We propose to develop a novel dual FRET molecular beacons technology for the early detection of cancer in living cells with high specificity, sensitivity and efficiency. Molecular beacons (MBs) are single-stranded oligonucleotides with a stem-loop hairpin structure and dual-labeled with a fluorophore at one end and a quencher at the other. Delivering MBs into cells will result in a fluorescence signal if the MBs hybridize to target mRNAs. Thus, when the target mRNAs corresponding to the molecular markers of a cancer are detected in cells, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of MBs would suffer from false positives in cancer cell detection due to degradation by cytoplasmic nucleases and nonspecific interactions. To overcome this difficulty, we have created the dual FRET MBs concept (i.e., to hybridize a pair of donor and acceptor MBs on the same target and detect the resulting FRET), and demonstrated its potential to significantly reduce or even eliminate the false positives. We have studied the energy transfer between MBs with different dye molecule pairs, developed lanthanide dyes and performed time-resolved FRET to further reduce the background noise. To guide the design of molecular beacons, we have synthesized MBs with various molecular structures and performed in-solution thermodynamic and kinetic studies of MB-target binding. We have also studied the feasibility of detecting K-ras codon 12 mutant mRNA and survivin mRNA in pancreatic cell lines. To further develop the new dual FRET molecular beacons technology for clinical applications, in this Phase I STTR project, we will enhance the intracellular stability of molecular beacons by modifying the backbone with 2'-O-methyl and phosphorothioate (PS) chemistry. We will synthesize 5 types of molecular beacons with a random sequence and compare their stability in both cell lysates and living cells. We will also study the effect of such modifications on hybridization kinetics and thermodynamics. We will determine the signal-to-noise ratio and specificity of dual FRET molecular beacons by detecting mRNA expression in both cell lysates and living cells. We will synthesize dual FRET MBs targeting K-ras codon 12 mutations and survivin, deliver the MBs into pancreatic cancer cell lines and HDF cells and establish the detection specificity and signal-to-noise ratio using fluorescence imaging and spectroscopy. We will determine the detection sensitivity by systematically varying the relative ratios of normal and cancerous cells in a mixture in vitro and seek out the cancer cells based on MB-induced fluorescence using a confocal microscope and a FACS cell sorter. The goals are to develop the dual FRET molecular beacons technology for early cancer detection and diagnosis, and to commercialize this technology for a wide range of biomedical applications including medical research, cancer analysis, drug discovery, and in vivo detection of gene expression.
Thesaurus Terms: fluorescence resonance energy transfer, fluorescent dye /probe, gene expression, neoplasm /cancer diagnosis, oligonucleotide, technology /technique development biosensor, early diagnosis, messenger RNA, neoplasm /cancer genetics, thermodynamics, thiophosphate cell line, confocal scanning microscopy, flow cytometry, polymerase chain reaction
