Detection of RNA Cancer Biomarkers in Living Cells and in Fresh Human Tissue by Peptide Nucleic Acids (PNAs)

PNAs are DNA mimics that consist of a neutral backbone allowing excellent hybridization to complementary DNA and RNA targets. Thiazole orange (TO) has been previously reported by Seitz and co-workers (1) as a surrogate base that when incorporated into a PNA sequence (a.k.a. FIT-PNA, forced intercalation-PNA), turns on its fluorescence upon hybridization to a complementary RNA/DNA sequence. We have used this approach by designing FIT-PNA molecules that targets the oncogenic form of kRAS (mutated kRAS) and were able to detect this mutation in living pancreatic cancer cells (2). We have recently developed these FIT-PNAs with a new surrogate base (BisQ) that emits at the red-region (613nm) and show that a fluorescent readout at SNP resolution in living cells is detected after 30 minutes by simple incubation of the FIT-PNA in cell culture (3). We have also targeted a long non-coding RNA (lncRNA) that has been found to be highly expressed in colorectal cancer (4, 5). By doing so, we could detect this cancer biomarker in living cells as well as in fresh (unfixed) tissue biopsies from grade 4 peritoneum cancer patients (submitted). We are now exploring other cancer biomarkers and in parallel designing and synthesizing new far-red and Near-IR fluorescent probes as surrogate bases with the vision of using such new PNA-FIT probes for non-invasive RNA biomarker detection in fluorescence guided surgery.

References:

1. Kohler, O. et al., ChemBioChem 2005, 6, 69-77.
2. Kam, Y. et al., Mol. Pharm. 2012, 9(3), 685-693.
3. Kolevzon, N. et al., Chem. Commun. 2016, 52, 2405-2407.
4. Nissan, A. et al. Int. J. Cancer 2012, 130, 1598-1606.
5. Kam, Y. et al., Cancer Lett. 2014, 352(1), 90-96.