title   
  

High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides

Halbmair, Karin and Seikowski, Jan and Tkach, Igor and Höbartner, Claudia and Sezer, Deniz and Bennati, Marina (2016) High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides. Chemical Science, 7 (5). pp. 3172-3180. ISSN 2041-6520 (Print) 2041-6539 (Online)

[img]
Preview
PDF (This is a RoMEO green journal -- author can archive publisher's version/PDF) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
964Kb

Official URL: http://dx.doi.org/10.1039/c5sc04631a

Abstract

Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson–Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the “ideal” A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an “ideal” A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.

Item Type:Article
Subjects:Q Science > QD Chemistry > QD001-65 General
ID Code:29740
Deposited By:Deniz Sezer
Deposited On:04 Nov 2016 14:59
Last Modified:04 Nov 2016 14:59

Repository Staff Only: item control page