# US 2025/0085276 A1 · Compositions and methods for serial target detection

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- Number: US 2025/0085276 A1
- Document ID: US20250085276A1
- Status: Published application
- Kind: A1
- Application number: 18/830,071
- Filed: 2024-09-10
- Published: 2025-03-13
- Earliest claimed: 2023-09-12
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Eli N. Glezer; Ronald Graham
- Web page: https://patentgrep.com/patent/US20250085276A1

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/56966: {Animal cells}
- G01N 33/582: {with fluorescent label}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed herein, inter alia, are methods and compositions useful for serially detecting multiple biological targets.

## Claims

### Claim 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
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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first probe has the formula:
wherein
R12 is hydrogen, halogen, —CCl3, —CBr3, —CF3, —CI3, —CHCl2, —CHBr2,
—CHF2, —CHI2, —CH2Cl, —CH2Br, —CH2F, —CH2I, —CN, —OH, —NH2, —COOH, —CONH2, —NO2,
—SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O)NH2,
—NHSO2H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl3, —OCF3, —OCBr3, —OCI3, —OC HCl2,
—OCHBr2, —OCHI2, —OCHF2, —OCH2Cl, —OCH2Br, —OCH2I, —OCH2F, —N3, —SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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
R6 is a third fluorescent moiety;
R7 is a third biomolecule-specific binding agent;
R8 is a quenching moiety;
Ring C is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
L6 and L8 are cleavable linkers and capable of cleaving under orthogonal cleaving conditions; L7 is a covalent linker;
R6 and R8 is a fluorescent-quencher pair;
W6 is O, NR6A, or S; W7 is O, NR7A, or S; W8 is O, NR8A, or S; and
R6A, R7A and R8A are independently hydrogen or substituted or unsubstituted alkyl;
(f) cleaving L8 thereby separating the third quenching moiety from Ring C; and
(g) detecting the third fluorescent moiety thereby detecting the third complex.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 1)

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

### Claim 18

A composition comprising:
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.

### Claim 19 (depends on 18)

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

### Claim 20 (depends on 18)

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.