Identifying Molecular Signatures of Tumors Using Novel Fluorescence Resonance Energy Transfer Networks
Poster Oct 07, 2014
Vishwa Nellore, Chris Dwyer
Fluorescence microscopy is one of the most widely used assays in biological systems. However, the technique suffers from limited multiplexing capability with previous attempts at detecting more than 11 fluorophores simultaneously resulting in barcodes that are too big for in vivo analysis, expensive and involve time-consuming detection schemes. Here, we introduce large DNA self-assembled FRET circuits that provide a unique, unpredictable optical output when probed by a series of light pulses. Markov and entropy modeling of the FRET sensors show that 125 fluorophores can be observed simultaneously. Furthermore, experimental analyses of over 1200 time-resolved fluorescence signatures on 300 prototypical circuits show that the optical responses are highly repeatable and minor variations between FRET networks can be discriminated resulting in a total of 10^375 unique responses. This increase in spatial information density enabled by FRET networks allowed us to identify molecular signatures in lung and breast cancer tumors.
It is now known that the presence of aberrant DNA/RNA secondary structure in the regulatory regions of genes involved in cell proliferation, cells growth and apoptosis can lead to cancer. The FRET sensor we designed, self-assembles DNA probes labeled with acceptor fluorophores to the target DNA/RNA secondary structure forming an optical network. A DNA strand labeled with a donor fluorophore triplex binds to a unique sequence adjacent to the secondary structure. When the donor fluorophore is excited, the optical network results in a different optical signal based on the presence of the wild type or the aberrant secondary structure, through which we identified lung and breast cancer cells with high specificity. Furthermore, the probe DNA strands may be loaded with cargo, such as photosensitizers, for therapy. The small size of fluorophores results in molecular scale spatial resolution while the optical sensing mechanism enables the in vitro and in vivo characterization of the structure at picosecond resolution.
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World Congress on Recent Advances in Aquaculture Research & Fisheries
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