Endogenous and artificial miRNAs explore a rich variety of conformations: a potential relationship between secondary structure and biological functionality

C M A Gangemi,A Ferro,C M Croce,G Oliviero,Sara García-Viñuales,G Piccialli,S Alaimo,D Milardi,M E Fragalà,A D’Urso,A P Falanga,N Borbone,R Purrello,A Pulvirenti

doi:10.1038/s41598-019-57289-8

C M A Gangemi, A Ferro + Show 12 more

Open Access

https://doi.org/10.1038/s41598-019-57289-8

Copy DOI

Abstract

Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.

Highlights

Over 2000 miRNAs have been identified in humans, and they target most of human protein coding genes
On the basis of this hypothesis, we decided to investigate by several techniques, such as electronic circular dichroism (ECD), differential scanning calorimetry (DSC), nuclear magnetic resonance (1H-NMR) and non-denaturing polyacrylamide gel electrophoresis (PAGE), the existence of secondary structures which could be correlated to the activity of these sequences
We performed a structural investigation on two top-ranked artificial miRNAs, which induced a significant inhibition of the luciferase activity for both c-MET and EGFR (a-miR-141 and artificial microRNAs (a-miR)-196), and as comparison we investigated the structure of two a-miRNAs which seemed to be inactive (a-miR-23 and a-miR-98)

Summary

Introduction

Over 2000 miRNAs have been identified in humans, and they target most of human protein coding genes. The goal was to develop the bioinformatics tool “miR-Synth” aimed at designing artificial microRNAs (a-miR) capable to target multiple genes in multiple sites[36]. Among the top-six ranked miRNAs displayed by “miR-Synth”, two of them (a-miR-23 and a-miR-98) did not show significant inhibition of the expression of c-MET and EGFR, indicating that these artificial miRNAs do not elicit efficient anticancer activity[36]. “miR-Synth” tool does not consider the role of possible secondary structures adopted by the miRNA sequences, which has been recently evaluated as an important factor likely affecting the miRNAs function[31,32,33,34]. As a result the prediction efficiency of computational resource (as “miR-Synth”) will be definitively improved, considering the potentiality of miRNA sequences to adopt several secondary structures as new constrain to design efficient miRNA sequences

Methods

Results

Conclusion