Peptidyl-oligonucleotide chemical ribonucleases: synthesis, characterisation and spectroscopic studies

Abstract

ABSTRUCT:The development of novel biocatalytic supramolecular structures mimicking the active centre of natural ribonucleases and capable of cleaving RNA targets can provide a basis for generating new useful biological tools and powerful therapeutics, affecting specific messenger RNAs and viral genomic RNAs. In spite of impressive advances made toward the development of sequence-specific artificial ribonucleases, the problem of insufficient efficiency of the existing metal-independent chemical ribonucleases, their poor selectivity and low catalytic turnover remains unmet. Recently, a new type of peptidyl-oligonucleotide chemical nucleases (PCNs), showing very unusual catalytic and structural properties, has been discovered. These novel oligonucleotide-mediated chemical nucleases were constructed by chemical conjugation of short, catalytically inactive oligopeptides containing alternating basic and hydrophobic amino acids with an oligonucleotide component, which are poorly or non-complementary to RNA target regions. The most remarkable feature of these novel biocatalysts was that the covalently attached oligonucleotide units induced catalytic activity of a previously inactive peptide and modulated its cleavage specificity towards RNA. However, the lack of knowledge of the structural aspects behind this discovery hinders the development of a new generation of peptidyl-oligonucleotide conjugates. Also, very little is known about the modes of interactions between peptidyl-oligonucleotide chemical ribonucleases and target RNA sequences, which create the additional barrier for improved design of new structural variants of PCN with an appropriate balance between sequence-specificity and high catalytic turnover.The aim of this research, therefore, was to investigate the structural aspects of this class of catalytic artificial ribonucleases using 1D and 2D NMR spectroscopy as well as their modes of interactions with the complementary and non-complementary RNA sequences using fluorescence, UV-visible spectroscopy, and Tm profile studies.To achieve this, the selected model conjugates (NH2-Gly-[ArgLeu]4-5'pTCAATC (17), NH2-Gly-[ArgLeu]4-5'pTTTTCAATC (18) and NH2-Gly-[ArgLeu]4-5'pTTTTCAATCp3'-Cy3 (19)) were synthesised and their identity confirmed using HPLC, NMR spectroscopy, mass spectrometry, UV-visible and fluorescence spectroscopies. The challenge here was to investigate how these functionally significant entities (peptide and oligonucleotide) interact with each other within the conjugate and cross-modulate their activities. The analysis of NOESY for 17 and 18 allowed the identification of inter-proton interactions within these structures and provide the information about mutual orientations of the fragments and interactions between the individual components. These structural studies provide sufficient evidence that both oligonucleotide and peptide cross-modulate each other's conformations leading to the formation of a new entity with unique structural properties. The final stage of this research was aimed to study the main modes of interactions between PCNs and different RNA sequences. To achieve this, binding experiments between the selected PCNs (non-fluorescent and fluorescently-labelled) and FAM-labelled RNA targets were carried out to follow the fluorescence and UV-visible response upon interaction. The oligonucleotide was shown to play the dominant role in the interactions between the conjugates 17-19 and the complementary target in the presence of a high level of counter cations, which were sufficient to prevent repulsion between the complementary strands. The peptide component seems to become the key player in these interactions, when the concentrations of counter cations are insufficient or negligible. It was also demonstrated that the conjugates 17-19 are capable of forming strong complexes with non-complementary RNA sequences at low concentrations of counter cations or in their absence, presumably due to electrostatic interactions between the negatively charged sugar-phosphate backbones of the RNA and the positively charged arginines of the conjugates. This mode of binding, which is mainly mediated by the peptide moiety via electrostatic interactions with the RNA strand, seems to represent the only possibility for interactions between the non-complementary RNA and the conjugate in water.

Date of Award	27 May 2011
Original language	English
Awarding Institution	The University of Manchester