COMPOSITIONS AND METHODS FOR SERIAL TARGET DETECTION

1 . A method of detecting multiple biomolecules, said method comprising:

(a) contacting a cell or tissue comprising a first biomolecule and a second biomolecule with a first probe and a second probe, thereby forming a first complex comprising said first biomolecule bound to said first probe and a second complex comprising said second biomolecule bound to said second probe, wherein

the first probe has the formula:

 and

a second probe has the formula:

 wherein

R 1 is a first fluorescent moiety;

R 2 is a first biomolecule-specific binding agent;

R 3 is a second fluorescent moiety;

R 4 is a second biomolecule-specific binding agent;

R 5 is a quenching moiety;

Ring A is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

Ring B is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

L 1 and L 5 are independently cleavable linkers capable of cleaving under identical cleaving conditions; L 3 is a cleavable linker capable of cleaving under orthogonal cleaving conditions relative to L 1 and L 5 ; L 2 and L 4 are independently covalent linkers;

R 3 and R 5 is a fluorescent-quencher pair;

W 1 is O, NR 1A , or S; W 2 is O, NR 2A , or S; W 3 is O, NR 3A , or S; W 4 is O, NR 4A , or S;

R 1A , R 2A , R 3A , and R 4A are independently hydrogen or substituted or unsubstituted alkyl; and

(b) detecting the first fluorescent moiety thereby detecting the first complex;

(c) cleaving L 1 and L 5 thereby separating the first fluorescent moiety from Ring A and separating the quenching moiety from Ring B; and

(d) detecting the second fluorescent moiety thereby detecting the second complex.

2 . The method of claim 1 , wherein the first probe has the formula:

 wherein

R 12 is hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 ,

—CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 ,

—SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 ,

—NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OC HCl 2 ,

—OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.

3 . The method of claim 1 , wherein the second probe comprises the formula:

4 . The method of claim 1 , wherein the first fluorescent moiety generates a first signal and the second fluorescent moiety generates a second signal, wherein said first signal and second signal are the same.

5 . The method of claim 1 , wherein the first fluorescent moiety generates a first signal and the second fluorescent moiety generates a second signal, wherein said first signal and second signal are the different.

6 . The method of claim 1 , further comprising

(e) contacting the cell or tissue with a third probe and binding the third probe to a third biomolecule thereby forming a third complex, wherein

the third probe has the formula:

 wherein

R 6 is a third fluorescent moiety;

R 7 is a third biomolecule-specific binding agent;

R 8 is a quenching moiety;

Ring C is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

L 6 and L 8 are cleavable linkers and capable of cleaving under orthogonal cleaving conditions; L 7 is a covalent linker;

R 6 and R 8 is a fluorescent-quencher pair;

W 6 is O, NR 6A , or S; W 7 is O, NR 7A , or S; W 8 is O, NR 8A , or S; and

R 6A , R 7A and R 8A are independently hydrogen or substituted or unsubstituted alkyl;

(f) cleaving L 8 thereby separating the third quenching moiety from Ring C; and

(g) detecting the third fluorescent moiety thereby detecting the third complex.

7 . The method of claim 1 , further comprising repeating steps (a)-(d).

8 . The method of claim 1 , further comprising (e) contacting the cell or tissue with a stain, wherein the stain binds to a third biomolecule.

9 . The method of claim 8 , wherein the stain is a fluorescent stain.

10 . The method of claim 1 , wherein detecting comprises directing an excitation light to the cell or tissue and detecting an emission light from the first fluorescent moiety, the second fluorescent moiety, and the stain.

11 . The method of claim 1 , wherein cleaving the cleavable linker comprises contacting the cleavable linker with a cleaving agent.

12 . The method of claim 1 , wherein the first biomolecule-specific binding agent and the second biomolecule-specific binding agent are each independently an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer.

13 . The method of claim 1 , wherein the first biomolecule-specific binding agent and the second biomolecule-specific binding agent are independently an antibody.

14 . The method of claim 1 , wherein the first biomolecule and the second biomolecule are different.

15 . The method of claim 1 , wherein the first biomolecule is a first protein and the second biomolecule is a second protein.

16 . The method of claim 1 , wherein said first biomolecule and second biomolecule are within the cell.

17 . The method of claim 1 , wherein said first biomolecule and second biomolecule are on the surface of the cell.

18 . A composition comprising:

the first probe has the formula:

 and

a second probe has the formula:

 wherein

R 1 is a first fluorescent moiety;

R 2 is a first biomolecule-specific binding agent;

R 3 is a second fluorescent moiety;

R 4 is a second biomolecule-specific binding agent;

R 5 is a quenching moiety;

Ring A is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

Ring B is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

L 1 and L 5 are independently cleavable linkers capable of cleaving under identical cleaving conditions; L 3 is a cleavable linker capable of cleaving under orthogonal cleaving conditions relative to L 1 and L 5 ; L 2 and L 4 are independently covalent linkers;

R 3 and R 5 is a fluorescent-quencher pair;

W 1 is O, NR 1A , or S; W 2 is O, NR 2A , or S; W 3 is O, NR 3A , or S; W 4 is O, NR 4A , or S;

R 1A , R 2A , R 3A , and R 4A are independently hydrogen or substituted or unsubstituted alkyl.

19 . A kit comprising a first probe and a second probe of claim 18 .

20 . A cell, comprising:

a first organelle bound to a first probe;

a second organelle bound to a second probe;

and a fluorescent stain bound to a nucleic acid molecule,

wherein the first probe and the second probe are the first probe and second probe of claim 18 .