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WO2025007154A1 - Procédés et compositions d'amplification du signal pour la détection de cibles moléculaires par dépôt itératif de sondes - Google Patents

Procédés et compositions d'amplification du signal pour la détection de cibles moléculaires par dépôt itératif de sondes Download PDF

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Publication number
WO2025007154A1
WO2025007154A1 PCT/US2024/036455 US2024036455W WO2025007154A1 WO 2025007154 A1 WO2025007154 A1 WO 2025007154A1 US 2024036455 W US2024036455 W US 2024036455W WO 2025007154 A1 WO2025007154 A1 WO 2025007154A1
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Prior art keywords
amplifier
probe
probes
primary
binding
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Inventor
Katsuya COLON
Carsten Tischbirek
Long Cai
Chuqi LU
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California Institute of Technology
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California Institute of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the present disclosure provides methods, compositions, and kits for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample.
  • This invention can be implemented for multiplex profiling of one or multiple ty pes of analytes such as, but not limited to, RNAs, DNAs, proteins, small molecule inhibitors, sugars, lipids, organelle, synthetic barcodes, in biological or clinical samples.
  • Imaging-based spatial transcriptomics methods have emerged as a powerful tool for understanding the spatial organization of gene expression within tissues and cells, providing valuable insights into cellular heterogeneity and tissue function (1).
  • a major limitation for imaging-based spatial transcriptomics methods is the low signal-to-noise ratio, particularly in thick tissue samples or non-ideal tissue samples such as FFPE or human derived tissue samples.
  • dim signals can be circumvented by using a larger number of hybridization probes per gene. Further, this approach will not work when targeting short RNAs species.
  • the present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.
  • a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample comprising contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte.
  • the method comprises contacting each of a plurality of the primary probes, each interacting wi th at least one analyte, with one or more amplifier probes.
  • the method comprises optionally, cross-linking one or more amplifier probes to a cellular component.
  • the method comprises optionally, separating each of one or more amplifier probes from its primary’ probe or from another amplifier probe. In some embodiments, the method comprises optionally, repeating any of the previous embodiments either alone or in combination. In some embodiments, the method comprises detecting one or more target analytes.
  • the method comprises contacting a plurality of the amplifier probes each with one or more readout probes. In some embodiments, the method comprises imaging the sample so that the interaction of the readout probes with their target analytes is detected.
  • the method comprises imaging the samples after contacting the amplifier probes with one or more readout probes.
  • the method further comprises amplifying the amplifier probes by contacting the amplifier probes from a previous contacting step with anew plurality of amplifier probes.
  • the method further comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes.
  • the method comprises optionally, separating an amplifier probe from the primary probe or another amplifier probe after imaging the sample.
  • the method comprises optionally, separating the readout probes from the amplifier probes.
  • a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample comprising contacting one or more analytes with a primary probe in a plurality of primary probes, wherein each probe in the plurality 7 of primary 7 probes interacts with at least one target analyte.
  • the method comprises contacting each primaryprobe interacting with at least one analyte, with one or more amplifier probes.
  • the method comprises cross-linking the amplifier probe to a cellular target.
  • the method comprises separating the amplifier probes from the primary probes.
  • the method comprises optionally, repeating any of the previous steps.
  • the method comprises contacting each amplifier probe with one or more readout probes.
  • the method comprises imaging the sample after contacting the amplifier probes with one or more readout probes so that the interaction of readout probes w ith their target analytes are detected.
  • Figure 1 depicts an exemplary design of a linear amplification process.
  • Figure 1 A illustrates an exemplary embodiment of a primary probe binding to a target molecule.
  • Figure IB illustrates an exemplary' embodiment of an amplifier probe binding to a unique region of the primary probe.
  • Figure 1C illustrates an exemplary embodiment of the amplifier probe binding to the sample through the reactive group.
  • Figure ID illustrates an exemplary embodiment of subsequent amplifier binding to the primary probe.
  • Figure IE illustrates an exemplary embodiment of multiple rounds of amplifier binding to the primary probe, binding to the sample, and unbinding from the primary- probe to allow the next amplifier to bind to the same primary’ probe site.
  • Figure IF illustrates an exemplary embodiment of readout probes binding to the amplifiers that are bound to the sample in order to detect the target molecule.
  • Figure 2 depicts an exemplary design of a process without binding probes to the sample.
  • Figure 2A illustrates an exemplary embodiment of a primary probe binding to a target molecule.
  • Figure 2B illustrates an exemplary embodiment of amplifier probe binding to a binding region of the primary probe.
  • Figure 2C illustrates an exemplary embodiment of the amplifier probe not binding to the sample.
  • Figure 2D illustrates an exemplary embodiment of readout probes not binding to the sample, as depicted by the dotted line.
  • Figure 3 depicts an exemplary design of a pieceyvise linear amplification process.
  • Figure 3A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample.
  • Figure 3B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary- probes that are bound to the sample.
  • Figure 3C illustrates an exemplary embodiment of binding the secondary amplifier probes to the sample.
  • Figure 3D illustrates an exemplary embodiment in which in a subsequent round of secondary amplifier addition, sample binding, and unbinding from the initial amplifier probe, more secondary amplifiers are added.
  • Figure 3E illustrates an exemplar ⁇ ' embodiment in which during another subsequent round of secondary amplifier addition, sample binding, and unbinding from the initial amplifier probe, more secondary amplifiers are added.
  • Figure 3F illustrates an exemplary embodiment in which each secondary amplifier probe can be bound by labelled readout probes that can be used to detect the molecular target in the sample.
  • Figure 4 depicts an exemplary design of a linear and exponential amplification process.
  • Figure 4A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample.
  • Figure 4B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary probes that are bound to the sample. In the example, each amplifier used for exponential amplification rounds has two binding sites for the next round of amplifiers.
  • Figure 4C illustrates an exemplary embodiment of binding an exponential amplifier probe to the sample. In this example, the amplifiers have two binding sites for the next round of amplifiers.
  • Figure 4D illustrates an exemplary embodiment of binding an exponential amplifier probe to the sample, with two amplifiers binding to each amplifier bound in the previous round.
  • the amplifier probes applied in exponential amplifier round 2 can be bound with the amplifier probes applied in round 1. which in turn can be bound by the amplifier probes applied in round 2 and so forth.
  • Figure 5 depicts an exemplary design of an exponential amplification process following an amplification factor according to a Pell number series.
  • Figure 5A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe.
  • Figure 5B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked and cleaved first amplifier at feature A+.
  • Figure 5C illustrates an exemplary embodiment of the next round in which the same amplifier is applied to the sample as in the initial round.
  • Figure 5D illustrates an exemplary embodiment of another round of amplification with secondary amplifier probes, which bind to the A+ sites as described in Figure 5B.
  • Figure 5E illustrates an exemplary embodiment of the next round of amplification, in which the first amplifier is added to the sample again. Binding is similar as described in Figure 5C.
  • Figure 6 depicts an exemplary design of an exponential amplification process without cleavage or displacement.
  • Figure 6A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe.
  • Components of the amplifier probe that are labelled in this example are a binding site R- for the primary probes, two A+ binding sites for subsequent amplifier probe binding, and a linkable feature that can bind to, for example, the sample.
  • Figure 6B illustrates an exemplary embodiment of two subsequent amplifier probes binding to the amplifier probe that was bound to the primary probe in the previous panel. Linkable features of the amplifiers in this illustration are stably bound to the sample.
  • Components of the amplifier probe that are labelled in this example are a binding site A- for the amplifier binding site A+ of the previous round, two R+ binding sites for subsequent amplifier probe binding, and a linkable feature.
  • Figure 6C illustrates an exemplary' embodiment of two subsequent amplifier probes binding to the amplifier probes bound in the previous round.
