PNA based florescence probes for a non-invasive detection of circulating miRNAs involved in Coeliac Disease.

Coeliac disease (CD) is an autoimmune enteropathy triggered by the interaction of environmental and genetic factors. The ingestion of dietary gluten induces an inflammatory process that results in the damage of the bowel mucosa (1). To date, the only way to confirm a celiac disease diagnosis is to have an intestinal biopsy, a very invasive method. The importance of early diagnosis for avoiding symptoms and complications underlines the need for tools that can detect CD as early as possible. Numerous researchers have tried to find novel molecular biomarkers to employ as alternative methods to avoid invasive biopsies. In the last two decades, microRNAs (miRNAs) have been widely studied and discovered to regulate many biological processes such as differentiation, development, and cell death. MiRNAs have been found circulating in different body fluids, such as serum and plasma, protected by a protein binding or enclosed in vesicles and could represent reliable and promising diagnostic, prognostic, and therapeutic biomarkers (2). In this work a group of circulating miRNAs deregulated in celiac disease have been identified (miRNA-449a, miRNA-492, miRNA-21, miR-486) and two types of probes based on peptide nucleic acid (PNA), fully complementary to miRNA sequences and labelled with two different fluorophores were developed. In order to obtain an innovative, non-invasive kit for the early diagnosis of this disease: a) PNA "light up" probes were synthesized and tagged with the fluorophore thiazole orange (TO), which lights-up upon hybridization with the target (3); b) a PNA oligomer labelled with Methylene Blue (MB) fluorophore was realized and its physical-chemical properties was compared with that of light up probes (4). CD and fluorescence experiments were carried out to demonstrate the formation of the PNA-RNA complexes and validate the feasibility of this new approach.