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Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron–electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

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Section: Discussionmentioning

confidence: 65%

“…As a result, distances between a pair of nitroxides can be measured more precisely and can be explicitly correlated to the native structure. 37,38 …”

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confidence: 99%

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

Zhang

Felice

et al. 2015

Biochemistry

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“…1; Hubbell & Altenbach, 1994). SDSL has been demonstrated as a powerful tool for investigating structure and dynamics of biomolecules, as evidenced by accompanying articles in this volume, as well as a number of excellent reviews (Ding et al, 2015; Fedorova & Tsvetkov, 2013; Hubbell & Altenbach, 1994; Hubbell, Cafiso, & Altenbach, 2000; Hubbell, López, Altenbach, & Yang, 2013; Krstic, Endeward, Margraf, Marko, & Prisner, 2012; Shelke & Sigurdsson, 2012; Sowa & Qin, 2008). …”

Section: Introductionmentioning

confidence: 98%

An Integrated Spin-Labeling/Computational-Modeling Approach for Mapping Global Structures of Nucleic Acids

Tangprasertchai

Zhang

Ding

et al. 2015

Methods in Enzymology

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The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modeling to map conformations of nucleic acids. This approach builds upon a SDSL tool kit previously developed and validated, which includes three components: (i) a nucleotide-independent nitroxide probe, designated as R5, which can be efficiently attached at defined sites within arbitrary nucleic acid sequences; (ii) inter-R5 distances in the nanometer range, measured via pulsed EPR; and (iii) an efficient program, called NASNOX, that computes inter-R5 distances on given nucleic acid structures. Following a general framework of data mining, our approach uses multiple sets of measured inter-R5 distances to retrieve “correct” all-atom models from a large ensemble of models. The pool of models can be generated independently without relying on the inter-R5 distances, thus allowing a large degree of flexibility in integrating the SDSL-measured distances with a modeling approach best suited for the specific system under investigation. As such, the integrative experimental/computational approach described here represents a hybrid method for determining all-atom models based on experimentally-derived distance measurements.

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“…SDSL monitors site-specifically attached stable radicals (e.g., nitroxide spin labels) using electron paramagnetic resonance (EPR) spectroscopy, and provides structural (e.g., distance constraints) and dynamic (e.g., motions at the labeling site) information on the parent molecule (20,21). SDSL can be applied to study high-molecular-weight systems under physiological conditions, and avoids a number of issues faced by crystallography (e.g., preparation of diffractable crystals, interference from lattice packing) and NMR (e.g., molecule size limitation).…”

Section: Introductionmentioning

confidence: 99%

“…SDSL can be applied to study high-molecular-weight systems under physiological conditions, and avoids a number of issues faced by crystallography (e.g., preparation of diffractable crystals, interference from lattice packing) and NMR (e.g., molecule size limitation). SDSL has matured as a method for investigating proteins (20,22,23) and membranes (24,25), and has been applied to study nucleic acids and, to a more limited extent, protein-nucleic acid complexes (21,26–30). …”

Section: Introductionmentioning

confidence: 99%

Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling

Reyes

Tangprasertchai

Yogesha

et al. 2016

Cell Biochem Biophys

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In a type II Clustered-Regularly-Interspersed-Short-Palindromic-Repeats (CRISPR) system, RNAs derived from the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet, details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic-acid dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain its function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

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Chapter 6. Nucleic acid structure and dynamics: perspectives from site-directed spin labeling

Cited by 17 publications

References 0 publications

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

An Integrated Spin-Labeling/Computational-Modeling Approach for Mapping Global Structures of Nucleic Acids

Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling

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