Xenon-129 NMR for biomolecular assays

The efficient use of conventional Nuclear Magnetic Resonance (NMR) spectroscopy for facile assays of complex biological mixtures is hindered by two features of NMR. The signal-to-noise ratio is generally low, requiring extensive signal-averaging and precluding the rapid detection of low-concentration analytes. Direct 1H NMR spectra of a biological compound and mixtures thereof are convoluted with the rich atomic-level information about the multitude of magnetically-distinct hydrogen atoms in the structure(s). Unique NMR properties of the 129 Xe atom can be exploited in order to overcome these difficulties. The ability of 129Xe to be optically pumped and its dramatic response to attributes of both chemical and bulk environments via a simplified chemical shift spectrum, allow for the increase in signal-to-noise and the spectral simplicity needed for the possible application of NMR as a rapid assay methodology. The subject of interactions between proteins in solution and unfunctionalized xenon (ordinary xenon dissolved in solution) is presented in the context of xenon-binding and conformation-sensitive assays. The functionalized xenon biosensor is developed as a means of specifically targeting the xenon atom to the protein by incorporating the biological ligand into the chemical structure of functionalized xenon. A detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin is presented. Binding of functionalized xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of the ligand-target interaction. The xenon chemical shifts for xenon encapsulated in different diastereomeric cage molecules are distinct. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor. Methods for enhancing the signal from functionalized xenon by making use of the laser-polarized xenon magnetization reservoir are presented. NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of remote-amplified detection.