  • the amplifier probes in this example have the same components as used in round 1 shown in Figure 6A. Additional rounds of exponential amplification beyond the 3 amplification rounds shown in this exemplary embodiment increase the number of amplifiers bound in proximity of the primary probe binding site.
  • Figure 7 depicts an exemplary design of amplifier probes that can be bound to the sample.
  • Figure 7A illustrates an exemplary embodiment of a primary probe bound with an amplifier probe.
  • Figure 7B illustrates an exemplary embodiment of an amplifier probe similar to the one described in example Figure 7A, but it shows two binding sites A+ instead of one binding site for designs that involve, for example, stronger linear amplification or non-linear amplification schemes.
  • the cleavage or displacement feature included in this example illustration can either be a dedicated part of the probe, or partially or completely overlap with the A+ example binding site adjacent to the R- primary probe binding site for multiple purposes as a displacement, cleavage, and/or amplifier and readout probe binding site.
  • Figure 8 depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe.
  • Figure 8A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe.
  • Figure 8B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+/R3-. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • Figure 8C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe.
  • FIG. 8D illustrates an exemplary embodiment of displacement probes remaining attached to the amplifier probe after washing away excess displacement probes. Displacement probes remain on the amplifier probe to avoid re-binding of the amplifier probe to the primary probe.
  • Figure 8E illustrates an exemplary embodiment of a subsequent amplification round with another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle). The amplification cycle numbers used for the linear signal amplification are shown above the example images.
  • Figure 9 depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe with displacers that partially bind to a second readout site on the amplifier probe.
  • Figure 9A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe.
  • Figure 9B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+/R3-.
  • the amplifier probe has two A3+ amplifier binding sites, instead of a dedicated toehold sequence.
  • the crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • Figure 9C illustrates an exemplar ⁇ ' embodiment of a displacement probe binding to the amplifier probe.
  • binding is aided by a toehold feature that partially covers amplifier binding site A3+ on the amplifier probe, which allows binding of the displacement probe to be favoured compared to the binding of R3- to R3+ on the primary probe.
  • the toehold consists of nucleotide 1 to nucleotide n, which can for example be a total length of 10 nt, of the amplifier binding site A3+. While the displacer probe in this exemplary' embodiment can remain bound to the amplifier probe, it allows for the same amplifier probe to be used in exponential amplification rounds when no displacer probe is applied to the sample.
  • Figure 9D illustrates an exemplary embodiment of displacement probes remaining attached to the amplifier probe after washing away excess displacement probes.
  • Figure 9E illustrates an exemplary’ embodiment of a subsequent amplification round with another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle).
  • Figure 10 provides an example design of a protocol for the iterative accumulation of probes near the primary probe binding site using photo-crosslinking, amplifier cleavage, and subsequent washing for unbinding from the primary probe.
  • Figure 10A illustrates an exemplary embodiment of a primary' probe binding to a target molecule and amplifier probes binding to the primary probe similar to Figure 7A.
  • Figure 10B illustrates an exemplary' embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+/R3-.
  • the crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • the white rectangle illustrates an example cleavable feature of the amplifier probe.
  • Figure 10C illustrates an exemplary embodiment of the amplifier probe that was cleaved at the cleavable feature.
  • the portion of the amplifier that includes binding feature A3+ remains in proximity to the primary probe, as it was linked to the sample through the linkable feature illustrated by the crossed-out circle.
  • Figure 10D illustrates an exemplary embodiment of the remaining portion of the cleaved amplifier which remains bound to example binding site R3+ through binding feature R3-. This portion of the cleaved amplifier can be removed, for example, through wash conditions suitable for the short remaining fragment.
  • Figure 10E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle), which has become available through the steps described in exemplary Figures 10B-D.
  • Figure 11 depicts exemplary images of a linear amplification process of the fluorescence signal intensity using diazirine modified oligonucleotides of a single gene in a cell culture sample.
  • Figure 11 A illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification.
  • Figure 1 IB illustrates an exemplary embodiment of a fluorescent intensity signal increase after five consecutive rounds of linear amplification.
  • Figure 11 C illustrates an exemplary embodiment of a fluorescent intensity signal increase after eleven consecutive rounds of linear amplification.
  • Figure 12 depicts exemplary images of a linear amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample. The scale bar is 20 microns.
  • Figure 12 illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.
  • Figure 13 illustrates an exemplary embodiment of a fluorescent intensity distribution change over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.
  • Figure 14 illustrates an exemplary embodiment of a combined linear and exponential amplification process of the fluorescence signal intensity of a single gene in a cell culture sample using benzophenone modified oligonucleotides.
  • Figure 14A illustrates an exemplary embodiment of a fluorescent intensity signal increase. Labelling above exemplary images shows number of total amplification rounds. Contrast is adjusted so that images look similar despite a fluorescence increase to illustrate the similarity of fluorescent signal features such as signal spot size and signal spot number-in different amplification rounds.
  • Figure 14B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.
  • Figure 15 illustrates additional designs for crosslinking amplification.
  • Figure 15 A illustrates schematics of a sacrificial layer design for amplification.
  • Figure 15B illustrates quantification of signal of individual amplified dot in cells implementing the scheme shown in Figure 15 A. The histogram shows the intensities of the single dots in cells compared to single molecule FISH (smFISH) imaging without amplification.
  • Figure 15C illustrates a bridge adapter design that generates signal only when two amplified balls are physically proximal to each other. The highlighted bridge probe with a thicken line shows the bridge adapter that bind across two amplified balls. The thicken line represents a readout probe binding site.
  • Figure 15D illustrates images of cells amplified using the scheme shown in Figure 15C.
  • non-specifically amplified dots in each of the amplified channels are not observed in the bridge adapter channel (right panel).
  • the lower right comers of each image show zoomed-in images shown in the white box.
  • the arrows in the insets on the lower right show regions where nonspecific dots appear in the individual amplified channel, but not in the bridge channel.
  • Figure 16 illustrates an exemplary embodiment of a combined linear and exponential amplification process of the fluorescence signal intensity of a single gene in a cell culture sample using benzophenone modified oligonucleotides.
  • Figure 16A illustrates an exemplary’ embodiment of a fluorescent intensity signal increase for a small part of a recorded image to visualize individual fluorescence intensity’ peaks. Labelling above exemplary images shows number of total amplification rounds. Contrast is matched for all example images, with a lOx shorter exposure time for the rightmost two images to illustrate the increase in signal intensity.
  • Figure 16B illustrates an exemplary embodiment of a fluorescent intensity' signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary' image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.
  • Figure 17 illustrates that probes can be crosslinked to the cell via CuAAC reaction.
  • Figure 17A is a schematic diagram of DNA oligo “clicked 7 ’ to the cell by the 3’ azide modification.
  • Figure 17B is a schematic diagram for the CuAAC crosslinking efficiency experiment. Primary’ probes and the amplifier tested were designed as show n, and only the amplifier was crosslinked to the cell.
  • Figure 17C illustrates examples of the smFISH signal of 1 cycle of crosslinked amplifier on 24 Eef2 primary probes. Pre- and post- 60% formamide wash, with and without click are shown in comparison, contrasts are adjusted to the same values for each channel.
  • Figure 17D illustrates a quantification of the signal intensity (peak value) for C. Images are Z projected for quantification.
  • Figure 18 illustrates how a split design can increase amplifier targeting specificity.
  • Figure 18A is a schematic diagram of the Split probes click-crosslinked amplification method.
  • Figure 17B illustrates an example of the Eef2 mRNA signal pre- (right, A488) and post- (left, Cy3B) 8 cycles of split probes amplification.
  • Figure 19 illustrates branch design enables rapid amplification with short amplifiers.
  • Figure 19A illustrates a schematic diagram of the Branch probes click- crosslinked amplification method.
  • Figures 19B-C Example of Eef2 signal from 24 primary probes after 6 cycles of amplification.
