A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA

Abstract

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 59 end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 59 end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 Å, the calculation of which is derived from the 1/r 3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 59 displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding

“…Finally, in addition to halogenation of uridines and cytosines for X-ray crystallography, other modifications are commonly used in structural studies of nucleic acids: ribose modification for EPR spectroscopy (Macosko et al 1999), 29-fluoro-nucleotides (Kreutz et al 2005(Kreutz et al , 2006 or 5-fluoropyrimidines (Marshall and Smith 1977;Rastinejad et al 1995) for NMR spectroscopy, introduction of various fluorescent bases for fluorescence studies (Qin and Pyle 1999;Yamana et al 1999;Ben Gaied et al 2005), or, more recently, incorporation of selenium for crystal structure determination (Wilds et al 2002;Hobartner and Micura 2004). Our results highlight the importance of checking the possible influence on RNA folding of even subtle nucleic acids modifications.…”

Site-directed Spin Labeling Studies on Nucleic Acid Structure and Dynamics

“…Shin et al reported a method for attaching nitroxides to the 5′ terminus of RNAs synthesized by the T7 RNA polymerase (53). In this method, a modified nucleotide, guanosine 5′-O-(3-thiomonophosphate) (GMPS), is added to a mixture of unmodified nucleoside triphosphates.…”

“…All reported cw-EPR distance measurements in nucleic acids are examples of statically disordered spins, with the dipolar interaction manifesting itself as an overall spectral broadening (24,53). To use cw-EPR to measure the dipolar interaction between sites A and B of a macromolecule, three spectra should ideally be measured.…”

“…Shin et al measured distances between the HIV Rev peptide and the Rev response element (RRE) RNA (53). One nitroxide was attached to a fixed residue within the Rev peptide, and the other nitroxide was attached to 5′ terminus of the RNA using the GMPS scheme (Section III.B.1).…”

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

“…SDSL utilizes a site-specifically attached nitroxide moiety that contains a stable, unpaired electron, and obtains local structural information by analyzing the electron paramagnetic resonance (EPR) spectrum of the nitroxide [8]. It is capable of providing information on high molecular weight assemblies under physiological conditions using a small amount of sample (∼5 μl of 50 μM of sample per measurement), and has been utilized to monitor solution structure and conformational changes at specific sites of RNA molecules [9][10][11][12][13][14][15][16] and to measure distances between two labeled sites within RNAs [17][18][19][20].An important source of information in RNA SDSL is the spectral lineshape of a singly labeled nitroxide [7]. In the conventional X-band measurements, the observed lineshape changes…”