FIT-PNA as a Diagnostic Probe to the KRT20 Cancer Biomarker

Maria Falchenko maria.falchenko@mail.huji.ac.il 1 Eylon Yavin 1 Dina Hashoul 1 Jeffrey Schlosshauer 1 Aviram Nissan 2 Rachel Shapira 2 Vera Paviov 2 Ganeshkumar Moorthy 1
1The School of Pharmacy, The Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel
2Oncological Surgery, Department of General and Oncological Surgery, The Chaim Sheba Medical Center, Tel Hashomer, Tel aviv, Israel

Abstract

Fluorescence-based imaging of RNA/DNA is considered as a powerful tool for the detection of distinct biomarkers for diagnosis and prognosis of diseases.

Peptide Nucleic Acid (PNA) is a synthetic DNA analog that is uncharged and achiral. These properties provide PNA with excellent binding to complementary DNA and RNA. In addition, PNA is not recognized and degraded by proteases, peptidases or nucleases. Therefore, it is very stable in biological fluids and may be used as a diagnostic tool in-vivo and ex-vivo. To investigate this ability, we synthesized a Forced Intercalation (FIT)-PNA probe to target KRT20- a new potential biomarker that was found to be elevated in colorectal cancer (CRC) patients. FIT-PNA1 has a unique feature to fluoresce only upon hybridization with the RNA biomarker. We introduced BisQ (a Cyanine dye), that acts as a "surrogate" base replacing one of the existing nucleotides in the PNA sequence. We have previously shown that using such a FIT-PNA, a single point mutation (SNP) of the K-ras gene is detected by simple incubation of the FIT-PNA with the suitable cell-lines2. Moreover, we have shown that FIT-PNA can detect the lncRNA CCAT1 that appears to act like an oncogenic lncRNA in CRC3 and others cancers.

We find that KRT20 FIT-PNA detects this mRNA in living cells as well as in fresh human tissue taken from a HIPEC (Hyperthermic intraperitoneal chemotherapy) surgical procedures.

Further experiments are planned to define the precise mRNA levels of KTR20 in the cell lines tested. Improving the characteristics of our FIT-PNA probe by elevating the quantum yield and shifting the fluorescence to the near-IR region, may provide a non-invasive and precise way to diagnose, and even guide in-vivo cytoreductive surgeries.

References

  1. Kohler, O. et al., ChemBioChem 2005, 6, 69-77.
  2. Kolevzon, N. et al., Chem. Commun. 2016, 52, 2405-2407.
  3. Nissan, A. et al. Int. J. Cancer 2012, 130, 1598-1606.








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