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

O A Patutina,N L Mironova,S K Miroshnichenko,D V Pyshnyi,V V Vlassov,M A Bazhenov,M A Zenkova

doi:10.1038/s41598-018-33331-z

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.

Highlights

Tumor development is accompanied by rapid proliferation, loss of differentiation, development of tumor cell resistance to death, reprogramming of energy metabolism, loss of adhesion between cells and tumor matrix, evasion of immune surveillance, immunosuppression, induction of angiogenesis, infiltration growth, and metastasis[1]
MicroRNAs are difficult objects for cleavage by sequence-specific artificial ribonucleases due to their short length and specific sequence often lacking linkages that are sensitive to hydrolysis in the 3′-region
The conjugates targeted miRNAs – miRNases - are built of two parts: oligonucleotide complementary to miRNA target and catalytic fragment which could be peptide[11,16] or other constructions displaying ribonuclease activity

Summary

Introduction

Tumor development is accompanied by rapid proliferation, loss of differentiation, development of tumor cell resistance to death, reprogramming of energy metabolism, loss of adhesion between cells and tumor matrix, evasion of immune surveillance, immunosuppression, induction of angiogenesis, infiltration growth, and metastasis[1]. Is application of artificial ribonucleases which are conjugates of oligonucleotides complementary targeted to miRNAs and catalytic constructions[10,11,12] This strategy was used for down-regulation of different viral RNAs13,14 and eukaryotic mRNAs15 and recently taking into account widespread of miRNA was applied for down-regulation of oncogenic miRNAs10,11. We developed metal-independent artificial ribonucleases — peptide-oligonucleotide conjugates (POCs) targeted to highly oncogenic mmu-miR-21(‘miRNases’), capable to cleave site- this miRNA exclusively at G-X linkages, and demonstrated specific inhibition of this miRNA in lymphosarcoma cells and significant reduction of cell proliferation[11]. The cleavage specificity was determined by the sequence of the peptide[16]

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