Fluorogen-binding RNA aptamers
Inventors
Ryckelynck, Michael • Autour, Alexis • Unrau, Peter • Dolgosheina, Elena • Jeng, Sunny Chiu Yuk • Panchapakesan, Shanker Shyam Sundhar • Abdolahzadeh, Amir • Cojocaru, Razvan • Ferré D'Amaré, Adrian • Trachman, Robert
Assignees
Simon Fraser University • Centre National de la Recherche Scientifique CNRS • Universite de Strasbourg • US Department of Health and Human Services
Publication Number
US-11434490-B2
Publication Date
2022-09-06
Expiration Date
2038-04-23
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Abstract
RNA aptamers are disclosed with distinct fluorescent properties, fluorophore binding affinities, and salt dependence. Also disclosed are corresponding fluorophores, with selected fluorophores evidencing high cellular permeability. The aptamer's high fluorophore affinities, the high brightness of the bound complexes, and their thermal and salt stability, provide distinct aspects of the disclosed aptamers.
Core Innovation
The invention discloses RNA aptamers with distinct fluorescent properties, fluorophore binding affinities, and salt dependence. Specific embodiments include Mango-II, Mango-III, and Mango-IV. Corresponding fluorophores with high cellular permeability are also disclosed. The aptamers exhibit high fluorophore affinities, brightness of bound complexes, and thermal and salt stability. These characteristics make the aptamers suitable for use with existing microscopy methodologies optimized for protein study, enabling the study of RNA function and dynamics both in vitro and in vivo.
The disclosed aptamers can be genetically encoded or directly transfected as in vitro transcribed RNAs, and have been demonstrated for imaging RNA molecules in fixed and live mammalian cells. Specifically, the aptamers can accurately image the sub-cellular localization of two small non-coding RNAs, 5S and U6, in both fixed and live cells. Certain aptamers are as bright or brighter than enhanced GFP when bound to TO1-Biotin, a fluorophore described herein.
The problem being addressed is the lack of fluorescent tools for imaging biologically important RNAs comparable to the fluorescent proteins used to study cellular proteins. Existing fluorogenic RNA aptamers have been limited by their inability to fold into tertiary structures that bind a fluorophore tightly and in an orientation that maximizes fluorescence. Optimization of both fluorophore binding and fluorescence brightness is desirable to improve fluorescent RNA imaging tags for cellular RNA studies.
Claims Coverage
The patent claims include multiple independent claims focusing on RNA aptamers with specific core sequences, their binding to selected fluorophores, and structural features mediating fluorophore binding and fluorescence.
RNA aptamer with defined active core sequence that forms a fluorophore binding conformation
An RNA aptamer comprising an active core sequence set forth in a defined nucleotide pattern with specific nucleotide allowances and prohibitions, wherein the aptamer adopts a tridimensional fluorophore binding conformation. The aptamer further comprises a 5′ leader sequence and a 3′ tail sequence contiguous with the core that mediate the juxtaposition of the core termini in the fluorophore binding conformation.
Interaction with fluorophores of Formula I and Formula II
The RNA aptamer interacts with a fluorophore selected from compounds of specified Formula I and Formula II, whose substituents and structural parameters are defined. Binding of these fluorophores by the aptamer increases fluorescence and brightness of the fluorophore-aptamer complex.
Core active sequence variants with defined G-quadruplex structures
The core active sequence of the aptamer comprises variants that form G-quadruplex structures and adopt specific nucleotide patterns and complementary pairings. Exemplary sequences include SEQ ID NO: 7 and SEQ ID NO: 8 and defined sets of sequences enumerated in the patent, excluding a specified sequence (SEQ ID NO: 6888).
Complementary 5′ leader and 3′ tail sequences mediating fluorophore binding conformation
The aptamer 5′ leader and 3′ tail sequences are complementary or complementary to a target sequence comprising leader and tail bait sequences. Binding of these complementary sequences mediate juxtaposition of the 5′ and 3′ termini of the core active sequence when in the fluorophore binding conformation.
High fluorophore binding affinity and brightness of fluorophore-aptamer complex
The aptamer in the fluorophore binding conformation has a fluorophore binding affinity of at least submicromolar to nanomolar levels (down to 0.5 nM). The fluorophore-aptamer complex has a brightness of at least 5,000 M−1cm−1, with some claims extending to at least 43,000 M−1cm−1, and a fluorescent lifetime in the range of 1 to 6 ns.
Fluorescence maintained in formaldehyde solution
The fluorophore-aptamer complex remains fluorescent in a formaldehyde solution, indicating stability compatible with fixation procedures.
Secondary aptamer sequence affinity for a secondary target moiety
The RNA aptamer includes a secondary aptamer sequence that has affinity for a secondary target moiety, enabling additional binding functionalities.
The claims cover RNA aptamers with defined active core sequences capable of adopting specific fluorophore binding conformations mediated by adjacent leader and tail sequences. These aptamers bind specific fluorophores of defined chemical formulas, resulting in highly fluorescent and bright complexes with strong binding affinities and stability, including functionality in fixed cell conditions. The claims also include bipartite systems involving complementary bait sequences and secondary affinity moieties, enabling versatile applications.
Stated Advantages
The aptamers demonstrate high fluorophore binding affinity and brightness, with some complexes being as bright or brighter than enhanced GFP.
The aptamer-fluorophore complexes exhibit thermal and salt stability, including Mg2+ resistance and fluorescence retention in formaldehyde solution, facilitating fixed cell imaging.
Selected fluorophores have high cellular permeability, supporting in vivo imaging applications.
The aptamers fold correctly both in the presence and absence of fluorophore, enabling consistent imaging in live cells with low background fluorescence.
Bipartite aptamer constructs provide high contrast ratios upon target binding, enabling RNA detection with low background.
Documented Applications
Imaging of small cellular RNAs such as 5S ribosomal RNA and U6 nuclear RNA in fixed and live mammalian cells.
Genetically encoded tags for live and fixed cell imaging of RNA localization and dynamics.
Use in bipartite detection systems as RNA Mango switch probes for targeting specific mRNAs, e.g., β-actin mRNA detection.
Fluorescence-based nucleic acid detection combined with rolling circle amplification for sensitive RNA quantification.
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