Monitoring folding pathways for large RNAs using site-directed spin-labeling techniques

The function of biomolecules is very sensitive to structure. Folding in proteins and nucleic acids is a hierarchical process progressing from primary to secondary, then tertiary, and finally, quaternary structures. RNA in its folded form performs a variety of biological activities. Obtaining intramolecular distance measurements makes it possible to generate structural models along the folding pathway that may be related to the overall function of the molecule. Distances can be measured by Site-Directed Spin-Labeling (SDSL), in which nitroxyl spin-label probes are attached and observed by EPR spectroscopy. Spin-labels can provide information concerning structure and conformational changes because they are particularly sensitive to molecular motion and interspin distances. Continuous-wave EPR spectroscopy has been commonly applied to detect and monitor nitroxide spin-label probes within biological systems. A previous published SDSL study from this laboratory investigated a 10-mer RNA duplex model system with spin-label probe succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate; however, an increased spin-labeling efficiency was observed with an isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO). In this thesis, a 4-isocyano TEMPO spin-label probe replaced the previously used succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate in 10-mer SDSL studies. The influence of labeling with the 4-iscocyano TEMPO spin-label in a 10-mer RNA model system was investigated with thermal denaturation, Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS), Electron Paramagnetic Resonance (EPR) spectroscopy, and reverse phase high performance liquid chromatography (RP-HPLC). In the 10-mer RNA duplex model system a 4-isocyano TEMPO spin-label is individually attached to one strand and two strands are annealed to measure distances. This methodology is limited to systems in which two oligonucleotides are annealed together. To circumvent this limitation and also to explore single-strand dynamics a new methodology was implemented, double spin-labeling. Double spin-labeled single-stranded RNA was investigated as a single-strand and within a duplex via MALDI-TOF-MS, EPR spectroscopy and RP-HPLC. A double spin-labeling strategy in this work will be applicable to large complex RNAs like Group I intron of Tetrahymena thermophilia