Catalytic de-crosslinking of samples for in situ analysis
Inventors
Delaney, Joshua • Gohil, Shalini • Gonzalez Muñoz, Veronica Emelina • GU, Joshua • Kim, Albert Dale • Luo, Yi • Mukherjee, Tathagata • Nagendran, Monica • Perna, III, James Francis • Pham, Kristen Nguyen • Tjandra, Meiliana
Assignees
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Publication Number
US-12516369-B1
Publication Date
2026-01-06
Expiration Date
Abstract
The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.
Core Innovation
The invention relates to an RCA-based detection system that includes a catalyst of formula (I), or a salt, zwitterion, or solvate thereof, together with a nucleic acid probe and a polymerase for rolling circle amplification. The nucleic acid probe comprises a target-binding sequence complementary to a portion of a target RNA analyte, and the polymerase performs RCA using the nucleic acid probe as a template to generate an RCA product.
The catalyst is defined by specific selections for A, X1, X2, X3, and X4, with Ra substituents, R' and R'' substitutions, and related structural constraints described for the formula (I) scaffold. The disclosed catalyst context includes catalyst embodiments and catalyst compound structures, together with salt, zwitterion, and solvate forms.
The description further states that the catalyst catalytically breaks down formaldehyde-induced aminal and hemi-aminal adducts in RNA and in a biological sample, and is compatible with RNA extraction and detection conditions. The biological sample is a tissue section in some refinements, and downstream processing includes sequencing a barcode sequence in the RCA product using sequencing by synthesis (SBS) or sequencing by ligation (SBL).
Claims Coverage
The consolidated claim coverage centers on an RCA-based detection system combining a defined catalyst of formula (I), or a salt, zwitterion, or solvate thereof, a nucleic acid probe complementary to a portion of a target RNA analyte, and a polymerase configured to perform RCA and generate an RCA product. Across the provided items, the independent inventive features are the catalyst structure, the target-complementary probe, and the RCA-generating polymerase, with dependent refinements adding catalyst function, sample constraints, and sequencing elements.
RCA-based detection system with defined catalyst of formula (I)
A rolling circle amplification (RCA)-based detection system comprising a catalyst of formula (I), or a salt, zwitterion, or solvate thereof, a nucleic acid probe comprising a target-binding sequence complementary to a portion of a target RNA analyte, and a polymerase for performing RCA using the nucleic acid probe as a template to generate an RCA product.
Catalyst structure defined by selection of A and X atoms
The catalyst of formula (I) is defined such that A is selected from COOH, P(=O)(OH)2, and S(=O)2OH, and X1, X2, X3, and X4 are each independently selected from CH, CRa, and N, with Ra and related substituents defined for the scaffold.
Catalyst-driven breakdown of hemi-aminal and aminal adducts
The catalyst catalyzes the breakdown of hemi-aminal adducts and/or aminal adducts in a biological sample, including formaldehyde-induced adducts.
Sequencing a barcode sequence in the RCA product
One or more reagents enable sequencing a barcode sequence in the RCA product using sequencing by synthesis (SBS) or sequencing by ligation (SBL).
Overall, the independent claim coverage consistently requires a catalyst of formula (I) in salt, zwitterion, or solvate form, a nucleic acid probe complementary to a target RNA analyte portion, and a polymerase that performs RCA to generate an RCA product. The dependent refinements in the provided items further define catalyst structure and function and add barcode sequencing elements.
Stated Advantages
The catalyst catalytically breaks down formaldehyde-induced aminal/hemi-aminal adducts in RNA.
The catalyst is stated to be compatible with RNA extraction and detection in the RCA-based detection system.
Reducing formaldehyde-derived aminal crosslinks and associated autofluorescence in fixed biological samples to improve detection.
Restoring accessibility for probe and antibody binding.
Improving signal density and signal-to-noise ratio after catalytic de-crosslinking versus non-catalytic approaches.
Documented Applications
RNA extraction and detection in an RCA-based detection system involving formaldehyde-treated RNA and formaldehyde-induced aminal/hemi-aminal adducts.
In situ detection of nucleic acids in fixed biological samples, including nucleic-acid detection involving RCA products or barcodes with optical detection such as fluorescent microscopy or imaging.
In situ detection of proteins in fixed biological samples using fluorophore-labeled antibodies or reporter oligonucleotide systems.
In situ detection workflows with sequencing readouts, including sequencing by synthesis (SBS), sequencing by ligation (SBL), and sequencing by hybridization (SBH).
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