Peptide nucleic acid conjugates as artificial ribonucleases : Cu2+ and Zn2+-dependent PNAzymes

Abstract

Synthetic modified oligonucleotides are gaining momentum as powerful therapeutic tools used in the treatment of life-threatening diseases. Degradation of disease-related RNA sequences is one of the goals achieved with oligonucleotide therapeutics, albeit the currently available technologies rely on the recruitment of endogenous enzymes, RNase H or RISC, limiting the chemical architecture of oligonucleotide drugs. Artificial ribonucleases based on conjugates of oligonucleotide analogues can be equipped with so-called “molecular scissors” to achieve catalytic RNA cleavage independently of endogenous enzyme action. If such artificial enzymes can be developed to a state where they accomplish rapid sequence-specific RNA cleavage under biological conditions, they could expand the arsenal of oligonucleotide-based tools available for nucleic acid manipulation for biological intervention.

Previously published Cu2+-neocuproine conjugates of peptide nucleic acid (PNA) are efficient site-specific artificial enzymes (PNAzymes) that degrade RNA bulges of 4 nucleotides in length. In this thesis, the activity of these Cu2+ PNAzymes was probed further by studying the dependence of the cleavage rate on the length and composition of the RNA bulge. Reduction in the bulge length resulted in significantly slower cleavage rates for 3-nucleotide bulge-forming RNA targets. Moreover, the fast cleavage of 4-nucleotide bulges was shown to require critical functional groups – the exocyclic amino group and the 2'-hydroxyl group of the adenosine nucleotide at the cleavage site, and the PNAzyme was shown to necessarily require a chelating group as part of its structure. Finally, RNA cleavage rates were shown to be unaffected by elongation of the RNA/PNAzyme complex.

While Cu2+-dependent PNAzymes could be impactful as research tools, they offer less hope for clinical applications due to the absence of free copper ions in biological fluids. As a more biocompatible alternative, Zn2+ is a desirable cofactor for artificial enzymes, although the previously published examples of such artificial enzymes have suffered from low activity. In this thesis, novel Zn2+-dependent dimethyl-dipyridophenazine-based PNAzymes were developed. The dependence of their activity on the RNA bulge sequence, pH and Zn2+ ion concentration was studied in detail. These Zn2+ dimethyl-dppz PNAzymes cleaved 3-nucleotide bulge-forming RNA target sequences at a single site with down to 10-minute half-lives at pH 7.4, thus outperforming all previously published artificial ribonucleases. Moreover, they were shown to be capable of cleaving clinically relevant RNA sequences, namely a Plasmodium falciparum (malaria parasite) mRNA model and a SARS-CoV-2 genomic RNA model.

The sequence of the RNA target was shown to be highly significant, both in the single-stranded bulge region and in the hybridised bulge-closing regions on both sides. The cleavage of 2, 3 and 4-nucleotide RNA bulges by Zn2+ dimethyl-dppz PNAzymes was studied and the sequence requirements for efficiently cleaved RNA targets were identified. The unprecedented efficiency and specificity of Zn2+ dimethyl-dppz PNAzymes will hopefully inspire future investigations to assess their efficacy in biological settings.