Novel CCAT1 and K-Ras Peptide Nucleic Acids with Specific Illumination at the Far-Red Spectrum

In the past decades much effort has been put forth in the discovery of biomarkers in cancer as means for designing novel drug targets and for diagnostic purposes. Using RNA as a biomarker has the advantage that detection could, in principle, be based not only on the over-expression of a given RNA molecule in malignant cells but also on the discrimination between mutated and non-mutated transcripts that are manifested in many types of malignancies.

The long non-coding RNA transcript, colon cancer associated transcript-1 (CCAT1) is highly expressed in human colorectal cancer with none or minimal expression in normal tissues [1]. Because CCAT1 is up-regulated in the vast majority of precancerous polyps and lymph nodes it could serve as an efficient biomarker for real time imaging and for screening purposes. In the context of early diagnosis mutated Kras oncogene was explored in our group as well. We have previously shown that cytosolic mutant K-ras as well as CCAT1 could be detected in living colon cancer cells by the forced intercalation (FIT) approach, using peptide nucleic acids (PNAs) in which the intercalated dye, thiazole orange (TO), served as a fluorescent base surrogate [2]. Alas, the specific emission spectra (ca. 530 nm) of TO is problematic for in vivo imaging due to the auto-fluorescence of biological specimens, including cell lines.

In this study we report on novel fluorescent probes (at the far red region), where the surrogate base TO is replaced by a novel fluorophore (BisQ) that emits light in the far-red region. The novel fluorophore is, in turn, incorporated into PNA sequences capable of hybridizing with cytosolic mutant K-ras mRNA and CCAT1 sequences in relevant cancer cells. The design of the new PNA also includes a cell penetrating peptide (dK₄) enabling the PNA to enter the cancer cells without the aid of a transfection agent.
BisQ labeled PNAs were shown to detect both biomarkers in living cancer cells as well as the CCAT-1 biomarker in mildly fixed human biopsies from patients with colorectal cancer.