  • Figure 19B illustrates that all amplifier binding sites were designed as 13 nucleotide (nt) (13nt BS).
  • Figure 19C illustrates that all amplifier binding sites were designed as 15 nt (15nt BS).
  • Figure 19D illustrates quantification of the peak intensity of dots for standard smFISH, 13nt BS amplified and I5nt BS amplified.
  • the amplification fold for 13nt BS is 19.35 (mean) and 16.64 (median); the amplification fold for 15nt BS is 39.43 (mean) and 37.36 (median).
  • Images are Z projected for quantification.
  • Figure 20 illustrates that a padlock design can reduce noise by exonuclease digestion.
  • Figure 20A illustrates a schematic diagram of the Padlock probes click-crosslinked amplification method.
  • Figure 20B is an example of the single Ee£2 primary probe signal after 14 cycles of amplification.
  • Figure 20C illustrates quantification of the peak intensity of dots for standard smFISH (24 primary probes) versus 1 primary probe amplified.
  • the amplification fold for the mean is 8.41 and for the median is 7.67.
  • Primary probes are expected to have 80% binding efficiency, therefore the amplification fold for the single probe is estimated to be ⁇ 150x.
  • oligonucleotide refers to a polymer or oligomer of nucleotide monomers, containing any combination of nucleobases. modified nucleobases, sugars, modified sugars, phosphate bridges, or modified bridges.
  • Oligonucleotides can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 500 nucleotides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, and triple-stranded, can range in length from about 12 to about 20 nucleotides, from about 10 to about 60 nucleotides, from about 10 to about 90 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 120 nucleotides in length. In some embodiments, the oligonucleotide is from about 4 to about 39 nucleotides in length.
  • the oligonucleotide is at least 4 nucleotides in length. In some embodiments, the oligonucleotide is at least 5 nucleotides in length. In some embodiments, the oligonucleotide is at least 6 nucleotides in length. In some embodiments, the oligonucleotide is at least 7 nucleotides in length. In some embodiments, the oligonucleotide is at least 8 nucleotides in length. In some embodiments, the oligonucleotide is at least 9 nucleotides in length. In some embodiments, the oligonucleotide is at least 10 nucleotides in length.
  • the oligonucleotide is at least 11 nucleotides in length. In some embodiments, the oligonucleotide is at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 21 nucleotides in length.
  • a probe refers to any molecules, synthetic or naturally occurring, that can attach themselves directly or indirectly to a molecular target (e.g., an mRNA sample, DNA molecules, protein molecules, RNA and DNA isoform molecules, single nucleotide polymorphism molecules, and etc.).
  • a probe can include a nucleic acid molecule, an oligonucleotide, a protein (e.g.. an antibody or an antigen binding sequence), or combinations thereof.
  • a protein probe may be connected with one or more nucleic acid molecules to form a probe that is a chimera.
  • a probe itself can produce a detectable signal.
  • a probe is connected, directly or indirectly via an intermediate molecule, with a signal moiety (e.g., a dye or fluorophore) that can produce a detectable signal.
  • a signal moiety e.g., a dye or fluorophore
  • a sample refers to a biological sample obtained or derived from a source of interest, as described herein.
  • a source of interest comprises an organism, such as an animal, plant, microorganism or human.
  • a biological sample comprises biological tissue or fluid.
  • a biological sample is or comprises bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc.
  • a biological sample is or comprises cells obtained from an individual.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g.. blood, lymph, feces etc.), etc.
  • body fluid e.g.. blood, lymph, feces etc.
  • sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary' sample. For example, filtering using a semi-permeable membrane.
  • sample may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary' sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
  • sample refers to a nucleic acid such as DNA, RNA. transcripts, or chromosomes.
  • sample refers to nucleic acid that has been extracted from the cell.
  • target analyte refers to transcripts, RNA, DNA loci, chromosomes, DNA, exogenous DNA, proteins, peptides, lipids, glycans, cellular component, small molecules, metabolites, primary’ probe, amplifier probe, organelles and any combinations thereof.
  • the term “cellular components” refers structures in a cell that regulating movement, maintain cell shape, producing proteins, or any combination thereof.
  • the cellular components are selected from a nucleolus, a nucleus, ribosomes, vesicles, a rough endoplasmic reticulum, a golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, mitochondrion, vacuoles, cell cytosol, lysosomes. centrosomes, cell membranes, and any combination thereof.
  • the “cellular components” refers to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probe, amplifier probe, organelles, and any combinations thereof.
  • the term “primary 7 probe” refers to a probe that interacts with a target analyte.
  • the probe is nucleic acid molecule.
  • the probe is an oligonucleotide.
  • the term ’‘amplifier probe” refers to a probe that interacts with a primary' probe or another amplifier probe.
  • a “primary 7 amplifier” probe is an amplifier probe that interacts with a primary probe.
  • a “secondary amplifier probe” is an amplifier probe that interacts with a primary amplifier probe or with a primary probe.
  • a “tertiary amplifier probe” is an amplifier probe that interacts with a secondary amplifier probe or with a primary amplifier probe.
  • a “quaternary amplifier probe” is an amplifier probe that interacts with a tertiary amplifier probe, or w ith a secondary amplifier probe.
  • the amplifier probe is a nucleic acid sequence.
  • the amplifier probe is an oligonucleotide.
  • the amplifier probe has a motif that crosslinks to a cellular component.
  • the term “readout probe” refers to a probe that interacts with an amplifier probe.
  • the readout probe is a nucleic acid sequence comprising one or more fluorophores.
  • the readout probe is an oligonucleotide.
  • cellular components refers to a target selected from transcripts, RNA, DNA loci, chromosomes, DNA, protein, antibodies, lipids, glycans, cellular components, organelles, synapses, cell-to-cell junctions, cellular component boundaries and any combinations thereof.
  • the term “distribution” refers to the location of a cellular component w'ithin a cell. In certain embodiments, the term “distributions” also refers to the interactions between cellular components and a cell, between other cellular components. [0049] As disclosed herein, the term “mapping” refers to detecting a barcode linked to a cellular component and identifying its position intracellularly or extracellularly. [0050] As disclosed herein, the term “interacting’' or “interacts”’ refers to the binding of two or more molecules.
  • binding- may occur by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces, between two or more molecules.
  • binding may refer to the formation of covalent bonds between two or more molecules.
  • binding may refer to the interaction of two or more molecules directly or indirectly.
  • two or more molecules that bind indirectly have one or more molecules that interact between the two or more molecules. As example, molecule A and molecule B interact indirectly if molecule C interacts with molecule A and molecule B. but molecule A and B do not directly interact.
  • binding may refer to the hybridization of two or more nucleotide sequences. In certain embodiments, “binding” may refer to a protein-nucleotide interaction. In certain embodiments, “binding” may refer to a protein-protein interaction.
  • the term “close proximity” refers to the distance between two objects wherein the first object is about 5. 10. 20, 30, 40, 50, 60, 70, 80, 90, 100. 150, 200. 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, or 3000 nanometers from the other.
  • the present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.
  • a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample comprising contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte.
  • the method comprises contacting each of a plurality of the primary probes, each interacting with at least one analyte, with one or more amplifier probes.
  • the method comprises optionally, cross-linking one or more amplifier probes to a cellular component.
  • the method comprises optionally, separating each of one or more amplifier probes from its primary’ probe or from another amplifier probe. In some embodiments, the method comprises optionally, repeating any of the previous embodiments either alone or in combination. In some embodiments, the method comprises detecting one or more target analytes.
  • the method comprises at least one step of cross-linking one or more amplifier probes to a cellular component.
  • the method comprises imaging the samples after contacting the amplifier probes with one or more readout probes.
  • the method further comprises amplifying the amplifier probes by contacting the amplifier probes from a previous contacting step with anew plurality of amplifier probes.
  • the method further comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes.
