A Distance Ruler for RNA Using EPR and Site-Directed Spin Labeling

Abstract

As a basic model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. The spin labels were attached to the 2'-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MD) and Fourier deconvolution methods (FD). The 10-mer duplexes have interspin distances ranging from 10 A to 30 A based on MD; however, dipolar line broadening of the CW EPR spectrum is only observed for the RNAs for predicted interspin distances of 10-21 A and not for distances over 25 A. The conformational changes in TAR (transactivating responsive region) RNA in the presence and in the absence of different divalent metal ions were monitored by measuring distances between two nucleotides in the bulge region. The predicted interspin distances obtained from the FD method and those from MD calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules.

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

“…Therefore, RNA mutants were synthesized with 29-aminouridine and subsequently spin labeled (Edwards et al 2001;Kim et al 2004). A 100 mM solution of the RNA in 100 mM sodium borate buffer (pH 8.5) (50% v/v), DMF (20% v/v) and formamide (30% v/v) was cooled down to À8°C and incubated for 2 h with a 15-fold excess of TEMPO.…”

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

“…Therefore, the observed nitroxide lineshape, and the resulting SDSL library, is directly correlated to the structural state of the labeled base. Methods for attaching nitroxides at RNA backbone [11] and sugar positions [10,19] have been reported, and the SDR system can be utilized for establishing SDSL lineshape libraries for these nitroxides.…”

“…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…”

Forty Years of the d1/d Parameter

“…The predicted interspin distances obtained from the FDM method and those from MDS calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules (Kim et al, 2004).…”

“…A distance ruler for RNA using EPR and site-directed spin labeling (SDSL) has been suggested by Kim et al, 2004. The site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined.…”