Red-emitting FIT-PNAs: “On site” detection of RNA biomarkers in fresh human cancer tissues
Introduction
Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts of >200 nucleotides. A variety of lncRNAs have been found to be differentially expressed in cancer (Huarte, 2015); with those acting as either oncogenic e.g. HOTAIR, MALAT1 (Huarte, 2015; Ji et al., 2003) or tumor suppressor genes e.g. GAS5 (Ma et al., 2016). It had become apparent in recent years that such lncRNAs have defined molecular mechanisms that influence the progression of the disease and thus may be excellent biomarkers for cancer diagnosis and prognosis. One of such lncRNA is CCAT1 (colon-associated transcript 1) (Guo and Hua, 2017; Nissan et al., 2012; Xin et al., 2016). CCAT1 is a 2682 nucleotides lncRNA that maps to chromosome 8q24.21. It is associated with a variety of cancers, including colon (Alaiyan et al., 2013), lung (Luo et al., 2014), gastric (Zhang et al., 2014), breast (Zhang et al., 2015), gallbladder (Ma et al., 2015), ovarian (Liu et al., 2013), and hepatocellular (Deng et al., 2015) cancers. Several studies have shown the prognostic value of CCAT1 for several indications (Luo et al., 2014; Nissan et al., 2012; Zhang et al., 2014).
KRT20 belongs to a family of proteins that form the intermediate filament cytoskeleton of epithelial cells. Its expression has been found in tumor cells located in peripheral blood (Wyld et al., 1998), bone marrow (Soeth et al., 1996), or lymph nodes (Futamura et al., 1998) originating from metastasis of patients with colon cancer.
The Seitz laboratory introduced the FIT-PNA concept in which one of the nucleobases (usually purines) is replaced with a “surrogate base” such as Thiazole Orange (TO) (Bethge et al., 2008; Koehler et al., 2005; Kummer et al., 2011). When TO is placed in the PNA strand, it is quenched due to intramolecular twisting of the dye in the excited state but gains fluorescence only after RNA/DNA hybridization. This is due to the change in the environment (viscosity) surrounding the probe (Silva et al., 2007). TO has also been used as a surrogate base in DNA as well as in DNA-LNA oligomers (Hoevelmann et al. 2014, 2016), where the surrogate base is flanked by LNAs leading to a dramatic increase in signal to background. In this regard, the Seitz group (Hoevelmann et al., 2016) and ours (Kolevzon et al., 2016) have recently developed a red-emitting surrogate base in DNA-LNA (QB = Quinoline Blue) and in PNA (BisQ = Bis Quinoline) oligomers, respectively.
We have used this approach as means for detecting point mutations in cancer by designing FIT-PNAs that target the KRAS oncogene and showed that this mRNA transcript can be detected and discriminated at a single nucleotide resolution in living cells (Kolevzon et al., 2016). This concept was further investigated by Wickstrom and co-workers in lung cancer (Sonar et al., 2014). Apart from detecting SNPs, FIT-PNAs have the capacity of detecting RNA in living cells as demonstrated for cells infected with viral mRNA (Kummer et al. 2011, 2012), miRNA (Torres et al., 2012), and lncRNAs (Kam et al., 2014).
Herein, we have designed and synthesized FIT-PNAs that turn on their fluorescence upon sequence-specific RNA hybridization. These probes were designed to target two RNA cancer biomarkers: CCAT1 and KRT20. We show the detection of CCAT1 and, to a lesser extent, KRT20 by their complementary FIT-PNAs in living cells. Importantly, we have been able to unveil that spraying the specific CCAT1 FIT-PNA probe directly on fresh (un-fixed) cancerous human tissue from surgical procedures (cytoreductive surgery) results in a bright fluorescent signal in a short time interval (minutes). KRT20 FIT-PNA shows a weaker fluorescence and slower response. No fluorescent signal is observed after spraying these FIT-PNAs with healthy fresh tissue taken from bariatric surgeries or when spraying a non-specific FIT-PNA on cancerous fresh tissue.
This approach opens a simple and straight forward methodology to detect RNA biomarkers in fresh tissues simply based on the design of the FIT-PNA probe that complements the RNA biomarker of choice. The detection is observed within minutes after spraying a buffered solution of the FIT-PNA directly on the malignant fresh tissue.
Section snippets
General
Manual solid-phase synthesis was performed by using 5 mL polyethylene syringe reactors (Phenomenex) that are equipped with a fritted disk. All column chromatography was performed using 60A, 0.04–0.063 mm Silica gel (Biolab, Israel) and manual glass columns. TLC was performed using Merck Silica Gel 60 F254 plates. HPLC purifications and analysis were performed on a Shimadzu LC-1090 system using a semi-preparative C18 reversed-phase column (Jupiter C18, 5μ, 300Å, 250 × 10mm, Phenomenex) at 50 °C.
FIT-PNA design and photophysical properties
The synthesis of a Fmoc-protected PNA monomer where the natural base is replaced with a surrogate base allows the selective introduction of the fluorophore at a desired location on the PNA sequence. The red-emitting BisQ surrogate base was introduced on a PNA backbone (Supplementary Scheme S1) consisting of Fmoc and allyl protecting groups (Sonar et al., 2014). Subsequently, FIT-PNAs targeting CCAT1 and KRT20 were synthesized on the solid phase as previously reported (Supplementary Table S1 and
Discussion
The presence of residual tumor tissue (defined as ‘occult residual disease’) after surgery has been reported in a variety of surgical procedures with high-grade glioma resections reaching 65% of cases (Stummer et al., 2006). This highlights the need for an accompanying diagnostic procedure to detect these occult tumor deposits. Most FGS procedures rely on systemic administration of the fluorophore; whether targeted or non-targeted. This, in turn, may result in systemic toxicity and requires
Conclusions
In summary, we have developed FIT-PNAs with a red-emitting base surrogate (BisQ) targeting the oncogenic CCAT1 lncRNA and KRT20 mRNA. Both KRT20 CCAT1 FIT-PNAs are brightly fluorescent after direct topical application (spraying) of these molecules on fresh cancerous tissue, with CCAT1 FIT-PNA generating a fluorescent signal in a matter of minutes. Such molecular probes may be promising candidates for clinical translation.
Conflicts of interest statement
The authors declare that they have no conflicts of interest.
CRediT authorship contribution statement
Dina Hashoul: Investigation, Methodology. Rachel Shapira: Formal analysis. Maria Falchenko: Investigation, Methodology. Odelia Tepper: Methodology. Vera Paviov: Methodology. Aviram Nissan: Conceptualization. Eylon Yavin: Conceptualization, Writing - original draft, Writing - review & editing.
Acknowledgments
This work was supported by the Israel Science Foundation (grant No. 476/17) and the Israel Innovation Authority (grant No. 55330). We thank Prof. Abraham Rubinstein for his useful insights. EY acknowledges the David R. Bloom Center for Pharmacy and the Alex Grass Center for Drug Design and Novel Therapeutics for financial support.
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The authors contributed equally to this article.