Nanometer Distance Measurements in RNA Using Site-Directed Spin Labeling

The method of site-directed spin labeling (SDSL) utilizes a stable nitroxide radical to obtain structural and dynamic information on biomolecules. Measuring dipolar interactions between pairs of nitroxides yields internitroxide distances, from which quantitative structural information can be derived. This study evaluates SDSL distance measurements in RNA using a nitroxide probe, designated as R5, which is attached in an efficient and cost-effective manner to backbone phosphorothioate sites that are chemically substituted in arbitrary sequences. It is shown that R5 does not perturb the global structure of the A-form RNA helix. Six sets of internitroxide distances, ranging from 20 to 50 Å, were measured on an RNA duplex with a known X-ray crystal structure. The measured distances strongly correlate (R2 = 0.97) with those predicted using an efficient algorithm for determining the expected internitroxide distances from the parent RNA structure. The results enable future studies of global RNA structures for which high-resolution structural data are absent.