Chemically Modified Thiazole Orange FIT-PNAs: Superior Nucleic Acid Sensing Molecules

Abstract

RNA and DNA biomarkers serve as diagnostic molecules for detecting a specific disease by a variety of methods and technologies. A common approach is based on a fluorescence readout signal generated after hybridization to a target nucleic acid sequence. One such technology is termed Forced-Intercalation Peptide Nucleic Acid (FIT-PNA). In the FIT-PNA design, one of the nucleobases of the PNA sequence is replaced by a fluorescent molecule termed as “surrogate base.” One of the more common fluorophores explored to date is the cyanine dye, Thiazole Orange (TO). In this report, we have designed TO-based FIT-PNAs that are chemically modified with a cyclopentane backbone (cpTO). In addition, we have introduced to the FIT-PNA design either a cyclopentane T (cpT) or a tetrahydrofuran T (THFT) flanking cpTO. In a model system (11-mer FIT-PNA), we observe a dramatic increase in fluorescence (with DNA or RNA complementary sequences) for both cpT-cpTO and THFT-cpTO FIT-PNAs in comparison to the unmodified counterpart (T-TO FIT-PNA). Moreover, sequence specificity for an RNA sequence with a single mismatch is dramatically improved for both cpT-cpTO and THFT-cpTO FIT-PNAs. Molecular simulations of both cpT-cpTO FIT-PNA and TO (unmodified) FIT-PNA further support the superiority of these chemically modified nucleic-acid probes, as corroborated by a greater π–π stacking of cpTO in the PNA-RNA duplex. Lastly, a cpT-cpTO FIT-PNA targeting the oncogenic long noncoding RNA ANRIL (antisense noncoding RNA in the INK4 locus) was shown to detect this RNA biomarker in ovarian cancer cells (OVCAR-8). This probe was superior to the unmodified TO-based FIT-PNA, highlighting the added value of chemically modified TO FIT-PNAs as means for obtaining highly sensitive and sequence-specific nucleic acid sensors.