Mimicking the action of ribonucleases : studies on RNase A and design of PNA based artificial enzymes

Abstract: A 3’-deoxy-3’-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. Despite that both the vicinal 2-hydroxy nucleophile and the 5’-oxyanion leaving group are intact, the 3’-methylenephosponate RNA modification is also highly resistant towards the action of RNase A. Several different approaches were explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Using a post PNA-assembly procedures oligoether attachment to both N-terminal and sidechain amino groups was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis, allowing combination of methods for attachment of different oligoethers and co-conjugation of neocuproine cleaver. We have previously shown that PNA-neocuproine conjugates can act as artificial RNA restriction enzymes (PNAzyme). In the present study we have additionally conjugated the PNA with different entities and also constructed systems where the PNA is designed to clamp the target RNA forming a triplex. Some conjugations are detrimental for the activity while most are silent which means that conjugation can be done to alter physical properties without losing activity. Conjugation with a single oligoether close to the neocuproine does enhance the rate almost two folds compared to the system without the oligoether. The systems designed to clamp the RNA target by forming a triplex are effective if the clamping part is not too long. Changing the direction of a closing base pair, from a GC to a CG pair, enhances the rate of cleavage with a clamping PNAzyme and without compromising the selectivity, leading to the so far most efficient artificial nuclease reported. Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific metal-free RNA cleavers. Here we investigate conjugates with peptide nucleic acids (PNA). In a first study we show that RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates. In a second study we show that tris(2-aminobenzimidazole) based artificial nucleases cleave RNA substrates, which form a bulge upon binding to the PNA, with turnover of substrate and a cleavage rate that is also dependent on the bulge sequence. Two methods of analysis for the kinetics, based on IE-HPLC separation of oligonucleotide fragments and analysis of Cy5-labelled oligonucleotide fragments by denaturating PAGE on a DNA sequencer respectively are also compared. To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have looked at BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.