Chemical ribonucleases mimics represent an important class of artificial enzymes, which can be generated by conjugation of short, catalytically inactive oligopeptides and oligonucleotides to produce biologically active conjugates. The most remarkable attribute of these hybrid structures is that the covalently attached oligonucleotide moiety seems to trigger catalytic activity of a previously inactive peptide, which becomes capable of cleaving RNA sequences. These biocatalytic structures mimicking the active centres of natural ribonucleases may provide a basis for the development of novel sequence-specific gene silencing approaches. This discovery may open new exciting avenues for advanced therapeutic interventions, where a disease caused by overexpression of a pathogenic protein could be treated through controlled down-regulation of disease-relevant mRNA, viral genomic RNA or micro-RNA. However, very little is known about structural mechanisms behind the biocatalytic activity of these artificial enzymes and their interactions with RNA sequences. The research presented in this thesis attempted to investigate structural and dynamics properties of these enzyme mimetics, both alone and in the complex with the target RNA. The first part of this research was focused on a high-resolution structural analysis of these enzyme mimics using high-field NMR spectroscopy and molecular dynamics of the model conjugates with the aim to provide a complete 3D description of their structure and dynamic behaviour. In order to overcome the challenges associated with the highly repetitive nature of the peptide component, we used site-specific 13C/15N isotopic labelling of the model conjugates, which allowed us to considerably increase the number of the NMR derived distance constraints and thus provide a more accurate description of the most populated conformational ensembles. The second part of this research was focused on the investigation of the structural aspects of RNA targeting by this class of peptidyl-oligonucleotide congugates, which were carefully designed to hybridise and cleave the highly oncogenic microRNA-21 in a sequence-specific manner. To gain structural insights into specific interactions between these catalytic conjugates and their bio-targets, we carried out 1 Î¼s molecular dynamics simulations of the hybridised complex between miR-21 and one of the most efficient chemically engineered miRNase. The results of this thesis are vital for the future design of such conjugates, which may guide future development of novel, more efficient therapeutic interventions based on the RNA targeting.
|Date of Award||31 Dec 2018|
- The University of Manchester
|Supervisor||Harmesh Aojula (Supervisor) & Elena Bichenkova (Supervisor)|