  • the method comprises optionally, separating an amplifier probe from the primary probe or another amplifier probe after imaging the sample.
  • the method comprises optionally, separating the readout probes from the amplifier probes.
  • the method comprises single molecule resolution of target analytes. In some embodiments, the method comprises single molecule resolution of cellular components.
  • the target analytes comprise transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probe, amplifier probe, organelles, membranes, and any combinations thereof.
  • the target analytes are obtained from bacterial cells, archaeal cells, eukaryotic cells, or a combination thereof.
  • the target analytes comprise molecular targets that are selected from proteins, modified proteins, transcripts, RNA, DNA loci, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, biologically active molecules, and combinations thereof.
  • the targets comprise subcellular features.
  • the target analytes comprise RNA-DNA interactions, RNA- protein interactions, DNA-protein interactions, protein-protein interactions, or nucleic acidsmall molecule interactions.
  • the cellular components comprise structures in a cell that regulating movement, maintain cell shape, producing proteins, or any combination thereof.
  • the cellular components are selected from a nucleolus, a nucleus, ribosomes, vesicles, a rough endoplasmic reticulum, a golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, mitochondrion, vacuoles, cell cytosol, lysosomes, centrosomes, cell membranes, and any combination thereof.
  • the cellular component is a target analyte. In some embodiments, a cellular component and a target analyte are the same. In some embodiments, a cellular component and a target analyte are different. In some embodiments, the cellular component is at about 10, 20, 30, 40, 50, 60, 70, 80. 90. 100, 150, 200, 250, 300. 350, 400. 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nanometers from the target analyte.
  • the primary probe is selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof. In some embodiments, the primary probe comprises an oligonucleotide.
  • the primary probe comprises oligonucleotides that are at least 5 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 6 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 7 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 8 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 9 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 10 nucleotides long.
  • the primary probe comprises oligonucleotides that are at least 11 nucleotides long. In some embodiments, the primary' probe comprises oligonucleotides that are at least 12 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 13 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 14 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 15 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 16 nucleotides long.
  • the primary’ probe comprises oligonucleotides that are at least 17 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 18 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 19 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 20 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 21 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 22 nucleotides long.
  • the primary' probe comprises oligonucleotides that are at least 29 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 30 nucleotides long. In some embodiments, the primary probes of any of the previous embodiments comprises oligonucleotides that are less than 35, 40, 45, 50. 100, 150, 200, 250, or 300 nucleotides in length.
  • the primary’ probe comprises a sequence that is complementary' to the target analyte. In some embodiments the sequence complementarity’ comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or 100%.
  • the primary probe comprises one or more amplifier probe binding sites. In some embodiments, the primary probe comprises two or more amplifier probe binding sites. In some embodiments, the primary probe comprises three or more amplifier probe binding sites. In some embodiments, the primary probe comprises four or more amplifier probe binding sites. In some embodiments, the primary probe comprises five or more amplifier probe binding sites. In some embodiments, the primary probe comprises six or more amplifier probe binding sites. In some embodiments, the primary probe comprises seven or more amplifier probe binding sites. In some embodiments, the primary probe comprises eight or more amplifier probe binding sites.
  • the one or more amplifier probe binding sites are the same sequences. In some embodiments, at least one of the amplifier probe binding sites in the one or more amplifier sequences are the same. In some embodiments, the one or more amplifier probe binding sites are different from each other.
  • the amplifier probe binding sites comprise a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 16 nucleotides long.
  • the amplifier probe binding sites comprise a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 22 nucleotides long.
  • the primary' probe comprises one or more analyte binding sites.
  • the analyte binding sites on the primary probe are the same.
  • the analyte binding sites on the primary’ probe are different.
  • the analyte binding site comprises a sequence that is complementary to the target analyte.
  • the sequence complementarity' comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the analyte binding site comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 9 nucleotides long.
  • the analyte binding site comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 19 nucleotides long.
  • the analyte binding site comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100. 150. 200, 250, or 300 nucleotides in length.
  • the amplifier probe interacts with a primary probe, the primary probe is a protein, by interacting with a nucleotide binding site on the protein. In some embodiments, the amplifier probe interacts with a primary probe, the primary probe is an antibody. In some embodiments, the amplifier probe interacts with a primary probe, the primary’ probe is a protein-oligonucleotide conjugate, by interacting with the oligonucleotide bound to the conjugate. In some embodiments, the amplifier probe interacts with a primary probe, the primary probe is an antibody-oligonucleotide conjugate, by interacting with the oligonucleotide bound to the conjugate.
  • the amplifier probe comprises one or more primary probe binding sites.
  • one or more amplifier probes bind to a contact site on at least one primary' probe.
  • one or more amplifier probes comprise reverse complementary binding sites to one or more primary probe binding sites.
  • one or more amplifier probes comprise one or more unique readout sites, or repeats of the same readout site on a primary probe or another amplifier.
  • the primary probe comprises one or more primary' probe binding sites. In certain embodiments, the primary probe comprises one or more moieties that allow unbinding of the amplifier probe from the primary’ probe. In certain embodiments, the primary probe comprises one or more of the same amplifier probe binding sites. In certain embodiments, the primary probe comprises one or more different amplifier probe binding sites. In certain embodiments, the primary' probe comprises one or more of the same readout probe binding site. In certain embodiments, the primary probe comprises one or more different readout probe binding site. In certain embodiments, the primary probe comprises one or more of different crosslinking sites. In certain embodiments, the primary probe comprises one or secondary probe binding sites. In certain embodiments, the primary probe comprises one or more tertiary' probe binding sites.
  • the amplifier probe is at least 5 nucleotides long. In some embodiments, the amplifier probe is at least 6 nucleotides long. In some embodiments, the amplifier probe is at least 7 nucleotides long. In some embodiments, the amplifier probe is at least 8 nucleotides long. In some embodiments, the amplifier probe is at least 9 nucleotides long. In some embodiments, the amplifier probe is at least 10 nucleotides long. In some embodiments, the amplifier probe is at least 11 nucleotides long. In some embodiments, the amplifier probe is at least 12 nucleotides long. In some embodiments, the amplifier probe is at least 13 nucleotides long.
  • the amplifier probe is at least 14 nucleotides long. In some embodiments, the amplifier probe is at least 15 nucleotides long. In some embodiments, the amplifier probe is at least 16 nucleotides long. In some embodiments, the amplifier probe is at least 17 nucleotides long. In some embodiments, the amplifier probe is at least 18 nucleotides long. In some embodiments, the amplifier probe is at least 19 nucleotides long. In some embodiments, the amplifier probe is at least 20 nucleotides long. In some embodiments, the amplifier probe is at least 21 nucleotides long. In some embodiments, the amplifier probe is at least 22 nucleotides long. In some embodiments, the amplifier probe is at least 23 nucleotides long.
  • the amplifier probe is at least 24 nucleotides long. In some embodiments, the amplifier probe is at least 25 nucleotides long. In some embodiments, the amplifier probe is at least 26 nucleotides long. In some embodiments, the amplifier probe is at least 27 nucleotides long. In some embodiments, the amplifier probe is at least 28 nucleotides long. In some embodiments, the amplifier probe is at least 29 nucleotides long. In some embodiments, the amplifier probe is at least 30 nucleotides long. In some embodiments, the amplifier probe is of any of the previous embodiments comprises nucleotides sequences that are less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.
  • the amplifier probe comprises a primary probe binding site.
  • the primary probe binding site interacts with the primary probe.
  • the primary probe binding site comprises a sequence that is complementary to the primary probe.
  • the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the primary probe binding site comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 10 nucleotides long.
  • the primary’ probe binding site comprises a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments. the primary probe binding site comprises a nucleotide sequence that is at least 16 nucleotides long.
  • the primary’ probe binding site comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 22 nucleotides long.
  • the primary' probe binding site comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.
