Structural Insight into Chemical Ribonucleases: High-field NMR and Molecular Modelling

Abstract

The development of novel Nucleic Acid-based artificial ribonucleases (ARs) imitating the active centre of natural enzymes that are capable of damaging messenger RNA provides a basis for generating useful tools for modern chemical biology or development of new therapeutics, affecting specific messenger RNAs and viral genomic RNAs. Recently, a new type of chemical ribonucleases, showing unusual biological properties, was discovered. These catalytic molecules were constructed by chemical fusion of short, synthetic oligopeptide with synthetic oligonucleotide fragments. The most remarkable feature of these chemical ribonucleases was that the short DNA fragment enormously enhanced the biological activity of a previously inactive peptide component. This research shows that the merger of these two chemical entities seems to produce a new, hybrid-like molecule that can synergistically combine the individual properties of the two components to yield a new and unusual biological ability. The oligonucleotide component seems to induce an 'active' structure of the catalytic peptide and hence significantly enhances its catalytic performance. However, neither structural aspects of the active conformation(s) nor the fine mechanisms of cross-modulated conformational re-arrangement and subsequent cleavage have yet been studied. This research focuses on the structural aspects of peptidyl-oligonucleotide ARs (POA) and their interactions with the complementary and non-complementary RNA targets using high-field 1H- and 31P-NMR spectroscopy, and restrained molecular modelling calculations to identify structural rules that govern their cleavage activity and base specificity. The 3D structures of the unhybridised NH2-Gly-[Arg-Leu]4-5'pTCAATC3' (POA2) and NH2-Gly-Arg-Leu-Arg-Lag-Arg-Val-Arg-Leu-5'pTCAATC3' (POA13) were resolved. The oligonucleotide strands of the free POA2 and POA13 seem to form right-handed DNA with a mixture of A and B forms. Both POA2 and POA13 form folded β-hairpin-like structures with exposed hydrophilic surfaces. Although the fine structure of the peptide parts was not resolved due to overlap of 1H signals, the mutual orientation of oligonucleotide and oligonucleotide were determined in POA2 and POA13, which induced different orientation upon replacing the two amino acid residues in POA13. DOSY experiments revealed that these constructs exist as monomers and dimers in D2O. The 3D structure of the POA13:RNA1 hybrid was resolved, and the oligonucleotide double-stranded region of the hybrid is in agreement with the right-handed DNA with a mixture of A, and B forms. The interactions between POA13 and RNA1 seem to be driven by two major binding modes, which involve electrostatic interactions and Watson-Crick base-pairing. On the other hand, interactions between POA13 and the non-complementary target RNA2 were qualitatively confirmed to be driven via electrostatic interactions only mediated by the oligopeptide fragment.The mode of aggregation seems to affect the specificity of RNA cleavage, which could explain difference in base specificity between POA2 and POA13 at high concentrations. The hypothetical mechanism of the cleavage event was then suggested based on the modes of interactions of POAs and RNA, and aggregation properties of the conjugates.

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Elena Bichenkova (Supervisor)