Zn²⁺-Dependent peptide nucleic acid-based artificial ribonucleases with unprecedented efficiency and specificity

Abstract

We present Zn2+-dependent dimethyl-dipyridophenazine PNA conjugates as efficient RNA cleaving artificial enzymes (10-minute RNA cleavage half-lives). These PNAzymes display site-specificity and cleavage of clinically relevant RNA models

Influence of sequence variation on the RNA cleavage activity of Zn2+-dimethyl-dppz-PNA-based artificial enzymes

“…the long recognition arm. 15 In such systems, the AAA bulge is cleaved site-specifically with an approximately 1 h half-life ( Fig. 1c ).…”

“…The present study takes a particular interest in the bulge-closing base pairs on both sides of the bulge, as well as the sequence and length of the shorter recognition arm of the RNA/PNAzyme complex. Moreover, in addition to the cleavage of 3-nucleotide RNA bulges, which have been studied in detail, 15 we demonstrate the cleavage of 2-nucleotide RNA bulges and study the sequence-dependence in 4-nucleotide bulges.…”

“… 16,18,19 Nonetheless, the recently reported novel Zn 2+ -dependent dimethyl-dipyridophenazine (dppz)-based PNAzymes are a breakthrough, as they accomplish rapid RNA cleavage with down to 16 min half-lives at pH 7.0 and 10 min at pH 7.4. 15 In addition, these artificial enzymes can be tailored to disease-related RNA targets. 15 These PNAzymes are designed to be partially complementary to the RNA target sequences in order to force the formation of a bulge in the RNA adjacent to the dimethyl-dppz “molecular scissors”, thus taking advantage of the inherent reactivity of phosphodiester linkages within single-stranded RNA bulges.…”

“… 15 In addition, these artificial enzymes can be tailored to disease-related RNA targets. 15 These PNAzymes are designed to be partially complementary to the RNA target sequences in order to force the formation of a bulge in the RNA adjacent to the dimethyl-dppz “molecular scissors”, thus taking advantage of the inherent reactivity of phosphodiester linkages within single-stranded RNA bulges. 23,24 Fast site-specific cleavage was shown for 3-nucleotide bulge-forming RNAs, including clinically relevant RNA target sequences ( i.e.…”

“…a malaria parasite Plasmodium falciparum mRNA model target and a SARS-CoV-2 genomic RNA model target). 15 While this significant development has successfully demonstrated rapid RNA cleavage of clinically relevant RNA models, the extent to which the target sequence can be modified while still maintaining efficient cleavage activity is still not fully understood. A thorough understanding of the intricacies of the sequence–activity relationships in the RNA/PNAzyme complexes can shed light on various aspects of PNAzyme action, while also being critical for the effective identification of novel RNA targets and for understanding possible off-target effects.…”