Method of characterizing interactions and screening for effectors

Patent metadata

Field	Value
Publication number	US-9459207-B2
Application number	US-201514838258-A
Country	US
Kind code	B2
Filing date	Aug 27, 2015
Priority date	Mar 3, 2010
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

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, r avg , as a function of concentration. The series of r avg 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 r avg 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.

First claim

We claim:

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; and
D. 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; and
C. 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; and
B. 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.

2. The method of claim 1 where said concentration series is produced by the successive dilution of an initial highest concentration of said molecular species, said dilution achieved using a stock solution of said solvent.

3. The method of claim 2 further comprising the step of adding a corresponding concentration of a second molecular species to some members of said concentration series.

4. The method of claim 1 further comprising the step of modeling each parameterized hydrodynamic radius of each complex by using the hydrodynamic radii of said complex's constituent molecules.

5. The method of claim 1 wherein said concentration series consists of 10 to 20 members.

6. The method of claim 1 wherein said postulated hydrodynamic radii are fixed values determined a priori.

7. The method of claim 1 wherein said postulated concentrations are fixed values determined a priori.

Assignees

• Wyatt Tech Corp

Inventors

• Hanlon Amy D
• Larkin Michael I

Classifications

• G01N2201/12

  Circuits of general importance; Signal processing · CPC title

• G01N2015/0216

  from fluctuations of diffraction pattern · CPC title

• G01N2015/0222

  from dynamic light scattering, e.g. photon correlation spectroscopy · CPC title

• G01N21/4738

  Diffuse reflection (precedence is given to G01N21/55 - G01N21/57 if specular component is taken into consideration), e.g. also for testing fluids, fibrous materials · CPC title

• G01N2015/0038

  Investigating nanoparticles · CPC title