Abstract
The development of Zn2+-dependent dimethyl-dppz-PNA conjugates (PNAzymes) as efficient site-specific artificial ribonucleases enables rapid sequence-specific degradation of clinically relevant RNA target sequences, but the significance of the RNA/PNAzyme sequence and structural demands for the identification of novel RNA targets are not fully understood. In the present study, we investigated the influence of sequence variation in the recognition arms of the RNA/PNAzyme complex on the RNA cleavage activity of the artificial enzymes. The base pairs closing the 3-nucleotide bulge region on both sides of the bulge as well as the neighbouring nucleobases were shown to significantly influence the RNA cleavage activity. Elongation of the RNA/PNAzyme complex was shown to be tolerated, although potentially prohibitive for catalytic turnover. The specificity of PNAzyme action was clearly demonstrated by the significantly reduced or absent cleavage activity in complexes containing mismatches. Further investigation into 2- and 4-nucleotide RNA bulges indicated that formation of 3-nucleotide bulges in the target RNA gives the optimal cleavage rates, while some potential off-target cleavage of formed 4-nucleotide bulges of select sequences should be considered.
Highlights
The development of efficient arti cial enzymes capable of sequencespeci c cleavage of RNA has been a long-standing goal of nucleic acid chemistry.[4,5,6,7,8]. Such arti cial ribonucleases have the potential to achieve degradation of disease-related RNA targets without the assistance of endogenous enzymes, thereby offering an alternative to gapmer antisense oligonucleotides (ASOs) and small interfering RNAs,[3,9] both of which are used in the clinic today
Sequence–activity relationships in the long recognition arm The reported RNA/PNAzyme complexes (Fig. 1) contain a CG base pair closing the bulge in the longer hybridised stem, i.e. the long recognition arm.[15]
This study has shown that the RNA cleavage activity is critically dependent on the sequence of the RNA/PNAzyme complex
Summary
Nucleic acid manipulation is important in molecular biology[1] as well as for therapeutic interventions in the clinic.[2,3] The development of efficient arti cial enzymes capable of sequencespeci c cleavage of RNA has been a long-standing goal of nucleic acid chemistry.[4,5,6,7,8] Such arti cial ribonucleases have the potential to achieve degradation of disease-related RNA targets without the assistance of endogenous enzymes, thereby offering an alternative to gapmer antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs),[3,9] both of which are used in the clinic today. The effective identi cation of novel RNA targets and for understanding possible off-target effects. 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 sequencedependence in 4-nucleotide bulges
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