Multiplexed single molecule RNA visualization with a two-probe proximity ligation system
Inventors
Samusik, Nikolay • Bava, Felice Alessio • Goltsev, Yury • Nolan, Garry P.
Assignees
Leland Stanford Junior University
Publication Number
US-12338490-B2
Publication Date
2025-06-24
Expiration Date
2037-02-24
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Abstract
SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.
Core Innovation
The invention provides compositions and methods, termed SNAIL-RCA (Splint Nucleotide Assisted Intramolecular Ligation followed by Rolling Circle Amplification), for the analysis of mRNA species at a single cell level. The core methodology utilizes two oligonucleotides per target nucleic acid: a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), each comprising regions complementary to adjacent sequences on the target RNA. Upon hybridization, the PO is circularized by ligation, allowing rolling circle amplification (RCA), with detection achieved through hybridization of a specific labeled detection probe.
The key problem addressed is the limitation of conventional single-cell RNA detection methods, which are either restricted in multiplexing capability due to the number of distinguishable fluorophores, or require laborious, costly, and inefficient procedures such as cDNA generation and multi-probe hybridization. Previous methods, including padlock probe ligation and branched DNA technology, require multiple steps or complex probe designs, restricting their use in high-throughput or highly multiplexed analyses, particularly in conjunction with cytometry platforms.
SNAIL-RCA simplifies the probe design by using only two probes per target, reducing the cost and complexity of analysis while allowing a high degree of multiplexing. This approach enables cost-efficient, specific, and sensitive detection of nucleic acids in single cells and is compatible with flow cytometry, mass cytometry, and microscopy, permitting simultaneous analysis of large numbers of cells and multiple nucleic acid targets.
Claims Coverage
The patent contains two independent claims, each specifying inventive features of the multiplexed, single-cell nucleic acid detection methods using SNAIL oligonucleotide technology.
Single target nucleic acid detection in a fixed and permeabilized single cell using a SNAIL two-probe system
A method for determining the level of a target nucleic acid in a fixed and permeabilized single cell, comprising: - Contacting the cell with a pair of SNAIL oligonucleotide primers (Splint Primer Oligonucleotide and Padlock Oligonucleotide), each with first and second complementarity regions, which bind to adjacent sequences on the target nucleic acid. - The Padlock Oligonucleotide (PO) hybridizes with its ends positioned directly adjacent to each other after binding. - Washing away unbound primers, then ligating the PO ends to generate a closed circle. - Performing in situ rolling circle amplification using the closed circle as a template and the SPO as a primer, generating an amplification product. - Contacting the amplification product with a labeled detection probe (fluorophore, isotope, or mass tag), under conditions for specific hybridization, to generate a labeled cell. - Detecting the level of the detection probe bound to the amplification product to determine the level of the target nucleic acid, and analyzing the labeled cell by mass cytometry or fluorescence-activated flow cytometry.
Simultaneous detection of different target nucleic acids in a single cell using multiplexed SNAIL primer pairs
A method for determining the level of a target nucleic acid from different nucleic acids in a fixed and permeabilized single cell, comprising: - Contacting the cell with a plurality of pairs of SNAIL oligonucleotide primers, each pair specific for a different target nucleic acid and each designed with adjacent complementarity regions as above. - Washing to remove unbound primer pairs, ligating the PO of each pair to form closed circles upon specific hybridization. - Performing rolling circle amplification in situ for each target, generating amplification products. - Contacting the amplification products with detection probes labeled with fluorophore, isotope, or mass tag, for specific hybridization. - Determining the levels of detection probes bound to amplification products representing the different target nucleic acids, and subjecting the labeled cell to mass cytometry or fluorescence-activated flow cytometry analysis.
The independent claims cover the innovative use of a two-probe SNAIL system for specific, multiplexed detection and quantitation of nucleic acids at the single-cell level, enabling analysis by mass or fluorescence cytometry.
Stated Advantages
Reduces the number of probes required per target, lowering analysis cost and simplifying probe design.
Allows a high degree of multiplexing, enabling simultaneous analysis of many nucleic acids in single cells.
Provides high specificity, low background, and high signal-to-noise ratios due to the requirement for adjacent probe hybridization and ligation.
Enables cost-efficient and sensitive detection of nucleic acids in single cells.
Compatible with flow cytometry, mass cytometry, and microscopy platforms for rapid, high-throughput analysis.
Allows simultaneous detection of nucleic acids and proteins in single cells.
Suitable for analysis of large numbers of cells at rates of up to 1000 or more cells per second.
Documented Applications
Determining the abundance of mRNA or other nucleic acids in single cells within complex cell populations, including tissues, biopsies, and blood samples.
Multiplexed detection and quantification of specific splice variants of mRNA transcripts in single cells.
Simultaneous detection and quantification of nucleic acid molecules and protein-protein interactions by combination with Proximity Ligation Assay (PLA).
Detection and quantification of specific DNA sequences, gene copy number variations, and genomic translocation/fusion events, including single-cell genotyping.
Simultaneous analysis of multiple nucleic acids and proteins in single cells using flow cytometry, mass cytometry, or microscopy.
Diagnostic methods for detecting aberrant cells (cancer, viral infection) by assessing nucleic acid expression profiles in single cells from clinical samples.
Non-diagnostic profiling of gene expression variability, heterogeneity, and regulatory networks in research samples and cultured cells.
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