64Cu and fluorescein labeled anti-miRNA peptide nucleic acids for the detection of miRNA expression in living cells

Stefania Croci,Roberto Corradini,Gianfranco Cicoria,Michele Iori,Pier Cesare Capponi,Carlo Salvarani,Annibale Versari,Martina Bonacini,Sara Rubagotti,Maria Parmeggiani,Alex Manicardi,Mattia Asti

doi:10.1038/s41598-018-35800-x

Abstract

MiRNAs are single stranded RNAs of 18–22 nucleotides. They are promising diagnostic and prognostic markers for several pathologies including tumors, neurodegenerative, cardiovascular and autoimmune diseases. In the present work the development and characterization of anti-miRNA radiolabeled probes based on peptide nucleic acids (PNAs) for potential non-invasive molecular imaging in vivo of giant cell arteritis are described. MiR-146a and miR-146b-5p were selected as targets because they have been found up-regulated in this disease. Anti-miR and scramble PNAs were synthesized and linked to carboxyfluorescein or DOTA. DOTA-anti-miR PNAs were then labelled with copper-64 (64Cu) to function as non-invasive molecular imaging tools. The affinity of the probes for the targets was assessed in vitro by circular dichroism and melting temperature. Differential uptake of fluorescein and 64Cu labeled anti-miRNA probes was tested on BCPAP and A549 cell lines, expressing different levels of miR-146a and -146b-5p. The experiments showed that the anti-miR-146a PNAs were more effective than the anti-miR-146b-5p PNAs. Anti-miR-146a PNAs could bind both miR-146a and miR-146b-5p. The uptake of fluorescein and 64Cu labeled anti-miR-146a PNAs was higher than that of the negative control scramble PNAs in miRNA expressing cells in vitro. 64Cu-anti-miR-146a PNAs might be further investigated for non-invasive PET imaging of miR-146 overexpressing diseases.

Highlights

MicroRNAs are single stranded RNAs of 18–22 nucleotides
A Peptide nucleic acids (PNAs) with scrambled sequence was designed as control; this was chosen aiming at minimizing its interaction with off-target sequences, as evaluated by the BLAST search on human transcriptome
In order to enable the uptake of the probes by the cells, a cell-penetrating peptide (CPP) composed of eight arginine residues (R8)[18,19] was linked to the PNA sequences

Summary

Introduction

MicroRNAs (miRNA, or miR) are single stranded RNAs of 18–22 nucleotides. They do not code proteins but regulate multiple mRNAs through base pairing mainly inhibiting translation or inducing mRNA degradation. The main advantages of the use of PNAs in this type of applications derive from their high affinity and sequence specificity in the interactions with complementary DNA and RNA, and from their great resistance to chemical and enzymatic degradation, making them suitable for the use in biological fluids and in vivo[15] Though their cellular uptake is rather limited as such, conjugation with cell-penetrating peptides (CPP), cationic backbones or nanoparticles can improve their delivery. MiR-146a and miR-146b-5p were selected as targets because they have been found up-regulated in inflamed temporal arteries from patients with giant cell arteritis (GCA) compared to normal, non-inflamed temporal arteries[17], with the aim of providing a molecular imaging approach for the early detection of GCA Such miRNAs have been found up-regulated in other autoimmune diseases and have a prognostic value in thyroid and lung cancers. The development of anti-miR PNAs based imaging could be readily applied to any other disease characterized by these and other miRNA overexpression

Methods

Results

Conclusion