Methods and compositions for rapid functional analysis of gene variants

Patent metadata

Field	Value
Publication number	US-11397180-B2
Application number	US-201715819221-A
Country	US
Kind code	B2
Filing date	Nov 21, 2017
Priority date	Jan 8, 2013
Publication date	Jul 26, 2022
Grant date	Jul 26, 2022

Abstract

Methods and compositions are disclosed for rapid functional analysis of gene variants based on analysis of protein-protein and protein-nucleic acid interactions.

First claim

What is claimed is:

1. A method of analyzing genetic pathways comprising the following steps in the recited order:
(a) providing a control and an experimental group of cells, wherein the control group of cells express at least four distinct wild type primary proteins, primary proteins A, B, C and D respectively, and wherein the experimental group of cells express variants of primary proteins A, B, C and D, wherein the variants have at least 97% or greater but less than 100% sequence identity with the primary proteins;
(b) lysing the control and the experimental group of cells;
(c) contacting each cell lysate in parallel with (i) a first primary agent attached to a surface of a magnetic bead that is not conjugated to an optically-active label, wherein the first primary agent is a binding partner capable of capturing the first protein A or the variant of primary protein A, (ii) a second primary agent attached to a surface of a magnetic bead that is conjugated to a first optically-active label, (iii) a third primary agent attached to a surface of a non-magnetic bead that is not conjugated to an optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is conjugated to a second primary optically-active label, wherein the second, third and fourth primary agents are different binding partners each capable of capturing the distinct primary proteins B, C, and D, or variants of primary proteins B, C, and D, respectively;
(d) contacting the protein-captured beads with (i) a first secondary optically-active label conjugated with a secondary agent, a binding partner that is capable of interacting with a first secondary protein, (ii) a second secondary optically-active label conjugated with a secondary agent that is capable of interacting with a second secondary protein, (iii) a third secondary optically-active label conjugated with a secondary agent that is capable of interacting with a third secondary protein, and (iv) a fourth secondary optically-active label conjugated with a secondary agent that is capable of interacting with a fourth secondary protein, wherein the second, third and fourth secondary agents are different binding partners each capable of interacting with a distinct secondary protein;
(e) passing the beads through a flow cytometer or optical plate reader;
(f) adjusting the forward scatter amplitude gain and side scatter voltage on a flow cytometer with a control un-complexed bead population prior to initiating the method of detecting complexed beads;
(g) detecting each optical signal of each bead, wherein each optical signal represents data from a single bead;
(h) aggregating each optical signal from each bead that have bound primary proteins A, B, C and D and variants of primary proteins A, B, C and D to identify the binding of primary proteins A, B, C and D and variants of primary proteins A, B, C and D;
(i) aggregating each optical signal from each bead and assessing the expression levels of the secondary proteins, wherein the aggregated optical signal from the first secondary optically-active label is used to identify the binding of the first secondary protein, the aggregated optical signal from the second secondary optically-active label is used to identify the binding of the second secondary protein, the aggregated optical signal from the third secondary optically-active label is used to identify the binding of the third secondary protein, and the aggregated optical signal from the fourth secondary optically-active label is used to identify the binding of the fourth secondary protein;
(j) recovering magnetic bead complexes by applying a magnetic field;
(k) recovering non-magnetic bead complexes;
(1) passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader;
(m) passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader;
(n) detecting the optical signal(s) of the recovered magnetic bead complexes; and
(o) detecting the optical signal(s) of the recovered non-magnetic bead complexes
(p) determining protein-protein interactions between the primary proteins A, B, C, and D or the variants of primary proteins A, B, C, and D, and the secondary proteins;
wherein the presence on a magnetic bead complex of only a secondary optically-active label indicates the interaction between the primary protein A or the variant of primary protein A and a second protein corresponding to the secondary optically-active labeled secondary agent;
and wherein the presence on a magnetic bead complex of both (i) a first primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein B or the variant of primary protein B and a second protein corresponding to the secondary optically-active labeled secondary agent;
and wherein the presence on a non-magnetic bead complex of only a secondary optically-active label indicates the interaction of the first protein C or the variant of primary protein C and a second protein corresponding to the secondary optically-active secondary labeled agent;
and wherein the presence of a non-magnetic bead complex of both (i) a second primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein D or the variant of primary protein D and a second protein corresponding to the secondary optically-active labeled secondary agent; and
(q) comparing the protein-protein interactions detected from the control group of cells with the protein-protein interactions detected from the experimental group of cells, thereby determining whether the experimental group of cells has any functional variation in the variants of primary proteins A, B, C or D as compared to the control group of cells.

2. The method of claim 1 , wherein recovering non-magnetic bead complexes in step (j) is achieved by centrifugation.

3. The method of claim 1 , wherein each primary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.

4. The method of claim 1 , wherein each secondary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.

5. The method of claim 3 , wherein the antibodies are monoclonal or polyclonal antibodies.

6. The method of claim 3 , wherein the antibody fragments are F(ab′)2 fragments, Fab′ fragments or ScFvs.

7. The method of claim 1 , wherein the magnetic beads are epoxy-coated magnetic beads.

8. The method of claim 1 , wherein the non-magnetic beads are carboxyl modified beads.

9. The method of claim 1 , wherein forward scatter amplitude gain and side scatter voltage on a flow cytometer are set to register populations of bead events to on scale, followed by applying an inclusion gate where selected linear populations of beads form collective clusters containing interrogation targets and can be analyzed in their entirety by flow cytometry.

10. The method of claim 1 , further comprising:
(r) quantifying the optical signal(s) detected so as to thereby quantify the amount of primary protein-secondary protein interaction on the bead and comparing the quantified amount against a control curve, wherein the control curve is generated by a set of standard optically active reference beads created using said first through fourth primary agents.

11. A method of analyzing genetic pathways comprising the following steps in the recited order:
(a) providing a control and an experimental group of cells, wherein the control group of cells express at least four wild type distinct primary proteins, primary proteins A, B, C and D respectively, and wherein the experimental group of cells express variants of primary prote

Assignees

• Albert Einstein College Medicine Inc
• Albert Einstein College Medicine

Inventors

• Loke Johnny C
• Ostrer Harry

Classifications

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N33/54326 Primary

  Magnetic particles · CPC title