  • the amplifier probe comprises a moiety that allows unbinding of the amplifier probe from the primary probe.
  • the unbinding is a disruption of the interaction of the amplifier probe from the primary probe.
  • the amplifier probe comprises a moiety’ that allows unbinding of the amplifier probe from a readout probe.
  • the unbinding is a disruption of the interaction of the amplifier probe from the readout probe.
  • the moiety is a displacement probe binding site.
  • the moiety comprises a sequence that is complementary to the primary' probe or amplifier probe.
  • the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%.
  • the moiety comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the moiety' comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 10 nucleotides long.
  • the moiety comprises a nucleotide sequence that is at least 1 1 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 16 nucleotides long.
  • the moiety comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 22 nucleotides long.
  • the moiety comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is less than 35. 40, 45, 50, 100, 150. 200, 250, or 300 nucleotides in length.
  • the amplifier probe comprises one or more readout probe binding sites.
  • the readout probe comprises a sequence that is complementary to the amplifier probe.
  • the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the readout probe comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 10 nucleotides long.
  • the readout probe comprises a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 16 nucleotides long.
  • the readout probe comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 22 nucleotides long.
  • the readout probe comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.
  • the amplifier probe comprises one or more of different crosslinking sites.
  • the crosslinking sites comprise a region of the amplifier probe that can be crosslinked to a cellular component.
  • one or more amplifier probes bind to other amplifier probes.
  • the amplifier probe comprises a secondary probe binding site.
  • the secondary probe binding site comprises a region that interacts with another amplifier probe thereby linking a primary amplifier probe to a secondary amplifier probe.
  • the amplifier probe comprises a tertiary probe binding site.
  • the tertiary probe binding site comprises a region that interacts with another amplifier probe, linking a primary' amplifier probe interacting with a secondary' amplifier probe to a tertiary' amplifier probe.
  • the amplifier probe comprises a quaternary probe binding site.
  • the quaternary probe binding site comprises a region that interacts with another amplifier probe, linking a primary amplifier probe interacting wi th a secondary' amplifier probe, the secondary' amplifier probe interacting with a tertiary' probe, to a quaternary amplifier probe.
  • the tertiary amplifier probe interacts with the secondary amplifier probe, and the secondary probe amplifier probe interacts with the primary probe.
  • the quaternary' amplifier probe interacts with the tertiary' amplifier probe, and tertiary probe interacts with the secondary amplifier probe, and the secondary probe interacts with the primary probe.
  • a quaternary amplifier probe is identical to the secondary amplifier probe.
  • the quaternary' amplifier probe interacts with the secondary amplifier probe.
  • the quaternary' amplifier probe comprises one or more binding sites for the secondary' amplifier probe, one or more binding sites for the tertiary' amplifier probe, or any combination thereof.
  • the tertiary amplifier probe comprises one or more binding sites for the secondary amplifier probe, one or more binding sites for the quaternary amplifier probe, or any combination thereof.
  • the secondary amplifier probe comprises one or more binding sites for the tertiary' amplifier probe, one or more binding sites for the quaternary' amplifier probe, one or more binding sites for the primary probe, or any combination thereof.
  • the secondary’ and tertiary probe binding sites comprise a sequence that is complementary to the primary amplifier probe.
  • the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%.
  • the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 8 nucleotides long.
  • the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 12 nucleotides long.
  • the tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the secondary, tertiary', or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 16 nucleotides long.
  • the secondary', tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the secondary', tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the secondary', tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 20 nucleotides long.
  • the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the secondary 7 , tertiary', or quaternary 7 amplifier probes each comprise a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the secondary 7 , tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 24 nucleotides long.
  • the secondary, tertiary’, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the secondary, tertiary’, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the secondary 7 , tertiary, or quaternary 7 amplifier probes each comprise a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.
  • the primary probe is amplified by contacting the primary 7 probe with a secondary 7 amplifier probe.
  • the method comprises contacting the secondary amplifier probe with a tertiary amplifier probe.
  • the method comprises contacting the tertiary’ amplifier probe with a quaternary amplifier probe, wherein the quaternary amplifier probe is the same as the secondary amplifier probe.
  • the method further comprises amplifying one or more amplifier probes by contacting one or more amplifier probes from a previous contacting step with a new plurality’ of amplifier probes.
  • the new plurality 7 of amplifier probes is the same as a previous plurality of amplifier probes.
  • the method comprises cross-linking one or more amplifier probes to one or more cellular components.
  • one or more amplifier probes are cross-linked to one or more cellular components.
  • one or more amplifier probes are cross-linked to proteins in close proximity to one or more cellular components.
  • one or more amplifier probes are cross-linked to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, lipids, glycans, cellular component, organelles and any combinations thereof in close proximity 7 to one or more cellular components.
  • one or more amplifier probes are crosslinked to one or more cellular components by optically dependent chemical moieties. In certain embodiments, one or more amplifier probes are crosslinked to proteins in close proximity’ to one or more cellular components by optically dependent chemical moieties. In certain embodiments, one or more amplifier probes are crosslinked to proteins in close proximity to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, lipids, glycans, cellular component, organelles and any combinations thereof to one or more cellular components by optically dependent chemical moi eties.
  • the optically dependent chemical moieties are selected from diazirines, benzophenones, aryl-azides, or any combination thereof.
  • optically dependent chemical moieties are photo-crosslinkers.
  • an amplifier probe comprises one or more optically dependent chemical moieties.
  • one or more amplifier probes are crosslinked to one or more cellular components by exposure to light at a set wavelength, a set intensity, a set duration, or any combination thereof.
  • one or more amplifier probes are crosslinked to one or more cellular components by esters.
  • the esters are NHS-esters.
  • one or more amplifier probes are crosslinked to one or more cellular components by cysteine reactive reagents.
  • the reactive agents are maleimides.
  • one or more amplifier probes are crosslinked to one or more cellular components using peptide coupling reagents.
  • the peptide coupling reagents are selected from carbodiimides, uronium salts, phosphonium salts, or any combination thereof.
  • one or more amplifier probes are crosslinked to one or more cellular components by in situ carbene generation.
  • one or more amplifier probes are crosslinked to one or more cellular components by contacting one or more cellular components with one or more rounds of crosslinkers.
  • the crosslinkers are selected from paraformaldehyde (PF A), PEGylated bis (sulfosuccinimidyl) suberate (BSPEG), or combinations thereof.
  • PF A paraformaldehyde
  • BSPEG PEGylated bis (sulfosuccinimidyl) suberate
  • one or more amplifier probes are crosslinked to one or more cellular components by crosslinking cysteines by having thiol modified oligo.
  • one or more amplifier probes are reacted with BM(PEG) n .
  • one or more amplifier probes are crosslinked to one or more cellular components by amine oligo or thiol oligo. In certain embodiments, one or more amplifier probes are crossed-linked to the cellular component by reacting the lysines, cysteines, or combinations thereof with using SM(PEG)n. [00115] In some embodiments, one or more amplifier probes are cross-linked to one or more cellular components, the one or more cellular components are functionalized with various reactive moieties on biomolecules (e.g., amines, hydroxyls, carboxylic acids, thiols, and the like), wherein the reactive moieties provide reactive handles for chemical deposition of probes. In some embodiments, the cellular components are primed to react with reactive moieties on the one or more amplifier probes.
  • one or more amplifier probes are crosslinked to one or more cellular components by a cross metathesis reaction.
  • the cross metathesis reaction conjugates an alkene probe and an NHS-alkene handles.
  • one or more amplifier probes are crosslinked to one or more cellular components by via A-halosuccinimides. and an alkene amplifier probe.
  • the crosslinking is via a Heck coupling using a palladium catalyst.
  • one or more amplifier probes are crosslinked to one or more cellular components by small molecule substrates coupled to a readout probe.
  • one or more amplifier probes can be read out using riboswitches or proteins.
