Fluorogenic nucleic acid molecule and target RNA fluorescent labeling method
Inventors
Okada, Yasushi • ARIYOSHI, Tetsuro
Assignees
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Publication Number
US-12371730-B2
Publication Date
2025-07-29
Expiration Date
Abstract
The present invention provides a fluorogenic RNA capable of visualizing mRNA in cells, particularly, living mammalian cells, and a target RNA fluorescent labeling method using the fluorogenic RNA. The present invention provides a fluorogenic nucleic acid molecule containing a base sequence having two or more fluorescent molecule-binding regions linked via a linker sequence, in which one or more fluorescent molecule-binding aptamer sequences are inserted into a scaffold sequence of each of the fluorescent molecule-binding regions.
Core Innovation
The disclosed invention relates to a fluorogenic nucleic acid molecule for fluorescence visualization in living mammalian cells. The molecule includes a base sequence having 2 or more fluorescent molecule-binding regions linked via a linker sequence, and 1 or more fluorescent molecule-binding aptamer sequences are inserted into a scaffold sequence of each region.
The linker sequence is a base sequence represented by any of SEQ ID NOS: 18 to 20. The fluorescent molecule-binding aptamer sequences are fluorogenic aptamer constructs, including stem-loop architectures and G-quadruplex structures flanking the aptamer region, and the tandem scaffold/aptamer architecture and linker design suppress quenching/FRET while supporting DFHBI-family fluorescence.
The document further describes labeling and detection concepts for visualizing mRNA and transcript dynamics using the fluorogenic nucleic acid molecule. Target RNA fluorescent labeling is described by incorporating the fluorogenic construct into the 3′ untranslated region of a target gene, or by probe hybridization to the target RNA, and fluorescence detection is described as photo-dependent on/off fluorescence associated with DFHBI structural switching and brightness subtraction between illumination start and illumination end points.
Claims Coverage
The partial claims provided include one independent claim directed to the fluorogenic nucleic acid molecule composition, with dependent refinements covering aptamer and fluorescent molecule types, vector architecture for expression, and fluorescence detection by brightness change. Across these claims, the inventive coverage centers on a tandem fluorogenic nucleic acid architecture with inserted aptamer sequences into scaffolded binding regions, where the linker sequence is restricted to SEQ ID NOS: 18 to 20.
Tandem fluorogenic binding regions linked by SEQ ID NOS: 18 to 20 linker
A base sequence having 2 or more fluorescent molecule-binding regions linked via a linker sequence, wherein the linker sequence consists of a base sequence represented by any of SEQ ID NOS: 18 to 20, and wherein each fluorescent molecule-binding region is part of the fluorogenic nucleic acid molecule.
Inserted fluorescent molecule-binding aptamer sequences in a scaffold sequence
1 or more fluorescent molecule-binding aptamer sequences are inserted into a scaffold sequence of each of the fluorescent molecule-binding regions.
Stem-loop aptamer architecture flanked by G-quadruplex structures
The fluorogenic nucleic acid molecule includes fluorescent molecule-binding aptamer sequences made from single-stranded nucleic acid that form a stem-loop structure flanked by G-quadruplex structures.
Broccoli family aptamer insert options
The fluorescent molecule-binding aptamer sequences are Broccoli, Broccoli3, or dBroccoli.
DFHBI-family fluorescent molecule binding
The fluorescent molecules binding to the aptamer sequences are fluorescent DFHBI-family molecules.
Vector architecture with fluorogenic construct in the 3′UTR
A DNA vector includes a promoter for expressing a foreign gene, a downstream restriction enzyme site for inserting the foreign gene coding region, and a downstream 3′ untranslated region containing a base sequence encoding a fluorogenic nucleic acid molecule as defined in claim 1.
Brightness-change fluorescence detection using DFHBI-family dye
Detecting a fluorescence signal from a DFHBI-family fluorescent molecule bound to the fluorogenic nucleic acid molecule by illuminating continuously with excitation light and computing the brightness change between an illumination start point and an illumination end point.
The claim set provided broadly covers a tandem fluorogenic nucleic acid molecule with aptamer insertions into scaffolded fluorescent molecule-binding regions, constrained by a linker sequence defined by SEQ ID NOS: 18 to 20. Dependent coverage provided here specifies stem-loop/G-quadruplex aptamer architectures, allowed Broccoli-family aptamer variants, DFHBI-family dye binding, delivery via a vector with the construct in the 3′ untranslated region, and a fluorescence readout based on brightness change between illumination start and end points.
Stated Advantages
Supports fluorescence visualization of mRNA and transcript dynamics in living mammalian cells.
Improves fluorescence performance in the disclosed experimental context.
Enables quantitative tracking of transcript dynamics compared with RT-qPCR in the disclosed example.
Supports single-cell linearity and enhanced sensitivity using time-gated FLIM as described.
Enables inhibitor or screening readouts using fluorescence signal, including outcomes related to G4 resolvase screening.
Documented Applications
Fluorescent labeling of target RNA by incorporating the fluorogenic nucleic acid into the 3′ untranslated region of a target gene for in-cell transcript labeling in living mammalian cells.
Fluorescent labeling of target RNA by probe hybridization, producing fluorescence readout based on DFHBI structural switching.
In-cell transcript dynamics tracking using photo-dependent on/off fluorescence with brightness subtraction, including averaging over multiple illumination cycles as described.
Screening for inhibitors using a fluorescence readout, including screening associated with G-quadruplex unfolding and G4 resolvase.
Transcript dynamics example using a CD83 3′UTR knock-in in stimulated cells as described.
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