Mechanistic characterization of RNA-ligating deoxyribozymes by chemical and biophysical methods and Spin-labeled RNA for EPR spectroscopy

Deoxyribozymes are increasingly recognized as practical tools for diverse applications in vitro and potentially in vivo, in particular for the modification and manipulation of RNA. Very little is currently known about the underlying mechanistic principles of DNA catalysis. The first part focuses therefore on the mechanistic characterization of RNA-ligating deoxyribozymes. Two combinatorial methods for probing functional DNA based on solid-phase synthesis were developed and successfully applied to the analysis of essential nucleotides and nucleotide functional groups in deoxyribozymes and DNA aptamers. For both methods, ribonucleotides are statistically incorporated into DNA sequences, with the 2’OH group acting as chemical tag. The second part focuses on the synthesis of spin labeled RNA for EPR spectroscopy. A new spin labeling technique for RNA was developed based on the convertible nucleotide approach. This technique enables the fast and efficient postsynthetic and site-specific installation of nitroxide labels at the exocyclic amino groups of A and C in the major groove and G in the minor groove. This labeling strategy was applied to different hairpin and duplex RNAs. Characterization of double labeled RNA systems by PELDOR EPR spectroscopy revealed a narrow distance distribution. The new spin labels are therefore suitable candidates for reliable distance measurements in RNA.