Gamma-FIT-PNAs as sensitive RNA probes

Abstract

A variety of oligonucleotide-based probes have been developed for specific and selective sensing of RNA and DNA. Among these, FIT-PNAs (forced intercalation-peptide nucleic acids) and FIT probes (DNA- and RNA-based sensors) have been studied for a variety of RNA biomarkers in cell culture and tissues, and in vivo. FIT-PNAs and FIT probes are RNA/DNA sensors that exhibit fluorescence upon sequence-specific RNA/DNA hybridization. Several synthetic approaches have been successfully applied to increase the brightness and selectivity of these molecules, including the introduction of cyclopentane (cp) modified PNA monomers (cpPNA) as well as locked nucleic acids (LNAs—for FIT probes). In this report, we have explored the biophysical properties of FIT-PNAs that are modified with gamma-L-serine PNAs (γPNAs). We found that introducing a single γ-PNA flanking the fluorophore (BisQ) in the FIT-PNA sequence is sufficient to achieve a 46-fold increase in fluorescence for the PNA:RNA duplex, similarly to cpPNA. Interestingly, when two γ-PNAs flank BisQ on both sides, a significant increase in RNA affinity is observed (over an 8 °C increase in melting temperature, T_m). Altogether, γ-PNAs are a beneficial chemical modification that leads to brighter FIT-PNAs with improved binding affinities to targeted RNA.