There is significant interest in developing methods that visualize and detect RNA in live cells. Bioorthogonal template driven tetrazine ligations are quickly becoming a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens and existing tetrazine-reactive fluorogenic probes are quenched by through‐bond energy transfer (TBET) or Fӧrster resonance energy transfer (FRET) between the donor fluorophore and acceptor tetrazine.
Chemists from UC San Diego have developed a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near‐infrared (NIR) emitting cyanine dye. While traditional fluorogenic tetrazine probes rely on TBET or FRET mechanisms, the inventors’ novel methodology cages the fluorophores through an Internal Charge Transfer (ICT) quenching pathway. Using this alternative fluorogenic mechanism, this invention can employ a wide range of fluorophore scaffolds, including NIR emitting cyanine dyes that are not well‐quenched by tetrazines via energy transfer mechanisms. Vinyl ether caged fluorophores and tetrazine partners are conjugated to high affinity antisense nucleic acid probes, which show highly selective fluorogenic reactivity when annealed to their respective target RNA sequences.
Given the expansion of tetrazine fluorogenic chemistry to NIR dyes, we believe highly selective proximity induced fluorogenic tetrazine reactions could find broad uses in illuminating endogenous biomolecules in living cells. The technology inherent in these highly fluorogenic probes can be used for diverse applications such as no‐wash live‐cell imaging, endogenous oncogenic miRNA detection, and systemic fluorescence imaging in vivo.
Can be used for diverse applications such as no‐wash live‐cell imaging, endogenous oncogenic miRNA detection, and systemic fluorescence imaging in vivo.
UCSD inventors have successfully demonstrated the use of these probes to detect expressed mRNA in live cells and will proceed to focus efforts on monitoring single RNA molecules in real time. This technology can be applied to a number of endogenous RNA target sequences. These NIR fluorogenic bioorthogonal probes will have direct application for RNA imaging in live cells and tissues as well as RNA diagnostics.
A provisional patent has been submitted
tetrazine-reactive fluorogenic probes, live cells, detect RNA, live-cell imaging, miRNA detection