The project is described at www.ccrnp.ncifcrf.gov/toms/patent/medusa/ The MedusaTM Sequencer is a single-molecule sequencing device that consists of a DNA (or RNA) polymerase attached to a set of four flexible arms. The tip of each arm carries a nonhydrolyzable nucleotide triphosphate analog and a spectrally distinct Forster Resonance Energy Transfer (FRET) acceptor fluorophore. A donor fluorophore attached to the polymerase excites an acceptor fluorophore by FRET if the fluorophores are close to each other. A MedusaTM Sequencer binds to a DNA primer hybridized to the DNA or RNA to be sequenced. The four arms with nucleotide tips """"""""test"""""""" the polymerase pocket by diffusing in and out. The arm tip that has the nucleotide complementary to the unknown base of the template will dwell longer in the pocket than the other three that are not complementary. However, the polymerase will not incorporate the nucleotide on the tip of the arm into the nascent strand because the nucleotide triphosphate α-βbond is nonhydrolyzable. FRET between the donor on the polymerase and the acceptor at the arm tip produces a characteristic spectrum that identifies the bound base. Free hydrolyzable dNTPs (or NTPs) allow the MedusaTM Sequencer to step forward. The series of FRET signals reveals the unknown nucleotide sequence. A MedusaTM Sequencer could also be injected into a cell to read mRNA sequences inside a living organism. Coded versions of the MedusaTM Sequencer can signal when the device has been damaged. Our collaborator is now determining whether single nucleotides can be distinguished using a single molecule microscope.
