IMPROVED DISTANCE ANALYSIS IN RNA USING NETWORK-EDITING TECHNIQUES FOR OVERCOMING ERRORS DUE TO SPIN-DIFFUSION

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determi nation of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances wit h greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are ap plied to a lead-dependent ribozyme (LZ2). We demonstrate the measureme nt of both aromatic proton-aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton-ribose proton NOEs free of the efficient cross-relaxation within the ribose r ing. In LZ2, the accuracy and precision of the resulting distances ale significantly improved. We also find that, by allowing the use of lon ger mixing times with greater sensitivity, the experimental attenuatio n of spin diffusion in RNA increases the distance range of interaction s that can be analyzed. This effect permits measurement of important l ong-range distances in LZ2 that are not accessible with standard techn iques. Thus, these techniques allow the simultaneous optimization of t he number, accuracy, and precision of distance constraints used for RN A structure determinations.