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Scheme 1 (above) shows the function of DNA-specific homogenous assay. It begins with a solution containing a cationic conjugated polyelectrolyte (CCO, shown in glowing blue) and a peptide nucleic acid (PNA) strand (shown in yellow) labeled with a chromophore dye (C*). The optical properties of the CCP and C* can be optimized by chemical functionalization/structure so that Foster enerfy transfer (FRET) from CCP (donor) to C* (acceptor is favored. Sicne PNA is neutral, there are no electrostatic interactions between PNA-C* and the CCP. Consider next the interaction between PNA and a single stranded DNA (ssDNA). The situation on the right of the image corresponds to addition of a complementary ssDNA, which hybridizes with the target PNA. Hybridization endows the C* bearing macromolecule with multiple negative charges. Elctrostatic attraction causees the formation of a complex between CCP and the DNA/PNA-C* hybrid, thus allowing for FRET. WHen ssDNA is added that does not match the PNA sequence (shown on the left), hybridization does not take place. Electrostatic complexation occurs only between the CC and DNA. The distance between CCP and C* is to large for FRET. The overall coordination of elctrostatic froces characteristic of polyelctrolytes and the optical coupling step has been shound t be general, the number of charges between CCP and DNA/PNA-C* may or may not be the same. By using the self-assemnling fluorescence amplifying process, one can obtain 100-fold sensitivity increase in standard homogenous assays. It is important to note that the overall enhancement can be obtained without the need for sophisticated instrumentation and is readily incorporated into standard assay protocols. Furthermore, extensions of this method have been made to detect RNA/RNA and RNA/protein interactions. |
