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RNA dynamics: perspectives from spin labels

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Abstract Dynamics are important and indispensible physical attributes that play essential roles in RNA function. RNA dynamics are complex, spanning vast timescales, and encompassing a large number of physical modes. The technique of site‐directed spin labeling (SDSL), which derives information on local structural and dynamic features of a macromolecule by monitoring a chemically stable nitroxide radical using electron paramagnetic resonance spectroscopy, has been applied to monitor intrinsic dynamics at defined structural states as well as to probe conformational transition dynamics of RNAs. The current state of SDSL studies of RNA dynamics is summarized here. Further development and application of SDSL promise to open up many more opportunities for probing RNA dynamics and connecting dynamics to structure and function. WIREs RNA 2012, 3:62–72. doi: 10.1002/wrna.104 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry

Site‐directed spin labeling studies of nanosecond motions of the P1 duplex in the Tetrahymena group I ribozyme. (a) Schematic representation of two ribozyme states: the open and closed complexes. Asterisk indicates the nitroxide label shown in the R1 panel of Figure 2. (b) X‐band cw‐EPR spectra of open and closed complexes acquired in aqueous buffer at 25°C. The broader spectrum of the closed complex indicates slower motions, which arises from reduced P1 mobility in the closed complex. (Reprinted with permission from Ref 50. Copyright 2009 American Chemical Society)

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Representative examples of nitroxide attached to backbone (R1), 2′‐position of sugar (R2), or various positions of base (R3) of a specific nucleotide within a nucleic acid strand.

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General strategy of site‐directed spin labeling.

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SDSL studies of NCp7 protein binding to HIV‐I RNA stem loop 3. (a) Schematics of NCp7–RNA complex formation. (b) Detection of NCp7–RNA complex formation by NCp7 induced X‐band EPR spectral broadening, which reports reduction of the overall tumbling of the nitroxide‐labeled RNA stem loop. (c) Stopped‐flow measurements of EPR spectral amplitude reveal a bi‐phasic behavior during formation of the NCp7–RNA complex. (Reprinted with permission from Ref 73. Copyright 2008 American Chemical Society)

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SDSL studies of the TAR RNA. (a) The secondary structure of the TAR RNA construct used in the study, with the four labeled uridines shown in bold. (b) Chemical structure of the nitroxide‐labeled uridine. (c) An example of variations of dynamic signatures, which are represented by differences in the effective hyperfine splitting obtained from measured X‐band cw‐EPR spectra, upon TAR binding to various small molecule ligands (I: Hoechst; II: DAPI; III: berenil; IV: CGP40336A; V: neomycin; VI: guanidine neomycin; VII: arginiamide). (Reprinted with permission from Ref 19. Copyright 2004 Elsevier Limited)

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SDSL distance measurements reveal changes of conformational disordering in the hammerhead ribozyme (HHRz) upon addition of Mg2+. A schematic representation of the HHRz being studied is shown on the left. Measured distance distribution profiles at various Mg2+ concentrations are shown on the right, with the x‐axis being the interspin distance and the y‐axis representing the probability of finding a given distance. (Reprinted with permission from Ref 39. Copyright 2010 American Chemical Society)

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The J1/2 element modulates nanosecond dynamics of the P1 duplex in the open complex of the Tetrahymena group I ribozyme. Simulations of the measured spectra indicate that lengthening J1/2 (from 0, 3, to 8 adenosines) results in decreases in motional ordering (lower order parameter S) and slower rotational diffusion rate (smaller R). This reveals changes in intrinsic dynamics of the P1 duplex upon alteration of J1/2. (Reprinted with permission from Ref 50. Copyright 2009 American Chemical Society)

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