  • one or more amplifier probes are crosslinked one or more cellular components with a functionalized hydrogel comprising a functional group that reacts with one or more modified amplifier probes.
  • one or more amplifier probes are crosslinked to a primary probe. In some embodiments, one or more amplifier probes are crosslinked to one or more primary probes. In some embodiments, one or more amplifier probes are crosslinked in close proximity to one or more cellular components. In some embodiments, one or more amplifier probes are crosslinked in close proximity to a primary probe interacting with a target analyte.
  • one or more amplifier probes are crosslinked to the cellular component via click-chemistry.
  • the click-chemistry may include but is not limited to: strain-promoted azide-alkyne cycloadditions (SPAACs); tetrazine-trans- cyclooctene ligations (TCO-Tz; or inverse-electron demand Diels-Alder reactions); thiol-ene reactions; thiol-yne reactions; oxime ligations; hydrazone ligations; Diels-Alder reactions; and inverse-electron demand Diels-Alder reactions (lEDDAs).
  • SPAACs strain-promoted azide-alkyne cycloadditions
  • TCO-Tz tetrazine-trans- cyclooctene ligations
  • lEDDAs inverse-electron demand Diels-Alder reactions
  • the method comprises contacting cell samples with amine groups.
  • an amine in the cells is modified with Alkyne-PEGx-NHS esters, where N is the number of monomer units.
  • an amine in the cells is modified with Azide-PEGx-NHS esters.
  • cell functionalization is accomplished using thiols or mercaptans as nucleophiles to make corresponding thioesters.
  • alkynes are present in the cells, for example, 3'-azide modified probes that can crosslink to the cell via copper(I)-catalyzed azide-alkyne cycloadditions (CuAACs).
  • CuAACs copper(I)-catalyzed azide-alkyne cycloadditions
  • one or more amplifier probes comprise 5' or 3' cross linkable molecules.
  • the cross-linkable molecules are photo-cross - linkable molecules.
  • the method comprises crosslinking the probes to cellular components via treatment of hydrazides with carbonyls to form stable hydrazones.
  • the method comprises one or more displacement probes that separate one or more amplifier probes from one or more primary probe.
  • the method comprises displacing one or more amplifier probes from one or more primary probes by cleavage of the amplifier probe.
  • the method comprises separating one or more amplifier probes by washing, displacement, cleavage, photocleavage, chemical reduction, chemical degradation, enzymatic digestion, enzymatic reactions modifying the amplifier, or any combination thereof.
  • the method comprises separating one or more amplifier probes by one or more displacement probes. In certain embodiments, the method comprises one or more displacement probes competing with an interaction between one or more primary probes and one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by binding a high-affinity locked nucleic acid (LNA) or RNA probe.
  • LNA high-affinity locked nucleic acid
  • the method comprises separating one or more amplifier probes by binding one or more displacement probes.
  • the method comprises one or more displacement probes crosslinking to the target analyte.
  • the method comprises one or more displacement probes comprise a binding site, wherein the binding site has reverse complementarity to a sequence on one or more amplifier probes.
  • the method comprises one or more displacement probes interacting with the primary, secondary, tertiary, or quaternary binding sites of the amplifier probe.
  • one or more displacement probes comprise one or more binding sites for a secondary probe, a tertiary probe, and/or a quaternary probe binding.
  • the method comprises separating one or more amplifier probes by light-based cleavage of one or more amplifier probes.
  • the method comprises washing steps, wherein one or more cleaved amplifier probes are separated from the analyte.
  • the method comprises separating one or more amplifier probes by photocleavage of one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by reducing a disulfide bridge within a secondary, or a tertiary' probe interacting with an amplifier probe to cleave it.
  • the method comprises separating one or more amplifier probes by cleaving a pH-dependent moiety within a secondary, or a tertiary' probe interacting with one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by site-specific protease cleavages of one or more amplifier probes by TEV Protease, ribonuclease, TALEN, Zinc Finger protein, lipase, or endoglycosidase.
  • the method comprises separating one or more amplifier probes by electrochemical cleavage of one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by enzymatic cleavage of one or more amplifier probes at recognition sites, or restriction enzymes cleaving double-stranded probes.
  • the Uracil- Specific Excision Reagent (USER) enzyme cleaves one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by CRISPR Cas9 modification.
  • the method comprises separating one or more amplifier probes by target-specific endonuclease to cleavage of one or more amplifier probes.
  • the method comprises separating one or more amplifier probes by binding one or more displacement probes, the one or more displacement probes crosslinking to the cellular component.
  • the method comprises separating one or more amplifier probes a by partial or complete removal of a secondary, or a tertiary' probe interacting with one or more amplifier probes.
  • the readout probe is selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof.
  • the readout probe further comprises a detectably moiety.
  • the detectably moiety is a fluorophore.
  • the readout probe comprises an oligonucleotide with a detectable moiety.
  • the readout probe comprises oligonucleotides that are at least 5 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 6 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 7 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 8 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 9 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 10 nucleotides long.
  • the readout probe comprises oligonucleotides that are at least 11 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 12 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 13 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 14 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 15 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 16 nucleotides long.
  • the readout probe comprises oligonucleotides that are at least 17 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 18 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 19 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 20 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 21 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 22 nucleotides long.
  • the readout probe comprises oligonucleotides that are at least 23 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 24 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 25 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 26 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 27 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 28 nucleotides long.
  • the readout probe comprises oligonucleotides that are at least 29 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 30 nucleotides long. In some embodiments, the readout probes of any of the previous embodiments comprises oligonucleotides that are less than 35, 40, 45, 50, 100 nucleotides in length.
  • the readout probe comprises a sequence that is complementary to the primary probe.
  • the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the readout probes comprise oligonucleotides with the same sequence. In some embodiments, the readout probes comprise oligonucleotides with different sequences.
  • the method comprises generating signals from in situ sequencing by sequencing by ligation or sequencing by synthesis methods.
  • the signals are generated by hybridizing a sequencing primer to the primary probes, amplifier probes, or any combination thereof.
  • a sequencing reaction generates a fluorescent signal.
  • the signals are generated by in situ sequencing of amplifier probes.
  • the method comprises sequencing amplicons derived from the sequencing methods of the previous embodiments.
  • the sequencing of amplicons maps the spatial positions of one or more cellular targets, one or more target analytes, or any combination thereof.
  • the method comprises barcoding the spatial position of target analytes, cellular targets, or any combination thereof by directly capturing the amplified products or by diffusing spatial barcodes into samples to barcode the positions, generating barcoded amplicons that are extracted and sequenced in order to map the identity and position of the molecules.
  • any of the previous embodiments are performed using microscopy slide based techniques.
  • the method comprises detecting signals generated from the interaction of two amplicons.
  • an adapter or bridge probe generates a signal when two amplified products are physically adjacent.
  • the adapter or bridge probe is an oligonucleotide, protein, or any combination thereof.
  • one or more of the adapters generate a signal indicating the physical proximity of two or more analytes.
  • one or more of the adapters generate a signal indicating the physical proximity’ of two or more primary probes.
  • two or more primary probes target the same target analyte.
  • the amplified products from two or more primary probes generate a signal that functions as a coincidence detector.
  • the coincidence detector indicates a specific vs a nonspecific interaction between the primary probes.
  • a non-specific interaction of only one of primary probe does not produce a signal.
  • the method comprises two or more primary probes interacting with different target analytes.
  • the method comprises amplifying products from the primary probes.
  • the amplified products from the primary probes generate a signal enhancing specificity, the signal enhancing specificity functions as a coincidence detector, the coincidence detector functions as a indicator of the physical proximity of one or more target analytes, one or more cellular components, or any combination thereof.
  • the individual amplified products from one or more of the primary probes produces a signal if the target analytes are in physical proximity, the individual amplified products from one or more of the primary probes produces a signal.
