Single Molecule BioAssays at Elevated Concentrations
In order to advance single-molecule fluorescence spectroscopy to the next level, handling and analysis of single molecules has to become broadly available. A further quantum leap is required to proceed to commercially successful a...
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Descripción del proyecto
In order to advance single-molecule fluorescence spectroscopy to the next level, handling and analysis of single molecules has to become broadly available. A further quantum leap is required to proceed to commercially successful applications such as drug screening and medical diagnostics.
In this project, I suggest a strategy to overcome the fundamental gap between the nanomolar concentration regime of current optical single-molecule spectroscopy and the nano- to millimolar dissociation constants of typical biomolecular interactions. I will use nano-apertures, which confine the detection to sub-attoliter volumes and allow single-molecule studies at elevated concentrations. To overcome unspecific binding and deteriorated fluorescence signals in the nano-apertures, I will use tailor-made DNA nanostructures produced by DNA origami. These nanostructures will match the nano-apertures like a plug in a socket. Inserting molecules at programmed positions in the nanostructures will open up a new realm of applications by the ability to immobilize exactly one molecule per nano-aperture and by obtaining comparable signals from every nano-aperture. I will spectroscopically characterize the nano-apertures creating a fluorescence map of their inside. I will exemplarily use the new abilities for previously impossible applications such as several folds improvement of single-molecule DNA sequencing, direct single-molecule RNA sequencing by reverse transcriptase for cancer screening, for paralleled drug screening of HIV protease inhibitors and for studying the chemomechanical coupling of single helicases.
In summary, I envision a broadly applicable platform that has the potential to become a golden standard by enabling both ground breaking fundamental research and commercial applications.