IP Library › Granted Patent US 12,428,666
Granted Patent B2
US 12,428,666 · App. 17/250,023 · Granted Sep 30, 2025

Compositions and methods related to kethoxal derivatives

Inventors: Chuan He (Chicago, IL); Xiaocheng Weng (Chicago, IL); Tong Wu (Chicago, IL)
Assignee: THE UNIVERSITY OF CHICAGO
C12Q1/6806C07C69/003C12N15/11C12N2830/46C12Q2565/518
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Quick Facts
Patent No.
US 12,428,666
App. No.
17/250,023
Granted
Sep 30, 2025
Kind
B2
Abstract

Embodiments are directed to N 3 -kethoxal reagents and derivatives thereof, and related methods that allow fast and reversible labeling of single-stranded nucleic acids in live cells. By way of example, one aspect is directed to a process for reversible labeling of single-stranded guanine bases in live cells, which results in an effective in vivo method for transcriptome-wide RNA secondary structure mapping and RNA G-quadruplex prediction.

Claims (22)

1. A compound of the formula:

wherein Y is an alkyne, an azide, a strained alkyne, a diene, a dieneophile, an alkoxyamine, a phosphine, a hydrazide, a thiol, or an alkene; and

X is a C1 to C10 alkyl or a C4 to C10 polyethylene glycol linker.

2. The compound of claim 1 , wherein Y is an azide.

3. A compound of the formula:

wherein Y is an alkyne, an azide, a strained alkyne, a diene, a dieneophile, an alkoxyamine, a phosphine, a hydrazide, a thiol, or an alkene; and X is CH 2 .

4. The compound of claim 1 , wherein the compound is of formula:

5. A method for labeling a guanine base comprising contacting a guanine base to be labeled with a compound of claim 1 to form a reaction mixture and incubating the reaction mixture at 30 to 40° C. for at least 5 minutes.

6. The method of claim 5 , wherein the compound is N3-kethoxal.

7. The method of claim 5 , wherein the guanine base is comprised in a polynucleotide.

8. The method of claim 5 , wherein the polynucleotide is a ribonucleic acid (RNA).

9. The method of claim 5 , wherein the polynucleotide is a deoxyribonucleic acid (DNA).

10. A method for labeling a single stranded nucleic acid in a cell comprising

(i) contacting a target cell with a compound of claim 1 , wherein Y is an azide, to form a treated cell comprising a nucleic acid having kethoxal derivative-labeled guanine bases;

(ii) contacting the treated cell with a crosslinking moiety comprising at least two click chemistry reactive moieties and a tag, wherein the crosslinking moiety crosslinks two proximal kethoxal derivative-labeled guanines to form a crosslinked nucleic acid; and

(iii) fragmenting and isolating the crosslinked nucleic acid using a reagent with an affinity for the tag;

wherein the click chemistry reactive moieties are alkyne moieties.

11. The method of claim 10 , wherein the tag is biotin, and wherein the reagent with an affinity for the tag is streptavidin.

12. The method of claim 10 , wherein the click chemistry reactive moieties are dibenzocyclooctyne moieties.

13. The method of claim 10 , wherein the crosslinked nucleic acid is RNA.

14. A compound having the formula:

15. A composition comprising the compound of claim 14 .

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 6, 2020
From: HE, CHUAN; WU, TONG; WENG, XIAOCHENG
To: THE UNIVERSITY OF CHICAGO
Reel/Frame 054292/0958 →
Continuity (3)
Provisional Application 62668543 · May 8, 2018
Provisional Application 62668994 · May 9, 2018
Related Publication 20210214773A1 · Jul 15, 2021
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