  • the proximity between combinations of target analytes such as nucleic acids, proteins, glycans, or any combination thereof, generates a detectable signal.
  • the proximity distance is less than 1, 2, 4, 5, 6, 7, 8, 9, or 10 nanometers.
  • the proximity distance in which a signal is generated is tuned by the length of the adaptor or bridge probe.
  • the adaptor or bridge probe is a nucleic acid.
  • the adaptor or bridge probe is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length.
  • two or more primary' probes target different target analytes, wherein the proximity’ of the primary probes generate a new analyte for further detection, amplification, or any combination thereof.
  • the readout probe comprises a fluorophore.
  • fluorophore is any fluorophore deemed suitable by those of skill in the arts.
  • the fluorophores include but are not limited to fluorescein, rhodamine, Alexa Fluors, DyLight fluors, ATTO Dyes, or any analogs or derivatives thereof.
  • the detectable moi eties include but are not limited to fluorescein and chemical derivatives of fluorescein; Eosin; Carboxyfluorescein; Fluorescein isothiocyanate (FITC); Fluorescein amidite (FAM): Erythrosine; Rose Bengal; fluorescein secreted from the bacterium Pseudomonas aeruginosa; Methylene blue; Laser dyes; Rhodamine dyes (e.g.. Rhodamine, Rhodamine 6G, Rhodamine B, Rhodamine 123, Auramine O, Sulforhodamine 101, Sulforhodamine B, and Texas Red).
  • Rhodamine dyes e.g.. Rhodamine, Rhodamine 6G, Rho
  • the fluorphores include but are not limited to ATTO dyes; Acridine dyes (e.g., Acridine orange, Acridine yellow); Alexa Fluor; 7-Amino actinomycin D; 8-Anilinonaphthalene-l -sulfonate; Auramine-rhodamine stain; Benzanthrone; 5,12- Bis(phenylethynyl) naphthacene; 9,10-Bis(phenylethynyl)anthracene; Blacklight paint; Brainbow; Calcein; Carboxyfluorescein; Carboxyfluorescein diacetate succinimidyl ester; Carboxyfluorescein succinimidyl ester; 1 -Chi oro-9, 10-bis(phenylethynyl)anthracene; 2- Chl oro-9, 10-bis(pheny lethyny l)anthracene; 2-Chloro-9
  • Cyanine such as Cy3 and Cy5, DiOC6, SYBR Green I); DAPI, Dark quencher, Dy Light Fluor, Fluo-4, FluoProbes; Fluorone dyes (e.g., Calcein, Carboxyfluorescein, Carboxyfluorescein diacetate succinimidyl ester, Carboxyfluorescein succinimidyl ester. Eosin, Eosin B, Eosin Y, Erythrosine, Fluorescein, Fluorescein isothiocyanate. Fluorescein amidite. Indian yellow.
  • Rhodamine Rhodamine, Rhodamine 123, Rhodamine 6G, RiboGreen, RoGFP, Rubrene, SYBR Green I, (E)-Stilbene, (Z)-Stilbene, Sulforhodamine 101, Sulforhodamine B, Synapto-pHluorin, Tetraphenyl butadiene, Tetrasodium tris(bathophenanthroline disulfonate) ruthenium(II), Texas Red, TSQ. Umbelliferone, or Yellow fluorescent protein.
  • the fluorophores include but are not limited to Alexa Fluor family of fluorescent dyes (Molecular Probes, Oregon). Alexa Fluor dyes are widely used as cell and tissue labels in fluorescence microscopy and cell biology. The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend into the infrared. The individual members of the family are numbered according roughly to their excitation maxima (in nm). Certain Alexa Fluor dyes are synthesized through sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. In some embodiments, sulfonation makes Alexa Fluor dyes negatively charged and hydrophilic.
  • Alexa Fluor dyes are more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission, and to some extent the newer cyanine series.
  • Exemplary Alexa Fluor dyes include but are not limited to Alexa-350, Alexa-405, Alexa-430, Alexa-488, Alexa-500, Alexa-514, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-610, Alexa-633, Alexa-647, Alexa-660, Alexa-680, Alexa-700, or Alexa-750.
  • the fluorophores comprise one or more of the DyLight Fluor family of fluorescent dyes (Dyomics and Thermo Fisher Scientific).
  • Exemplary DyLight Fluor family dyes include but are not limited to Dy Light-350, Dy Light-405.
  • the fluorophore comprises a nanomaterial. In some embodiments, the fluorophore is a nanoparticle. In some embodiments, the fluorophore is or comprises a quantum dot. In some embodiments, the fluorophore is a quantum dot. In some embodiments, the fluorophore comprises a quantum dot. In some embodiments, the fluorophore is or comprises a gold nanoparticle. In some embodiments, the fluorophore is a gold nanoparticle. In some embodiments, the fluorophore comprises a gold nanoparticle.
  • the method comprises imaging the readout probes. In some embodiments, the method comprises imaging the barcodes. As understood by a person having ordinary skill in the art, different technologies can be used for the imaging steps.
  • the imaging methods comprise but are not limited to epifluorescence microscopy, confocal microscopy, the different types of super-resolution microscopy (PALM/STORM, SSIM/GSD/STED), and light sheet microscopy (SPIM and etc.).
  • the imaging methods comprise exemplary super resolution technologies include, but are not limited to I 5 M and 4Pi-microscopy, Stimulated Emission Depletion microscopy (STEDM), Ground State Depletion microscopy (GSDM), Spatially Structured Illumination microscopy (SSIM), Photo- Activated Localization Microscopy (PALM), Reversible Saturable Optically Linear Fluorescent Transition (RESOLFT), Total Internal Reflection Fluorescence Microscope (TIRFM). Fluorescence-PALM (FPALM), Stochastical Optical Reconstruction Microscopy (STORM), Fluorescence Imaging with One- Nanometer Accuracy (FIONA), and combinations thereof.
  • STEDM Stimulated Emission Depletion microscopy
  • GSDM Ground State Depletion microscopy
  • SSIM Spatially Structured Illumination microscopy
  • PARM Photo- Activated Localization Microscopy
  • REOLFT Reversible Saturable Optically Linear Fluorescent Transition
  • TIRFM
  • EM electron microscopes
  • an imaging step detects a target.
  • an imaging step localizes a target.
  • an imaging step provides three- dimensional spatial information of a target.
  • an imaging step quantifies a target.
  • the method comprises analyzing cell size and shape, markers, immunofluorescence measurements, or any combinations thereof.
  • signals are detected by imaging or sequencing.
  • the samples are imaged after contacting one or more amplifier probes with one or more readout probes.
  • the method comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes.
  • the targets that are selected from proteins, modified proteins, transcripts, RNA. DNA loci, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, biologically active molecules, and combinations thereof.
  • the targets comprise subcellular features.
  • the nuclear lamin can be one set of barcodes, and the nucleolus can be targeted with another set of barcodes. This allows each sample can be analyzed with a combination of barcodes on different subcellular compartments.
  • the method compnses barcoding targets, wherein the targets are different.
  • the method comprises fluorescence detection. In some embodiments, the method comprises fluorescence detection or other methods of detection. In some embodiments, the method comprises sequential hybridization to detect target analytes. [00172] In some embodiments, the probes are used in a method to barcode one or more molecular targets. See, for example, International PCT Patent Application No.
  • the probes are used in a method for linked amplification tethered with exponential radiance (LANTERN). See, for example, International Patent Application No. PCT/US2022/021826, FILED March 24, 2022, and titled LINKED AMPLIFICATION TETHERED WITH EXPONENTIAL RADIANCE, the entire contents of which are herein incorporated by reference in its entirety for all purposes.
