# US 12,216,121 B2 · Methods and compositions for spacial and temporal measurement of catalytic activity

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- Number: US 12,216,121 B2
- Document ID: US12216121B2
- Status: Granted
- Kind: B2
- Application number: 16/603,722
- Filed: 2018-04-12
- Published: 2021-04-01
- Granted: 2025-02-04
- Earliest claimed: 2017-04-12
- Assignees: The University of Chicago
- Applicants: The University of Chicago
- Inventors: Yamuna Krishnan; Krishna Dan; Aneesh T. Veetil; Kasturi Chakraborty
- Web page: https://patentgrep.com/patent/US12216121B2

## CPC

- G01N 33/573: for enzymes or isoenzymes
- C07H 21/04: with deoxyribosyl as saccharide radical
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies
- 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)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

Described herein are nucleic acid molecules and complexes useful for spatiotemporally mapping intra-endosmal thiol disulphide exchange. Aspects of the disclosure relate to a composition comprising a first nucleic acid conjugated to a normalization moiety; and a second nucleic acid conjugated to a catalytic substrate; wherein reaction of the substrate with a catalyst produces a detectable product; and wherein the first and second nucleic acids are complementary or substantially complementary. Further aspects relate to a composition comprising: a first nucleic acid conjugated to a normalization moiety and to a catalytic substrate; and a second nucleic acid; wherein reaction of the substrate with a catalyst produces a detectable product; and wherein the first and second nucleic acids are complementary or substantially complementary.

## Claims

### Claim 1

A composition comprising:
a first nucleic acid; and
a second nucleic acid;
wherein the first nucleic acid and the second nucleic acid are in a nucleic acid duplex;
and wherein the first nucleic acid comprises SEQ ID NO:1 and the second nucleic acid comprises SEQ ID NO:2, or wherein the first nucleic acid comprises SEQ ID NO:2 and the second nucleic acid comprises SEQ ID NO:1.

### Claim 2 (depends on 1)

The composition of claim 1 further comprising a catalytic substrate conjugated to the first nucleic acid or the second nucleic acid
wherein the first nucleic acid is conjugated to a normalization moeity.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the catalytic substrate is an enzymatic substrate, and wherein reaction of the catalytic substrate with an enzyme produces a detectable product.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the normalization moiety and the catalytic substrate are in a 1:1 ratio.

### Claim 5 (depends on 3)

The composition of claim 3, wherein the detectable product is fluorescent.

### Claim 6 (depends on 2)

The composition of claim 2, wherein the catalytic substrate comprises a disulfide bond.

### Claim 7 (depends on 2)

The composition of claim 2, wherein the catalytic substrate comprises a thioester moiety.

### Claim 8 (depends on 2)

The composition of claim 2, wherein the catalytic substrate comprises Gly-Phe or Cbz-Phe-Lys.

### Claim 9 (depends on 2)

The composition of claim 2, wherein the catalytic substrate comprises a protected fluorophore.

### Claim 10 (depends on 2)

The composition of claim 2, wherein the catalytic substrate is derived from 6′-O propargyl fluorescein.

### Claim 11 (depends on 2)

The composition of claim 2, wherein the catalytic substrate comprises:

### Claim 12 (depends on 3)

The composition of claim 3, wherein the reaction comprises a thiol disulfide exchange.

### Claim 13 (depends on 3)

The composition of claim 3, wherein the normalization moiety and the detectable product each comprise a fluorophore comprising an emission wavelength.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the fluorophore of the normalization moiety and the fluorophore of the detectable product have different emission wavelengths.

### Claim 15 (depends on 1)

The composition of claim 1, wherein the duplex directs a cell to localize the duplex to the endosome or lysosome.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the duplex directs a cell to target one of the endoplasmic reticulum or golgi.

### Claim 17 (depends on 1)

A kit comprising: the composition of claim 1.

### Claim 18 (depends on 2)

The composition of claim 2, wherein the first nucleic acid is conjugated to the catalytic substrate.