# US 2021/0277399 A1 · Methods of Imaging and Tracking Nucleic Acids in Cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0277399 A1
- Document ID: US20210277399A1
- Status: Published application
- Kind: A1
- Application number: 17/172,480
- Filed: 2021-02-10
- Published: 2021-09-09
- Earliest claimed: 2020-02-10
- Assignees: Emory University
- Applicants: Emory University
- Inventors: Tewoderos Ayele; Jennifer Heemstra
- Web page: https://patentgrep.com/patent/US20210277399A1

## CPC

- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/16: Aptamers
- C12N 2310/3513: Protein; Peptide
- C12N 2310/3517: Marker; Tag
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This disclosure relates to methods of imaging and tracking RNA or other nucleic acids in cells. In certain embodiments, this disclosure relates to methods of labeling intracellular nucleic acids by expressing in the cell RNA containing an aptamer sequence that specifically binds a label, e.g., fluorescent label, or placing in the cell nucleic acids containing an aptamer sequence that specifically binds a label, wherein the label comprises a carbene forming group, such as a diazirine group, under conditions such that the label specifically binds the aptamer sequence forming a complex; and exposing the complex to electromagnetic radiation sufficient for carbene formation resulting in a covalent bond to the aptamer sequence and the label.

## Claims

### Claim 1

A method comprising:
a) providing,
1) a conjugate comprising a fluorescent tag linked to a diazirine group and
2) a nucleic acid encoding RNA comprising a first aptamer segment that specifically binds the fluorescent tag and a second segment;
b) transfecting or inserting the nucleic acid into a cell such that RNA is expressed in the cell;
d) incubating the cell with the conjugate such that the fluorescent tag and the RNA form a complex inside the cell; and
e) exposing the complex to ultraviolet radiation such that a covalent bond between the RNA and the conjugate forms providing labeled RNA inside the cell with a fluorescent signal.

### Claim 2 (depends on 1)

The method of claim 1 further comprising the step of imaging the fluorescent signal.

### Claim 3 (depends on 1)

The method of claim 1 further comprising the step of tracking the location of the fluorescent signal.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second segment specifically binds to a protein in the cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first aptamer segment has a 5′ end consisting of the nucleic acid sequence of GGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC (SEQ ID NO: 1).

### Claim 6 (depends on 5)

The method of claim 5, wherein the conjugate is a salt of N-(4-((4-(dimethylamino)phenyl)(4-(3-(3-methyl-3H-diazirin-3-yl)propanamido)phenyl)methylene)cyclo hexa-2,5-dien-1-ylidene)-N-methylmethanaminium.

### Claim 7 (depends on 6)

The method of claim 6, wherein exposing the complex to ultraviolet radiation is irradiation at 365 nm.

### Claim 8 (depends on 7)

The method of claim 7, wherein exposing the complex to ultraviolet radiation is for not more than 15 min when the conjugate is at a concentration of 30 μM.

### Claim 9 (depends on 7)

The method of claim 7, wherein exposing the complex to ultraviolet radiation is for not more than 10 min when the conjugate is at a concentration of 100 μM.

### Claim 10

A method comprising:
a) providing,
1) a conjugate comprising a fluorescent tag linked to a diazirine group and
2) RNA comprising a first aptamer segment that specifically binds the fluorescent tag and a second segment;
b) transfecting or inserting the RNA into the cell;
c) incubating the cell with the conjugate such that the fluorescent tag and the RNA form a complex inside the cell; and
d) exposing the complex to ultraviolet radiation such that a covalent bond between the RNA and the conjugate forms providing labeled RNA inside the cell with a fluorescent signal.

### Claim 11 (depends on 10)

The method of claim 10 further comprising the step of imaging the fluorescent signal.

### Claim 12 (depends on 10)

The method of claim 10 further comprising the step of tracking the location of the fluorescent signal.

### Claim 13 (depends on 10)

The method of claim 10, wherein the second segment specifically binds to a protein in the cell or hybridizes to nucleic acid in the cell.

### Claim 14 (depends on 10)

The method of claim 10, wherein the first aptamer segment has a 5′ end consisting of the nucleic acid sequence of GGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC (SEQ ID NO: 1).

### Claim 15 (depends on 14)

The method of claim 14, wherein the conjugate is a salt of N-(4-((4-(dimethylamino)phenyl)(4-(3-(3-methyl-3H-diazirin-3-yl)propanamido)phenyl)methylene)cyclo hexa-2,5-dien-1-ylidene)-N-methylmethanaminium.

### Claim 16 (depends on 15)

The method of claim 15, wherein exposing the complex to ultraviolet radiation is irradiation at 365 nm.

### Claim 17 (depends on 16)

The method of claim 16, wherein exposing the complex to ultraviolet radiation is for not more than 15 min when the conjugate is at a concentration of 30 μM.

### Claim 18 (depends on 16)

The method of claim 16, wherein exposing the complex to ultraviolet radiation is for not more than 10 min when the conjugate is at a concentration of 100 μM.

### Claim 19

A salt of N-(4-((4-(dimethylamino)phenyl)(4-(3-(3-methyl-3H-diazirin-3-yl)propan amido) phenyl)methylene)cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium.