title: Fluorescence detection of KRAS2 mRNA hybridization in lung cancer cells with PNA-peptides containing an internal thiazole orange.
abstract: We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5' end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5' terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA.
authors.affiliation: Biochemistry & Molecular Biology, §Radiology, and ∥Kimmel Cancer Center Thomas Jefferson University , Philadelphia, Pennsylvania 19107, United States.
authors.forename: Mahesh V
authors.initials: MV
authors.lastname: Sonar
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Wampole
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Jin
authors.forename: Chang-Po
authors.initials: CP
authors.lastname: Chen
authors.forename: Mathew L
authors.initials: ML
authors.lastname: Thakur
authors.forename: Eric
authors.initials: E
authors.lastname: Wickstrom
mesh_terms: D052160:Benzothiazoles / Q000737:chemistry*; D045744:Cell Line, Tumor; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D011804:Quinolines / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D018631:ras Proteins / Q000235:genetics*
chemical_list: D052160:Benzothiazoles; C117307:KRAS protein, human; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D011804:Quinolines; D012333:RNA, Messenger; C051000:thiazole orange; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins