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Publication Number
US-11649486-B2
Publication Date
2023-05-16
Expiration Date
Abstract
Nucleic acid probes for detection of a target nucleic acid molecule by an RCA reaction in the presence of the target nucleic acid molecule, comprise a first circular template strand which is capable of acting as a template for RCA, and is protected from RCA in the absence of the target nucleic acid molecule by at least a second protector strand which comprises a region of complementarity to the first template strand and is hybridised thereto to form a double-stranded circular structure containing the first template strand inside the protector strand(s). At least one of the second and/or any further protector strands comprises a target binding site, such that upon binding of the probe to the target nucleic acid molecule the probe is able to undergo a strand displacement reaction which allows RCA of the first template strand. Methods of detecting target analytes use such probes.
Core Innovation
The invention provides double-stranded circular RCA nucleic acid probes with a first circular template strand that is protected from rolling circle amplification (RCA) in the absence of the target nucleic acid molecule. The protection is achieved by at least a second protector strand that is hybridised to form a double-stranded circular structure in which the first circular template strand is contained inside the protector strand(s), thereby inhibiting RCA.
A key feature is that the protector strand(s) include a target binding site with at least a first single-stranded region having an accessible part of the target binding site. Upon binding to a complementary binding site in the target nucleic acid molecule, the probe undergoes a strand displacement reaction that allows RCA of the first circular template strand.
Protector strand architectures include single protector loop and stem-loop forms and multi-protector envelope structures with partially double-stranded stem regions, accessible toehold regions, and opening or unfolding via strand displacement. In some embodiments, the probe is activatable by a separate activator molecule recruited by the target, and activation enables primer accessibility either by strand release or cleavage, or by primer binding to prime RCA.
The disclosed probes also support structural and sequence variants, including features for template size and nucleotide length constraints, mismatch or insertions to enable smaller circles, and reporter or barcode domains for multiplexing. Detection and kit formats are described, including solution or in situ detection approaches and examples such as target dose-response detection and proximity-based protein detection.
Claims Coverage
The independent claims cover activatable nucleic acid RCA probes in which a circular template strand is inhibited by protector strands forming a double-stranded circular structure, and RCA is initiated after target-dependent or target/activator-dependent strand displacement that exposes primer accessibility and allows RCA. Across the independent claims, the core coverage centers on two inventive features.
Double-stranded circular protector inhibition with target-dependent strand displacement
A nucleic acid probe for detection of a target nucleic acid molecule by a rolling circle amplification (RCA) reaction, comprising a first circular template strand and at least a second protector strand that protects the first circular template strand from RCA in the absence of the target nucleic acid molecule, wherein the protector strand(s) comprise a target binding site and a region of complementarity to the first circular template strand, are hybridised thereto to form a double-stranded circular structure containing the first circular template strand inside the protector strand(s), and further comprise at least a first single-stranded region with at least an accessible part of the target binding site allowing binding to the target nucleic acid molecule, such that a strand displacement reaction allows RCA of the first circular template strand.
Stem-loop opening and 3′-end release or primer binding for RCA
Binding of the target nucleic acid molecule or a separate activator molecule causes a strand displacement reaction that opens at least the stem of the stem-loop structure and releases the 3′ end of the second protector strand, where the released 3′ end is cleavable to provide a primer for RCA of the first circular template strand, or able to bind to the first circular template strand following the strand displacement reaction to prime RCA.
Overall, the claim set focuses on activatable RCA probes where protector strands form a double-stranded circular structure that inhibits RCA until target, and in some embodiments a separate activator molecule, triggers strand displacement. Activation exposes primer accessibility by cleavable release or primer binding to the first circular template strand, enabling RCA under target-dependent conditions.
Stated Advantages
Target-dependent control of RCA by inhibiting RCA in the absence of the target nucleic acid molecule and enabling RCA upon binding of the target nucleic acid molecule through a strand displacement reaction.
Optional activator-mediated activation where binding of the target nucleic acid molecule or separate activator molecule triggers strand displacement that releases a primer-capable 3′ end for RCA.
Documented Applications
Detection of rolling circle amplification (RCA) products, including RCA products generated by super-RCA or second-generation RCA, with signal readouts such as fluorescence, fluorescence microscopy, multiplex or sequential detection, and flow cytometry.
Proximity-based detection using proximity probes and proximity-dependent detection with an activator nucleotide, including activator-protected by a hairpin and activated by UNG, for detection of RCA products.
Detection on cells and in situ detection, including solid-phase and in situ detection involving antibody conjugation to probes and detection of protein or protein complexes on cells with labeled RCA products.
Immobilized probe detection on solid supports using amplification concepts with RCA-product-recognizing immobilized probes.
Target nucleic acid detection by rolling circle amplification (RCA) using activatable nucleic acid probe constructs.
Proximity-based detection of analytes, including protein detection via proximity probes.
Multiplexing using reporter or barcode domains.
In situ and solution formats for detection and demonstration examples including target dose-response detection and selectivity.
Use in a kit format including probe and additional RCA-related components, and detection of RCA products.
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