Abstract
Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3’UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3’UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.
Highlights
One in every 3000 newborn Caucasians is affected by Cystic Fibrosis (CF), an autosomal recessive genetic disorder caused by mutations in a gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride-conducting transmembrane channel expressed in most epithelial and blood cells
In a previous study, using an in vitro system based on the luciferase reporter system, we demonstrated that peptide nucleic acids (PNAs) can inhibit miRNA activity and rescue the CFTR expression [14,15]
The anti-miRNA approach suffers from a main limitation, i.e., it may interfere with other miRNA targets leading to unpredictable side effects. To overcome this problem and to counteract the effects of miRNAs binding to the CFTR messenger RNA (mRNA) [26], we propose the use of PNAs as miRNAs target protectors (TPs)
Summary
One in every 3000 newborn Caucasians is affected by Cystic Fibrosis (CF), an autosomal recessive genetic disorder caused by mutations in a gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride-conducting transmembrane channel expressed in most epithelial and blood cells. MicroRNAs are evolutionarily conserved single-stranded non-coding RNAs, 18–25 nucleotides in length, that regulate the expression of specific genes at a post-transcriptional level, inhibiting the protein production [11,12,13]. In this context, the anti-miRNA strategy represents a valid tool within basic research and clinical applications.
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