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

Merita Murtola,Alice Ghidini,Pasi Virta,Roger Strömberg

doi:10.3390/molecules22111856

Merita Murtola, Alice Ghidini + Show 2 more

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https://doi.org/10.3390/molecules22111856

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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.

Highlights

Therapeutic oligonucleotides (ONs) provide an opportunity for treating serious, life-threatening diseases with limited options using traditional low molecular weight molecules and antibody drugs [1]. there are many ongoing clinical trials and some ON drugs [2], ON therapeutics has not quite lived up to the early expectations, especially regarding the time frame for development
We report on how the size and was motivated by the fact that we found the bulge sequence to have a substantial effect on the rate sequence of formed 3- and 4-nt bulges affect the rate and site selectivity of RNA cleavage by a of cleavage in Cu2+ -based systems
Results may have a Metal structural role affecting the reactivity of phosphodiester bonds within the bulge, making ions, the stacking interactions within an RNA/PNA duplex, and the formed RNA bulge the activity of PNAzyme sensitive to the sequence ofwithin the RNA

Summary

Introduction

Therapeutic oligonucleotides (ONs) provide an opportunity for treating serious, life-threatening diseases with limited options using traditional low molecular weight molecules and antibody drugs [1]. there are many ongoing clinical trials and some ON drugs [2], ON therapeutics has not quite lived up to the early expectations, especially regarding the time frame for development. Therapeutic oligonucleotides (ONs) provide an opportunity for treating serious, life-threatening diseases with limited options using traditional low molecular weight molecules and antibody drugs [1]. Antisense oligonucleotide (AON) action based on a 1:1 binding stoichiometry may require a relatively high dosage of ON to silence the target mRNA transcript effectively [5]. Efficacy of therapeutic ONs is more readily achieved if catalytic cleavage of the target RNA is obtained, which can occur if native enzymes (e.g., RNAse H for antisense and RNA-induced silencing complex (RISC). Modified antisense oligonucleotides often do not retain the ability to activate native enzymes. It is not completely clear if in some circumstances the activity and/or concentration of the host enzymes can become limiting

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