Method of characterizing interactions and screening for effectors

摘要

This invention enables high throughput detection of small molecule effectors of particle association, as well as quantification of association constants, stoichiometry, and conformation. “Particle” refers to any discrete particle, such as a protein, nucleic acid, carbohydrate, liposome, virus, synthesized polymer, nanoparticle, colloid, latex sphere, etc. Given a set of particle solutions having different concentrations, dynamic light scattering measurements are used to determine the average hydrodynamic radius, ravg, as a function of concentration. The series of ravg as a function of concentration are fitted with stoichiometric association models containing the parameters of molar mass, modeled concentrations, and modeled hydrodynamic radii of the associated complexes. In addition to the ravg value analysis, the experimental data may be fit/analyzed in alternate ways. This method may be applied to a single species that is self-associating or to multiple species that are hetero-associating. This method may also be used to characterize and quantify the association between a modulator and the associating species.

主权项

1. A method to characterize and measure the equilibrium self-association constants of a molecular species and its underlying stoichiometries within a given solvent comprising the steps of
1. collecting dynamic light scattering data from a plurality of samples spanning range of varying concentrations of said molecular species in said solvent comprising the steps of
A. preparing a concentration series of a plurality samples of varying known concentrations of said molecular species in said given solvent;B. illuminating each member of said concentration series with a light beam from a laser source;C. measuring the intensity fluctuations of light scattered by each said member of said molecular concentration series; andD. deriving an experimentally derived scattered light correlation function from each said measured member; 2. modeling theoretical light scattering data comprising the steps of
A. postulating a plurality of possible stoichiometries of the self-associating species that may be present within each of said members of said concentration series;B. parameterizing the concentrations of each self-associating and non-associating species within each member of said concentration series; andC. parameterizing the hydrodynamic radii of each self-associating species; 3. comparing said experimentally derived scattered light correlation functions from said plurality of samples to said modeled theoretical light scattering data for each of said possible stoichiometries that may be present within each member of said concentration series comprising the steps of
A. calculating modeled theoretical scattered light correlation functions from said modeled theoretical light scattering data for each said postulated stoichiometry and said parametrized concentrations and said parametrized hydrodynamic radii; andB. obtaining a best fit of said experimentally derived scattered light correlation functions from each said concentration series member to each of said modeled theoretical light scattering correlation function, and 4. deriving from said best fit the equilibrium association constants of said self-associations and their corresponding stoichiometries.