Detection of endogenous K-ras mRNA in living cells at a single base resolution by a PNA molecular beacon

Detection of mRNA alterations is a promising approach for identifying biomarkers as means of differentiating benign from malignant lesions. By choosing the KRAS oncogene as a target gene, two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (TO-PNA-MB, where TO = thiazole orange) were synthesized and compared in vitro and in vivo. Their specificity was examined in wild-type KRAS (HT29) or codon 12 point mutation (Panc-1, SW480) cells. Incubation of both beacons with total RNA extracted from the Panc-1 cell line (fully complementary sequence) showed a fluorescent signal for both beacons. Major differences were observed, however, for single mismatch mRNA transcripts in cell lines HT29 and SW480. PS-DNA-MB weakly discriminated such single mismatches in comparison to TO-PNA-MB, which was profoundly more sensitive. Cell transfection of TO-PNA-MB with the aid of PEI resulted in fluorescence in cells expressing the fully complementary RNA transcript (Panc-1) but undetectable fluorescence in cells expressing the K-ras mRNA that has a single mismatch to the designed TO-PNA-MB (HT29). A weaker fluorescent signal was also detected in SW480 cells; however, these cells express approximately one-fifth of the target mRNA of the designed TO-PNA-MB. In contrast, PS-DNA-MB showed no fluorescence in all cell lines tested post PEI transfection. Based on the fast hybridization kinetics and on the single mismatch discrimination found for TO-PNA-MB we believe that such molecular beacons are promising for in vivo real-time imaging of endogenous mRNA with single nucleotide polymorphism (SNP) resolution.