PARTICLE-BASED ISOLATION OF PROTEINS AND OTHER ANALYTES
Methods, compositions and apparatus useful for individually manipulating and individually detecting analytes such as proteins. Analytes can be attached to particles to facilitate individual manipulation or detection of the particle-attached analytes. The particle-attached analytes can be composed of a single analyte attached to a single particle, such that no more than one analyte is attached per particle and no more than one particle is attached per analyte.
1 .- 25 . (canceled)
26 . A method comprising:
(a) forming a reaction mixture comprising:
(i) a plurality of proteins, wherein individual proteins of the plurality of proteins each comprise a plurality of first reactive moieties, and
(ii) a plurality of particles, wherein individual particles of the plurality of particles each comprise a second reactive moiety, wherein the second reactive moiety of the particles is reactive with the first reactive moieties of the proteins, and wherein each of the particles is immobilized on a solid support;
(b) reacting a first reactive moiety of a protein in the reaction mixture with a second reactive moiety of a particle in the reaction mixture to attach the protein to the particle via a bond formed between the first reactive moiety of the protein and the second reactive moiety of the particle, wherein the reacting yields no more than one protein attached to the particle, and wherein immobilization of the particles inhibits more than one of the particles in the reaction mixture from simultaneously contacting the protein, thereby forming an immobilized particle-protein conjugate on the solid support; and
(c) detaching the immobilized particle-protein conjugate from the solid support in solution-phase, thereby producing a solution-phase particle-protein conjugate.
27 . The method of claim 26 , wherein a particle of the plurality of particles comprises nucleic acid origami.
28 . The method of claim 27 , wherein the nucleic acid origami comprises a plurality of at least 50 oligonucleotides hybridized to a scaffold nucleic acid.
29 . The method of claim 27 , wherein the nucleic acid origami comprises a staple oligonucleotide hybridized to two regions of a scaffold nucleic acid that are separated from each other in the nucleotide sequence of the scaffold nucleic acid.
30 . The method of claim 26 , wherein the individual particles each comprise a nucleic acid linker attaching the individual particle to the second reactive moiety.
31 . The method of claim 26 , wherein each of the individual particles comprises a single second reactive moiety.
32 . The method of claim 26 , further comprising quenching reactivity of the second reactive moiety or the first reactive moiety after step (b).
33 . The method of claim 32 , wherein the quenching comprises attaching a ligand to the second reactive moiety.
34 . The method of claim 33 , further comprising separating particles from the particle-protein conjugate via binding of a receptor to the ligand.
35 . The method of claim 26 , further comprising (d) binding a probe to the protein of the particle-protein conjugate.
36 . The method of claim 35 , further comprising (e) detecting the binding of the probe to the protein of the particle-protein conjugate.
37 . The method of claim 35 , further comprising attaching the particle-protein conjugate to a solid support prior to step (d), thereby binding the probe to the protein of the particle-protein conjugate on the solid support.
38 . The method of claim 26 , further comprising, after step (c), separating the particle-protein conjugate from particles that lack an attached protein.
39 . The method of claim 38 , further comprising attaching the protein-attached particles to a second solid support, thereby forming an array of protein-attached particles.
40 . The method of claim 26 , wherein the plurality of proteins comprises at least 60% of the proteins encoded by a human proteome.
41 . The method of claim 26 , wherein, prior to step (a), the plurality of proteins is modified to add the first reactive moieties to the proteins.
42 . A composition, comprising:
(a) a plurality of proteins, wherein individual proteins of the plurality of proteins each comprise a plurality of first reactive moieties, and
(b) a plurality of particles contacted to the plurality of proteins, wherein individual particles of the plurality of particles each comprise a second reactive moiety, wherein the second reactive moiety of the particles is reactive with the first reactive moieties of the proteins, and wherein each of the particles is immobilized on a solid support;
wherein immobilization of the particles on the solid support inhibits more than one of the particles from simultaneously contacting a single protein of the plurality of proteins.
43 . The composition of claim 42 , wherein the first reactive moiety and the second reactive moiety comprise click reactants.
44 . The composition of claim 42 , wherein the solid support comprises a bead.
45 . The composition of claim 42 , wherein a particle of the plurality of particles comprises nucleic acid origami.