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

Abstract

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.

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance

“…Over recent years, interest in the development of oligonucleotide derivatives for the suppression of hyperexpressed pathogenic RNAs has grown significantly. In the last three years several novel types of oligonucleotide conjugates for targeted degradation of tumor-associated RNA targets were designed, which included Zn 2+ -and Cu 2+ -dependent conjugates of the short peptide nucleic acids (PNA) and neocuproine for degradation of Leukemia-related bcr/abl mRNA fragment [34,35]; metal-independent conjugates of DNA/LNA mixmers with tris(2-aminobenzimidazole), capable of cleaving the proto-oncogenic serine-threonine kinase PIM1 mRNA fragment [36]; and conjugates of hairpin DNA or 2 -OMe oligonucleotides and a peptide [LRLRG] 2 , so-called miRNAses, for selective inactivation of oncogenic miRNAs miR-17 and miR-21 [8][9][10].…”

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

“…Nowadays, this approach has been successfully applied for the synthesis of peptidyl-oligonucleotide conjugates of various design [ 34 , 36 , 42 , 47 , 48 , 49 , 50 ]. Another version of fragment conjugation, which was widely applied for the synthesis of various ss-aRNases, was also based on the post-synthetic coupling between the key players, when one of the reacting components (usually oligonucleotide) was still bound to the solid support, while the second component (usually catalytic moiety) was in solution [ 26 , 30 , 31 , 32 , 37 , 38 , 40 , 51 , 52 , 53 ]. The third major approach was solid-phase synthesis based on the sequential assembly of the oligonucleotide and peptide (or any other RNA cleaving groups) on a single solid support during standard synthesis to generate a complete conjugate structure [ 33 , 54 , 55 , 56 ].…”

“…In terms of RNA cleaving constructs, the most actively used groups were trisbenzimidazole [ 26 , 30 , 31 , 32 ], imidazole [ 33 ] and the peptide [(ArgLeu) 2 Gly] 2 [ 34 , 35 , 36 ], which represent metal ion-independent catalysts ( Figure 1 , Table 1). Another efficient catalytic group was dimethylphenanthroline, which chelates either Cu 2+ or Zn 2+ ( Figure 1 , Table 1), and thus represents metal-dependent catalysts [ 37 , 38 , 39 , 40 ]. Alongside these, there are several rarely used groups (acridine and azacrown) which also deserve some attention.…”

“…In that respect, ss-aRNases are similar to ASO, and many oligonucleotide analogues, which were initially introduced and tested as ASO, were later successfully used as an RNA-recognition domain within ss-aRNases. Amongst such analogues, PNAs deserve special attention, as they were used as structural elements within two highly successful series of aRNases; the first was based on the trisbenzimidazole catalyst [ 26 , 30 , 31 , 32 ], and the second was based on the dimethylphenanthroline cleaving group [ 37 , 38 , 39 , 40 ]. By switching from DNA oligonucleotides to the PNA backbone, not only was a considerable increase in the nuclease resistance of the ss-aRNase in vivo achieved, but the binding affinity and cleavage activity of the conjugate was also improved [ 26 ].…”

A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates

“…Although in modern life sciences gapmers [2,14,15] and siRNAs [16] are the preferred tools for achieving the site-specific cleavage of RNA strands, there is still interest in synthetic ribonucleases and recent years have seen important progress in the field. Effective catalysts that are based on metal ions [17][18][19][20][21][22][23], guanidines or polyamines [24][25][26][27], peptide conjugates [28][29][30][31][32], and deoxyribozymes [33,34] have been described. Starting from heterocyclic guanidine analogs [35], we have investigated the conjugates of tris(2-aminobenzimidazoles) and different types of oligonucleotides, such as DNA [36], PNA [37,38], or DNA-LNA mixmers [39], which were shown to act as sequence-specific metal-free RNA cleavers.…”