Compositions and methods related to kethoxal derivatives
Inventors
He, Chuan • WENG, Xiaocheng • Wu, Tong
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12428666-B2
Publication Date
2025-09-30
Expiration Date
Abstract
Embodiments are directed to N3-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.
Core Innovation
The disclosed subject matter provides compounds of a specified chemical formula in which Y is selected from 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 selected as a C1 to C10 alkyl group, a C4 to C10 polyethylene glycol linker, or CH2. The document further includes embodiments in which the compound is N3-kethoxal (azido-kethoxal), and references compounds having a specified formula.
The disclosure addresses labeling of guanine bases by contacting guanine bases with the claimed compound to form a reaction mixture and incubating under specified labeling conditions. It provides selective, fast, reversible labeling of guanine bases in single-stranded DNA/RNA in live cells and forms kethoxal-derivative-labeled guanine bases when the labeling compound includes the azide functionality.
Beyond labeling, the disclosure includes use of tagged click-chemistry crosslinkers having at least two click-chemistry reactive moieties to crosslink proximal kethoxal-derivative-labeled guanines. The crosslinked nucleic acid is then fragmented and isolated via tag-affinity capture, with the tag and affinity reagent described as biotin and streptavidin, respectively, and with additional embodiments specifying dibenzocyclooctyne reactive moieties.
The labeled nucleic acids enable transcriptome-wide RNA secondary structure mapping and RNA G-quadruplex prediction, and the disclosure also includes proximity and interaction mapping concepts including KARRI for RNA-RNA interaction mapping and CLIP-seq-style protein-RNA crosslinking concepts.
Claims Coverage
The patent document includes independent claims covering compound formulas and a tagged click-chemistry crosslinking workflow. The inventive features focus on the variable Y functional group, linker choices for X, and downstream labeling, crosslinking, and tag-affinity isolation of kethoxal-derivative-labeled nucleic acids.
Click-functional handle with alkyl or PEG linker
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.
Fixed methylene linker with click-functional handle
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 CH2.
Guanine base labeling using the Y-functional compound
A method for labeling a guanine base by contacting the guanine base with a compound of the formula wherein Y and X are as defined, to form a reaction mixture and incubating under specified labeling conditions.
Tagged click chemistry crosslinking and tag-affinity isolation of crosslinked nucleic acid
A method for labeling a single stranded nucleic acid in a cell using a Y-azide compound to generate kethoxal-derivative-labeled guanine bases, then using a tagged click-chemistry crosslinker with at least two click chemistry reactive moieties to crosslink two proximal kethoxal-derivative-labeled guanines, followed by fragmenting and isolating the crosslinked nucleic acid via tag-affinity capture.
Biotin/streptavidin affinity isolation for the tagged workflow
The method wherein the click chemistry workflow uses a biotin tag and a streptavidin reagent that has affinity for the tag.
Dibenzocyclooctyne reactive moieties for the click crosslinker
The method wherein the click chemistry reactive moieties are dibenzocyclooctyne moieties.
The inventive coverage centers on formula-defined compounds with variable Y functional groups and X linker choices, together with guanine labeling and a tagged click-chemistry crosslinking workflow that uses biotin/streptavidin affinity capture and, in some embodiments, dibenzocyclooctyne reactive moieties.
Stated Advantages
Selective, fast, reversible labeling of guanine bases in single-stranded DNA/RNA.
Enables transcriptome-wide RNA secondary structure mapping.
Enables RNA G-quadruplex prediction (rG4 detection).
Reports improved specificity versus DMS/SHAPE.
Documented Applications
Transcriptome-wide RNA secondary structure mapping (keth-seq) using reversible labeling of guanine bases with kethoxal-click derivatives.
RNA G-quadruplex prediction (rG4 detection) based on labeled guanine bases.
RNA-RNA interaction mapping using KARRI (kethoxal-assisted RNA-RNA interaction mapping) with kethoxal-assisted labeling and proximity/click crosslinking concepts.
Protein-RNA crosslinking concepts referenced as CLIP-seq-style crosslinking using kethoxal derivatives.
Interested in licensing this patent?