Systems and methods for high throughput screening of molecular interactions
AssigneeChildren's Medical Center Corporation
InventorsWesley Philip Wong, Clinton H. Hansen
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Abstract
This disclosure provides new and improved systems and methods for analyzing binding interactions and for identifying and measuring agents that modulate such binding interactions, including weak binding interactions. The methods may be used in high throughput screening assays.
Claims52
A system for analyzing modulation of a binding interaction, comprising
- a first member of a binding pair, conjugated to a first oligonucleotide, wherein the first oligonucleotide is conjugated to a FRET donor fluorophore, and
- a second member of the binding pair, conjugated to a second oligonucleotide, wherein the second oligonucleotide is conjugated to a FRET acceptor fluorophore,
- wherein the first and second members bind to each other with a member dissociation constant, Kd, in the range of about 100 pM to about 100 μM, and wherein the first and second oligonucleotides hybridize to each other with an oligonucleotide dissociation constant, Kd, that ranges from about 100 pM to about 1 μM.
The system of any of the foregoing claims, wherein the first member of the binding pair is a virus or viral component capable of binding to a mammalian cell and the second member of the binding pair is a mammalian cell or a component of the mammalian cell to which the viral component binds.
The system of any one of the foregoing claims, wherein once the first and second oligonucleotides are hybridized to each other, the donor and acceptor FRET fluorophores are spaced apart by 1 to 8 base pairs, optionally 4-7 base pairs, further optionally 4 or 5 base pairs.
The system of any one of the foregoing claims, wherein the donor FRET fluorophore is Alexa 555 and the acceptor FRET fluorophore is Alexa 647, or wherein the donor FRET fluorophore is Alexa 488 and the acceptor FRET fluorophore is Alexa 555, or wherein the donor FRET fluorophore is Alexa 594 and the acceptor FRET fluorophore is Alexa 647.
The system of any one of the foregoing claims, wherein the first member and/or second member is a protein.
A method for detecting an agent capable of modulating a binding interaction, comprising
- (a) providing (i) a first member of a binding pair, conjugated to a first oligonucleotide, wherein the first oligonucleotide is conjugated to a FRET donor fluorophore, and (ii) a second member of the binding pair, conjugated to a second oligonucleotide, wherein the second oligonucleotide is conjugated to a FRET acceptor fluorophore, wherein the first and second members bind to each other with a member dissociation constant, Kd, in the range of about 100 pM nM to about 100 μM, and wherein the first and second oligonucleotides hybridize to each other with an oligonucleotide dissociation constant, Kd, that ranges from about 100 pM to about 1 μM and is comparable to or greater than the concentration of oligonucleotides used in the assay,
- (b) combining the first member and the second member with a sample, under conditions that allow binding of the first member to the second member and that allow binding of the first oligonucleotide to the second oligonucleotide, optionally wherein the first member and second member are provided in concentrations ranging from about 100 pM to about 100 nM,
- (c) measuring test fluorescence from the FRET acceptor, and
- (d) (1) identifying the sample as containing an agent that
- (i) decreases binding of the first member to the second member as a test fluorescence that is less than a control fluorescence measured after combining the first member and the second member under the same conditions as (b) but in the absence of the sample, or
- (ii) increases binding of the first member to the second member as a test fluorescence that is greater than a control fluorescence measured after combining the first member and the second member under the same conditions as (b) but in the absence of the sample;
- or
- (2) identifying the sample as containing an agent that modulates binding of the first member to the second member, wherein a test fluorescence that is less than a control fluorescence, measured in the absence of the sample, is indicative of an agent that decreases binding of the first member to the second member, and a test fluorescence that is greater than a control fluorescence, measured in the absence of the sample, is indicative of an agent that increases binding of the first member to the second member.
A system for analyzing modulation of a binding interaction, comprising
- a first member of a binding pair, conjugated to a first oligonucleotide, wherein the first oligonucleotide is conjugated to a fluorophore, and
- a second member of the binding pair, conjugated to a second oligonucleotide,
- wherein the first and second members bind to each other with a member dissociation constant, Kd, in the range of about 100 pM nM to 100 μM and wherein the first and second oligonucleotides hybridize to each other with an oligonucleotide dissociation constant, Kd, that ranges from about 100 pM to about 1 μM.
A method for detecting an agent capable of modulating a binding interaction, comprising
- (a) providing (i) a first member of a binding pair, conjugated to a first oligonucleotide, wherein the first oligonucleotide is conjugated to a fluorophore, and (ii) a second member of the binding pair, conjugated to a second oligonucleotide, wherein the first and second members bind to each other with a member dissociation constant, Kd, in the range of about 100 pM to about 100 μM, and wherein the first and second oligonucleotides hybridize to each other with an oligonucleotide dissociation constant, Kd, that ranges from about 100 pM to about 1 μM and is about equal to or greater than the concentration of oligonucleotides used in the assay, optionally wherein said concentrations are about 100 pM to about 100 nM,
- (b) combining the first member and the second member with a sample, under conditions that allow binding of the first member to the second member and that allow binding of the first oligonucleotide to the second oligonucleotide,
- (c) measuring test fluorescence using fluorescence polarization, and
- (d) identifying the sample as containing an agent that
- (i) decreases binding of the first member to the second member as a test fluorescence that is less than a control fluorescence measured after combining the first member and the second member under the same conditions as (b) but in the absence of the sample, or
- (ii) increases binding of the first member to the second member as a test fluorescence that is greater than a control fluorescence measured after combining the first member and the second member under the same conditions as (b) but in the absence of the sample.
A system for analyzing modulation of a binding interaction, comprising a polymer conjugated to
- (i) a first member of a binding pair and a second member of a binding pair, wherein the first and second members bind to each other with a member dissociation constant, Kd, of 100 pM to 100 μM, and wherein the polymer adopts a looped conformation when the first and second member bind to each other, and
- (ii) a first FRET oligonucleotide conjugated to a first FRET fluorophore and a second FRET oligonucleotide conjugated to a second FRET fluorophore, wherein the first and second FRET oligonucleotides hybridize to each other with a FRET oligonucleotide dissociation constant, Kd, and wherein one of the FRET fluorophores is a FRET donor fluorophore and the other of the FRET fluorophores is a FRET acceptor fluorophore.
A system for analyzing modulation of a binding interaction, comprising
- a complex comprising a single-stranded scaffold nucleic acid hybridized to a plurality of single-stranded oligonucleotides thereby forming a linear partially double-stranded nucleic acid,
- wherein a first single-stranded oligonucleotide in the plurality is linked to a first member of a binding pair, and a second single-stranded oligonucleotide in the plurality is linked to a second member of a binding pair, wherein the first and second members bind to each other with a member dissociation constant, Kd, of 100 pM to 100 μM, and wherein the complex adopts a looped conformation when the first and second member bind to each other,
- wherein a first FRET oligonucleotide conjugated to a first FRET fluorophore is conjugated to the complex and a second FRET oligonucleotide conjugated to a second FRET fluorophore is conjugated to the complex, wherein the first and second FRET oligonucleotides hybridize to each other with a FRET oligonucleotide dissociation constant, Kd, optionally in the range of about 100 pM to 1 μM, and wherein one of the FRET fluorophores is a FRET donor fluorophore and the other of the FRET fluorophores is a FRET acceptor fluorophore.
Dependencies are inferred from the claim text.