Compositions and methods for serial target detection

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
• R1 is a first fluorescent moiety;
• R2 is a first biomolecule-specific binding agent;
• R3 is a second fluorescent moiety;
• R4 is a second biomolecule-specific binding agent;
• R5 is a quenching moiety;
• Ring A is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
• Ring B is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
• L1 and L5 are independently cleavable linkers capable of cleaving under identical cleaving conditions;
• L3 is a cleavable linker capable of cleaving under orthogonal cleaving conditions relative to L1 and L5;
• L2 and L4 are independently covalent linkers;
• R3 and R5 is a fluorescent-quencher pair;
• W1 is O, NR1A, or S;
• W2 is O, NR2A, or S;
• W3 is O, NR3A, or S;
• W4 is O, NR4A, or S;
• R1A, R2A, R3A, and R4A are independently hydrogen or substituted or unsubstituted alkyl;
• and
• (b) detecting the first fluorescent moiety thereby detecting the first complex;
• (c) cleaving L1 and L5 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.