Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets

Abstract

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein–peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

“…1d). We employed a microfluidic setup to facilitate measurement of affinities to minimize the sample quantities 27 : dose-response curves were set up by coupling 'droplet-on-demand' formation ( Fig. 1e) and a microfluidic fluorescence anisotropy imaging platform ( Fig.…”

“…Reagents. Preparation of BRC4 repeat peptide, N-terminally labeled with fluorescein (BRC4 fl , sequence CKEPTLLGFHTASGKKVKIAKESLDKVKNLFDEKEQ) was described previously 27 . CHES was from Sigma, Pico-Surf 1 was from Dolomite, HFE-7500 was from 3M.…”

“…Fluorescence polarization was measured in nanoliter droplets produced and analyzed in microfluidic devices to quantify bound vs unbound RAD51:BRC complexes and derive Kd values, essentially as described previously 27 . Briefly, nanoliter droplets were generated from a well in which an increasing amount of peptide is allowed to compete with BRC4 fl .…”

“…Flow rates were 3 μL/min for withdrawal and 40 μL/min for 30 seconds for peptide injection. Data was fit to a competitive binding model using 12 nM for the Kd of the monomeric RAD51:BRC4 fl interaction 27 . A crystal was cryo-cooled in liquid nitrogen without the application of a cryo-protectant and diffraction data were collected at Diamond Light Source (Harwell, UK) synchrotron radiation source.…”

High-Throughput, Lysis-free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

“…19,21,25 For the latter a range of optical signals reporting on reaction progress is now available, 26 based on fluorescence, 27 absorbance, 28 or anisotropy. 29 In many cases, directed evolution campaigns that screened for single variants required an intrinsic physical link between genotype and phenotype, 30 e.g. using phage display for antibody evolution.…”