Biophysical characterization of microRNA mixtures based on Molecular Beacons

Diverse studies have shown a relationship between dysregulated microRNAs (miRNAs), including miRNA-29b and miRNA-9, and several diseases. So, it is hypothesized that miRNAs can be studied as potential agents to be exploited in biomedical applications, due to their ability to take part in gene expression regulation at a post-transcriptional level. Considering the possibility of using miRNAs, it is important to characterize and validate this bioproduct, structurally and functionally. The goal of this work is to optimize an assay that can detect and biophysically characterize a miRNA sample without interference from the respective precursor form, by using molecular beacons (MB). MBs are hairpin-shaped probes composed of nucleic acid labeled with a quencher at the 3′ end and a fluorophore (reporter) at the 5’ end. Here, MB loops were designed so MB-9-1 and MB-29-1 would be complementary to the miRNA-9-1-5p and the miRNA-29b-1-3p, respectively. The MBs designed in this work specifically identified each target miRNA, even in artificial mixtures or complex samples, and the obtained fluorescence was directly proportional to miRNA concentration. Even if the precursor forms (pre-miRNAs) were present in the samples, no significant signal was shown, allowing the distinction between both forms. The outcomes of this work confirm the MBs potential to assess and characterize miRNA samples to be exploited in biochemical, biophysical, or biomedical fields.