Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions
Yan Wang, Venkatesan Kathiresan, Yaoyi Chen, Yanping Hu, Wei Jiang, Guangcan Bai, Guoquan Liu, Peter Z. Qin, Xianyang Fang
Abstract
under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs.