Kim, Yaewon (2018-12). Methods for Ligand Screening by Dissolution DNP Assisted NMR Spectroscopy. Doctoral Dissertation. | Thesis individual record

NMR spectroscopy is one of the front-line techniques in drug screening for binding
identification and affinity determination. A critical issue that limits the scope of NMR in
screening applications is low detection sensitivity. A solution can be provided by the
hyperpolarization technique of dissolution dynamic nuclear polarization (D-DNP). With
a several thousand-fold enhancement of NMR signals, the need for signal averaging and
the problems arising due to low protein or ligand solubility can be avoided.
v19F-NMR relaxometry for ligand screening using D-DNP is demonstrated. With a
well polarizable reporter ligand containing v19F atoms, binding affinities of non-fluorinated
ligands can be determined under competitive binding through transverse relaxation (Tv2)
measurements. The enhanced sensitivity by the D-DNP method allows lowering the
protein and ligand concentrations to the micromolar to sub-micromolar range.
Despite the substantial signal enhancement by DNP, the achievable throughput is
limited because the most commonly available instrumentation for D-DNP provides a
single hyperpolarized sample after each polarization process. We demonstrate that
multiplexed NMR detection can improve the throughput of D-DNP experiments by
permitting parallelized screening experiments with a single hyperpolarized aliquot of
ligand. In combination with a flow injection system capable of mixing the hyperpolarized
sample with several different secondary samples, Tv2 relaxation times of the reporter ligand
can be obtained from multiple channels simultaneously at desired concentration ratios of
the reporter to competitive ligand concentrations. This method extends the range of
binding affinity detectable in a single experiment to three orders of magnitude. It greatly
reduces the chance of missing the binding detection due to non-optimal sample
concentrations in every individual experiment. This multiplexed D-DNP approach may be
more broadly applied to chemical or biochemical problems requiring variable-dependent

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