Nucleic acid concatemers and methods for stabilizing and/or compacting the same
Inventors
Costa, Justin • HERNÁNDEZ NEUTA, Jorge Iván • MARKS, Patrick J.
Assignees
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Publication Number
US-12553079-B2
Publication Date
2026-02-17
Expiration Date
Abstract
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.
Core Innovation
The invention relates to circularized or circularizable RCA probes for in situ rolling circle amplification, where the circularizable probe includes secondary structure that supports intramolecular folding into a hairpin or dumbbell configuration. The circularizable probe includes a first hybridizing region and a second hybridizing region that form complementary hybrids, with a hairpin secondary structure formed by a hairpin stem region and a hairpin loop and a dumbbell secondary structure formed by a dumbbell stem region and dumbbell loops. The probe can further include primer binding sequences and/or anchor sequences, and can include barcode sequences for downstream analysis.
The disclosed approach uses nucleic acid concatemers, including rolling circle amplification products (RCPs), that are compacted and/or stabilized at a location in a biological sample. A circular probe, circularizable probe, or probe set that hybridizes to a target nucleic acid is contacted with the biological sample, and rolling circle amplification generates an RCP at the location using the circular probe or a circularized probe generated from the circularizable probe or probe set as a template.
The invention also describes compacting and stabilizing rolling circle amplification products (RCPs) by promoting inter-copy hybridization between complementary regions. In this framework, the RCPs include repeating sequence copies such that Copy 1 and Copy 2 hybridize via inter-copy hybridization to compact and stabilize the RCP, thereby reducing feature spread or increasing signal localization. The compacted RCPs are described as retaining feature density while reducing RCP size for both hairpin and dumbbell probe designs.
In further aspects, the invention describes embedding or immobilizing nucleic-acid concatemers, including RCPs, in porous hydrogel matrices to stabilize signals. The porous matrix is described with examples including polyacrylamide-based, alginate-based, and polyethylene glycol-based matrices, and can include functional moieties for attachment. The document further connects these stabilized or compacted products to in situ detection workflows involving probe hybridization, fluorescent detection, and decoding or readout from barcode sequences, including reduction of tissue autofluorescence using fluorescent label quenchers.
Claims Coverage
The partial content provides three independent claims. Across these claims, the inventive features center on compacting and/or stabilizing a rolling circle amplification product via hybridization between complementary sequences from different RCP copies, using circular/circularizable probes or circularized padlock probes, and optionally incorporating loop-linked hybridizing-region architecture and barcode sequences.
Compacting or stabilizing an RCP using first and second hybridizing regions
A method where a circular probe, circularizable probe, or probe set is contacted with a biological sample, rolling circle amplification generates an RCP using the circular probe or a circularized probe as a template, the RCP comprises multiple copies of complementary sequences of a first and a second hybridizing region, and complementary sequences of the first and second hybridizing regions from different copies hybridize to one another to compact and/or stabilize the RCP at the location, followed by detecting the RCP at the location in the biological sample.
Padlock probe with first and second hybridizing regions linked by a loop and one or more barcode sequences
A method where a padlock probe hybridizes to a target nucleic acid and includes first and second hybridizing regions capable of hybridizing to each other that are linked by a loop region and one or more barcode sequences, the padlock probe is circularized to generate a circularized padlock probe using the target nucleic acid and/or a splint as template, rolling circle amplification generates an RCP comprising multiple copies of complementary sequences of the first and second hybridizing regions, and complementary sequences from different copies hybridize to one another to compact and/or stabilize the RCP at the location in the biological sample.
Padlock probe with two loop-linked hybridizing-region pairs and barcode sequences
A method where a padlock probe hybridizes to a target nucleic acid and comprises first and second hybridizing regions linked by a first loop region, third and fourth hybridizing regions linked by a second loop region, and one or more barcode sequences in the first and/or second loop regions, the padlock probe is circularized to generate a circularized padlock probe using the target nucleic acid and/or a splint as template, rolling circle amplification generates an RCP comprising multiple copies of complementary sequences of the first, second, third, and fourth hybridizing regions, and complementary sequences of the first and second hybridizing regions from two or more different copies hybridize to one another and complementary sequences of the third and fourth hybridizing regions from two or more different copies hybridize to one another to compact and/or stabilize the RCP at the location in the biological sample.
Across the independent claims, compacting and/or stabilizing is achieved by hybridization among complementary sequences encoded in multiple copies within an RCP generated by rolling circle amplification from a circular/circularizable probe or from a circularized padlock probe, with probe architecture optionally including loop-linked hybridizing-region arrangements and barcode sequences.
Stated Advantages
Compacts and/or stabilizes the RCP at the location in the biological sample.
Reduced feature spread or increased signal localization.
Retaining feature density while reducing RCP size.
Reduced tissue autofluorescence using fluorescent label quenchers.
Reduced RCP size and/or reduced fractions associated with smaller size distributions.
Documented Applications
Detecting RCPs at a location in a biological sample using circular/circularizable probes or circularized padlock probes.
In situ rolling circle amplification.
In situ detection workflows involving probe hybridization, fluorescent detection, and decoding or readout from barcode sequences.
Spatial transcriptomics.
In situ sequencing contexts, including sequential hybridization and imaging approaches.
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