Abstract
Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses–miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide “shoulders” of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5–7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5′- and 3′-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.
Highlights
Rapid progress in the study of the role of non-coding RNAs in the functioning of genome has broadened the scope of therapeutic targets for a variety of human diseases
These miRNAs were chosen as targets for Dual conjugate (DC), since they represent multifunctional regulators of cellular processes and their expression is impeded in wide spectrum of pathologies [14,15,16,17,18,19,20] (Table S1)
The “dual” conjugate design of miRNA specific peptidyl-oligonucleotide conjugates involves placing of the catalytic peptide [LRLRG]2 in-between two short miRNA-targeting deoxyribo-oligonucleotide recognition motifs (Figure 1 and Table 1) to ensure that it is located opposite to the central region of the miRNA sequence
Summary
Rapid progress in the study of the role of non-coding RNAs in the functioning of genome has broadened the scope of therapeutic targets for a variety of human diseases. The main components of the miRISC are TNRC6A, TNRC6B or TNRC6A proteins from GW182 family functioning as scaffold and Ago that serves as a regulator of miRNA stability and activity and promotes mRNA cleavage [1]. Binding of Ago to miRNA divides it into three functional domains: (1) “seed chamber” corresponding to the seed region of miRNA (nucleotides 2–8 at the 50 -end), (2) “central gate” which matches the region from 9th to 13th nt of miRNA, and (3) “supplementary chamber” that comprises 30 -end of the molecule. As well as in the “central gate” of miRNA/Ago complex; and (3) miRNA degradation, if mRNA interacts with miRNA in the “seed region” and “supplementary chamber” [2]. Design of various oligonucleotide-based constructions for miRNA inhibition is the most actively developing area in miRNA-based therapy [3]
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