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

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

Further Probing of Cu²⁺-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target

Innovative developments and emerging technologies in RNA therapeutics

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.ISSN:1547-6286ISSN:1555-858