A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3'UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells.

Shaiq Sultan,Andrea Rozzi,Alessia Finotti,Ilaria Lampronti,Roberto Gambari,Roberto Corradini,Jessica Gasparello,Giulio Cabrini,Alex Manicardi,Eva Reali,Monica Borgatti,Chiara Papi

doi:10.3390/molecules25071677

Abstract

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.

Highlights

Peptide nucleic acids (PNAs) are DNA analogues of outstanding biological properties [1,2,3,4] since, despite a radical structural change with respect to DNA and RNA, they are capable of sequence-specificMolecules 2020, 25, 1677; doi:10.3390/molecules25071677 www.mdpi.com/journal/moleculesMolecules 2020, 25, 1677 and efficient hybridization with complementary nucleic acids, forming Watson–Crick double helices [1].In addition, they are able to generate triple helices with double stranded DNA and to perform strand invasion [4,5]
Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p
An alternative strategy for up-regulating cystic fibrosis transmembrane conductance regulator (CFTR) might be the masking of the miR-145-5p binding site with PNAs directed against this sequence (Figure 1, bottom)

Summary

Introduction

Molecules 2020, 25, 1677 and efficient hybridization with complementary nucleic acids, forming Watson–Crick double helices [1]. They are able to generate triple helices with double stranded DNA and to perform strand invasion [4,5]. They have been used as very efficient tools for pharmacologically-mediated alteration of gene expression, both in vitro and in vivo [6,7,8].

Objectives

Methods

Results

Conclusion