METHODS AND COMPOSITIONS FOR IN VIVO NON-COVALENT LINKING
Disclosed are methods of facilitating the interaction of a first and a second component, the method including the use of an antibody fragment and an epitope tag. The antibody fragment may be bound to a first component, while the epitope tag may be bound to a second component. The antibody fragment may have a binding specificity for the epitope tag sufficient to cause an interaction between the antibody fragment and the epitope tag.
1 . A method of facilitating the interaction of a first and a second component, comprising
a. providing an antibody fragment bound to a first component; and
b. providing an epitope tag bound to a second component;
wherein said antibody fragment comprises binding specificity for said epitope tag sufficient to cause a interaction between said antibody fragment and said epitope tag.
2 . The method of claim 1 wherein said antibody fragment and said epitope tag transiently interact.
3 . The method of claim 1 wherein said antibody fragment and said epitope tag transiently interact, wherein said transient interaction occurs in the interior of a cell.
4 . The method of claim 1 wherein said antibody fragment is covalently bound to said first component.
5 . The method of claim 1 wherein said antibody fragment comprises a single chain variable fragment (ScFv).
6 . The method of claim 1 wherein said antibody fragment comprises a single chain variable fragment (ScFv), a single domain antibody (sdAb), a domain antibody, a SMIP, or a combination thereof.
7 . The method of claim 1 wherein said first and second component comprise an epitope tag covalently attached to target protein and a ScFv covalently attached to a signal.
8 . The method of claim 1 wherein said first component is selected from a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, and combinations thereof.
9 . The method of claim 1 wherein said first component comprises an effector molecule.
10 . The method of claim 1 wherein said first component comprises an effector molecule selected from a transcription factor (activator or repressor), chromatin remodeling factor, exonuclease, endonuclease, transposase, methytransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, integrase, recombinase, ligase, topoisomerase, gyrase, helicase, fluorophore, and combinations thereof.
11 . The method of claim 1 wherein said first component comprises an effector molecule capable of modifying gene expression.
12 . The method of claim 1 wherein said second component comprises an effector protein.
13 . The method of claim 1 wherein said second component comprises an effector protein selected from a transcription factor (activator or repressor), chromatin remodeling factor, exonuclease, endonuclease, transposase, methytransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, integrase, recombinase, ligase, topoisomerase, gyrase, helicase, fluorophore, and combinations thereof.
14 . The method of claim 1 wherein said first component comprises an effector molecule protein capable of modifying gene expression.
15 . The method of claim 1 wherein said second component is selected from a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, and combinations thereof.
16 . The method of claim 1 wherein said second component comprises a protein capable of modifying gene expression.
17 . The method of claim 1 wherein said second component comprises a protein, wherein said protein modifies DNA.
18 . The method of claim 1 wherein said first and second component comprise a first protein and a second protein.
19 . The method of claim 1 wherein said first and second component comprise a DNA binding protein and an effector protein, wherein said interaction results in a change in gene expression or a modification of DNA.
20 . The method of claim 1 wherein said first and second component comprise a fluorophore and a protein, wherein said interaction permits real-time monitoring of protein expression and subcellular localization.
21 . The method of claim 1 wherein said first and second component comprise a first and second small molecule capable of interacting to activate a prodrug.
22 . A kit comprising an antibody fragment and an epitope tag;
wherein said antibody fragment is bound to a first component;
wherein said epitope tag is bound to said second component; and
wherein said antibody fragment comprises binding specificity for said epitope tag sufficient to cause an interaction between said antibody fragment and said epitope tag.
23 . The kit of claim 22 wherein said first and second component are selected from a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, and combinations thereof.
24 . A method of facilitating the interaction of a first and a second component, comprising
a. providing a scaffold protein bound to a first component; and
b. providing a corresponding binding site bound to a second component;
wherein said scaffold protein specifically binds to the corresponding binding site to cause an interaction between the scaffold protein and the corresponding binding site.
25 . The method of claim 24 wherein the scaffold protein is an antibody mimetic.
26 . The method of claim 24 wherein the scaffold protein and the corresponding binding site transiently interact.
27 . The method of claim 26 wherein the transient interaction occurs in the interior of a cell.
28 . The method of claim 24 wherein the scaffold protein is covalently bound to said first component.
29 . The method of claim 1 wherein the scaffold protein comprises an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain or a Kunitz domain peptide, a monobody, or a combination thereof.
30 . The method of claim 24 wherein the first component is a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, or a combination thereof.
31 . The method of claim 24 wherein the first component comprises an effector molecule.
32 . The method of claim 31 wherein the effector molecule is a transcription factor (activator or repressor), chromatin remodeling factor, exonuclease, endonuclease, transposase, methytransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, integrase, recombinase, ligase, topoisomerase, gyrase, helicase, fluorophore, or a combination thereof.
33 . The method of claim 24 wherein said first component comprises an effector molecule capable of modifying gene expression.
34 . The method of claim 24 wherein said second component comprises an effector protein.
35 . The method of claim 24 wherein said second component comprises an effector protein selected from a transcription factor (activator or repressor), chromatin remodeling factor, exonuclease, endonuclease, transposase, methytransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, integrase, recombinase, ligase, topoisomerase, gyrase, helicase, fluorophore, and combinations thereof.
36 . The method of claim 24 wherein said first component comprises an effector molecule protein capable of modifying gene expression.
37 . The method of claim 24 wherein said second component is selected from a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, and combinations thereof.
38 . The method of claim 24 wherein said second component comprises a protein capable of modifying gene expression.
39 . The method of claim 24 wherein said second component comprises a protein, wherein said protein modifies DNA.
40 . The method of claim 24 wherein said first and second component comprise a first protein and a second protein.
41 . The method of claim 24 wherein said first and second component comprise a DNA binding protein and an effector protein, wherein said interaction results in a change in gene expression or a modification of DNA.
42 . The method of claim 24 wherein said first and second component comprise a fluorophore and a protein, wherein said interaction permits real-time monitoring of protein expression and subcellular localization.
43 . The method of claim 24 wherein said first and second component comprise a first and second small molecule capable of interacting to activate a prodrug.
44 . A kit comprising a scaffold protein and a corresponding binding site;
wherein the scaffold protein is bound to a first component;
wherein the corresponding binding site is bound to said second component; and
wherein the scaffold protein specifically binds to the corresponding binding site to cause an interaction between the scaffold protein and the corresponding binding site.
45 . The kit of claim 44 wherein said first and second component are selected from a protein, a small molecule, a fluorophore, a signal peptide, a nanoparticle, a cellular component, and combinations thereof.
46 . The method of claim 12 or 34 , wherein the effector molecule is a nuclease.
47 . The method of claim 46 , wherein the nuclease is BfiI.
48 . The method of claim 46 , wherein the nuclease is BmrI.
49 . The method of claim 46 , wherein the nuclease is Clo051.
50 . The method of claim 46 , wherein the nuclease is FokI.
51 . A method for modifying a genome of an organism comprising the steps of
a. providing an antibody fragment bound to a first component, wherein the first component is a DNA binding molecule; and
b. providing an epitope tag bound to a second component, wherein the second component is an effector molecule capable of modifying gene expression;
wherein said antibody fragment comprises binding specificity for said epitope tag sufficient to cause an interaction between said antibody fragment and said epitope tag.
52 . The method of claim 51 , wherein the DNA binding molecule is a DNA, RNA, or protein and the effector molecule is an endonuclease, the interaction of which induces a change in gene expression or a modification of a genomic DNA sequence or base pair.
53 . A cell modified according to the method of claim 51 or 52 .