Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA—Bulge Size and Sequence Dependence

Abstract

In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7–8 h as compared to 11–12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50–60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21–24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7–8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.

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].…”

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.…”

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

“…Oligonucleotide-based artificial nucleases (OBANs) have been shown to catalytically cleave their RNA targets in a sequence- and site-selective manner [ 7 , 8 , 9 ]. Neocuproine, as a metal chelate, has also been incorporated into a peptide nucleic acid (PNA) backbone at a central position, giving rise to PNA-based artificial ribonucleases (PNAzymes) [ 10 , 11 , 12 , 13 , 14 ]. These PNAzymes are designed to be partially complementary to the RNA target in such a way as to force the formation of an RNA bulge at the position adjacent to the chelating moiety.…”