Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H

Abstract

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3′-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH2-Gly(ArgLeu)4-TCAA displaying ribonuclease activity to its 5′-end. Designed miRNases efficiently cleaved miRNA targets, exhibiting Pyr-X specificity, and cleavage specificity had strong dependence on the miRNA sequence in the site of peptide location. In vitro, designed miRNases do not prevent cleavage of miRNA bound with the conjugate by RNase H, and more than an 11-fold enhancement of miRNA cleavage by the conjugate is observed in the presence of RNase H. In murine melanoma cells, miRNase silences mmu-miR-17 with very high efficiency as a result of miR-17 cleavage by miRNase and by recruited RNase H. Thus, miRNases provide a system of double attack of the miRNA molecules, significantly increasing the efficiency of miRNA downregulation in the cells in comparison with antisense oligonucleotide.

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance

“…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. The location of the peptide at the internal position of the conjugates, rather than at the end of the 5 -end of the oligonucleotide recognition motifs [9,10,13,21], provides the opportunity to cleave miRNA approximately in the middle of the chain, leading to a complete loss of normal functioning of the molecule. However, such design requires addressing the key thermodynamic challenges, emerging from targeting relatively short miRNA substrates (≤25 nt), when two recognition motifs of the Dual conjugate need to be short enough (in order to allow 5-nt cleavage site in the middle of miRNA to remain single-stranded), but strong enough in terms of their hybridization ability.…”

“…The ribonuclease activity of DCs was studied under two different buffer conditions: buffer (1) contained 50 mM Tris-HCl, pH 7.0, 200 mM KCl, 1 mM EDTA, which imitates intracellular conditions (e.g., pH and potassium ions concentration) and was used in a number of studies previously [9,10,[26][27][28]; and buffer (2) contained 20 mM Tris-HCl, pH 7.8, 40 mM KCl, 8 mM MgCl 2 , 1 mM DTT, 0.02 mg/mL BSA, which is recommended for in vitro reactions with RNase H (Figures 3 and 4). The cleavage of synthetic miRNA targets at a concentration of 1 µM was carried out at a 20-fold excess of the corresponding DCs (20 µM), since in the previous studies these conditions were found to be optimal for comparison of cleaving activity of miRNases [9,10,13]. It was shown that the composition of the buffer could strongly affect the specificity and efficiency of miRNAs cleavage by DCs (Figures 3 and 4).…”

“…A new, promising strategy for the highly specific inhibition of pathologically overexpressed miRNAs is the use of miRNA-targeted sequence-specific artificial ribonucleases (aRNases) [8][9][10][11][12]. These RNA cleaving compounds are conjugates of an addressing oligonucleotide that selectively binds to an miRNA target and a covalently attached cleaving group that catalyzes the degradation of target RNA molecule.…”

“…Instead, long, continuous stretches of DNA or hairpin sequences could be employed to enable the precise recognition and efficient hybridization with short non-coding miRNA sequences [8,10,12]. We have recently designed miRNA-targeted sequence-specific artificial RNases, termed miRNases, intended to inactivate miR-17 and miR-21 oncogenic targets [8,9]. The miRNase represents a particular class of the peptidyl-oligonucleotide conjugates, consisting of a hairpin-like DNA (to enhance binding affinity) complementary to the 5 -region of miRNA target, and a cleaving peptide containing alternating arginine and leucine residues.…”

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.…”