  • LANTERN linked amplification tethered with exponential radiance
  • the probes are used in a method for ClampFISH. See, for example, ClampFISH detects individual nucleic acid molecules using click chemistry -based amplification, Rouhanifard S.H. et al.. Nature Biotechnology 37: 84-89 (2019), the entire contents of which are herein incorporated by reference in its entirety for all purposes.
  • the method comprises readout probes that are selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof.
  • the method comprises contacting each sample in the one or more samples with a first plurality of readout probes, so that the probes interact with one or more targets. In some embodiments, the method comprises imaging the sample after the first contacting step so that interaction of the readout probes with their targets is detected.
  • the method comprises a contacting step that differs from another contacting step in the labelling of at least one of the targets.
  • the method comprises a contacting step wherein each detectably labelled probe in the first plurality of probes is labelled with a detectably moiety.
  • the method comprises a contacting step wherein each detectably labelled probe comprises a detectable moiety and at least one contacting step differs from another contacting step by having a different detectable moiety for each target.
  • the method comprises a contacting step wherein at least two different readout probes that interact with a first target and wherein at least two different readout probes interact with a second target.
  • the readout probes comprise labels selected from two, three, or four different labels.
  • the barcode for the target in the sample comprises a signal that is amplified.
  • the barcode for the target in a sample comprises a signal that is amplified by rolling circle, padlock, branched DNA, ClampFISH, LANTERN, or any combination thereof.
  • the method comprises using readout probes wherein each detectably labelled probe comprises the same detectable moiety and the same sequence.
  • the method comprises readout probes wherein each readout probes interacts with its target through one or more intermediate probes each of which is hybridized to the target.
  • the method comprises repeating the contacting and imaging steps, each time with a new plurality of readout probes so that a target in the sample is described by a barcode, and can be differentiated from another target in the sample by a difference in their barcodes.
  • the method comprises an error correction round. See, for example. International Patent Application No. PCT/US2017/044994, FILED August 0, 2017, and titled SEQUENTIAL PROBING OF MOLECULAR TARGETS BASED ON PSEUDOCOLOR BARCODES WITH EMBEDDED ERROR CORRECTION MECHANISM, the entire contents of which are herein incorporated by reference in its entirety for all purposes.
  • the method comprises an error correction round performed by selecting from block codes such as Hamming codes, Reed-Solomon codes, Golay codes, or any combination thereof.
  • block codes such as Hamming codes, Reed-Solomon codes, Golay codes, or any combination thereof.
  • the method of any of the previous embodiments further comprises an error correction step.
  • the error correction step comprises performing additional rounds of contacting and imaging prior or in between or after steps (i)-(v).
  • the method comprises a step of removing the readout probes after one or more imaging steps.
  • the step of removing the readout probes comprises contacting the plurality of readout probes with an enzyme that digests a readout probes.
  • the step of removing comprises contacting the plurality of readout probes with a DNase, contacting the plurality of readout probes with an RNase, photobleaching, strand displacement, formamide wash, heat denaturation, or combinations thereof.
  • the step of removing comprises photobleaching to remove the readout probes.
  • the method comprises removing readout probes by using stripping reagents, wash buffers, photobleaching, chemical bleaching, and any combinations thereof.
  • the amplifier probe is separated by stringent wash conditions of the sample.
  • the stringent wash conditions comprise 30%, 40%, 50%, 55%, 60%, or 70% formamide in a buffered or aqueous solution.
  • the method comprises clearing the sample.
  • the sample is cleared by CLARITY.
  • the method of any of the preceding embodiments comprises optionally washing the sample after each step.
  • the sample is washed with a buffer that removes non-specific hybridization reactions.
  • formamide is used in the wash step.
  • the wash buffer is stringent.
  • the wash buffer comprises 10% formamide, 2xSSC, and 0. 1% triton X-lOOs.
  • Figure 1A illustrates an exemplary embodiment of a primary probe binding to a target molecule.
  • a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary 7 probe.
  • Figure IB illustrates an exemplary embodiment of amplifier probe binding to a unique region of the primary probe.
  • a ssDNA amplifier probe binds to a specific region of the ssDNA primary probe at a site where both probes have reverse complement sequences from each other.
  • the amplifier probe is functionalized with a reactive group.
  • Figure 1C illustrates an exemplary embodiment of the amplifier probe binding to the sample through the reactive group.
  • the amplifier group was modified with a photoreactive group that can be crosslinked by UV-light excitation to nearby reactive groups in the sample.
  • the amplifier probe is either cleaved and partially washed away or displaced from the primary probe so that the initial primary probe binding site for another amplifier is freed up again.
  • Figure ID illustrates an exemplary embodiment of subsequent amplifier binding to the primary probe.
  • the amplifier binds at the same, freed-up location where the previous amplifier was bound.
  • Figure IE illustrates an exemplary embodiment of multiple rounds of amplifier binding to the primary probe, binding to the sample, and unbinding from the primary probe to allow the next amplifier to bind to the same primary' probe site. With each round the number of amplifiers in close proximity to the target molecule can increase by one. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.
  • Figure IF illustrates an exemplary embodiment of readout probes binding to the amplifiers that are bound to the sample in order to detect the target molecule.
  • readout probes are ssDNA probes that can bind to the amplifier probes at a specific site at which both probes are reverse complement to each other.
  • This example depicts an exemplary design of a process without binding probes to the sample. Without repeated binding of amplification probes to the sample, no signal amplification can take place with the method described here.
  • Figure 2A illustrates an exemplary' embodiment of a primary probe binding to a target molecule.
  • a ssDNA primary' probe binds to an RNA target at a site that is defined by a reverse complement region of the primary probe. It is the same example setting as described for Figure 1A.
  • Figure 2B illustrates an exemplary ⁇ embodiment of an amplifier probe binding to a binding region of the primary' probe.
  • a ssDNA amplifier probe binds to a specific region of the ssDNA primary probe at a site where both probes have reverse complement sequences from each other.
  • the amplifier probe is functionalized with a reactive group. It is the same example seting as described for Figure IB.
  • Figure 2C illustrates an exemplary' embodiment of the amplifier probe not binding to the sample. As the amplifier probe is not linked to the sample, the example cleavage shown here results in amplifier probe removal from the sample.
  • Figure 2D illustrates an exemplary embodiment of readout probes not binding to the sample, as depicted by the dotted line. There are no amplifiers that are bound to the sample, and there are no available binding sites for the readout probes, so it is not possible to detect the target molecule.
  • This example depicts an exemplary design of a piecewise linear amplification process.
  • Figure 3A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. Following the binding, each amplifier probe was either cleaved and partially washed away or displaced from the primary probe so that the initial primary probe binding site for another amplifier is freed up again. Multiple rounds of adding amplifier rounds results in the presence of multiple amplifier rounds near the primary probe and molecular target of the probes. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.
  • Figure 3B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary probes that are bound to the sample.
  • the secondary amplifier probes have a region reverse complementary to the initially bound amplifier probes.
  • the number of secondary amplifier probes that can bind to the sample depends on the number of initial amplifier probes present in the sample. In this exemplary embodiment, the number of secondary amplifiers that can bind to the initial amplifier probes is 6.
  • Figure 3C illustrates an exemplary 7 embodiment of binding the secondary 7 amplifier probes to the sample. This can be induced in the same or different ways compared to binding the initial amplifier probes. In this exemplary 7 embodiment, 6 secondary amplifier probes are bound to the sample.
  • Figure 3D illustrates an exemplary 7 embodiment in which in a subsequent round of secondary 7 amplifier addition, sample binding, and unbinding from the initial amplifier probe, 6 more secondary 7 amplifiers are added.
  • Figure 3E illustrates an exemplary embodiment in which during another subsequent round of secondary 7 amplifier addition, sample binding, and unbinding from the initial amplifier probe, 6 more secondary 7 amplifiers are added. The exemplary 7 process can be repeated until a sufficient number of secondary amplifiers for the molecular target detection is present.
  • Figure 3F illustrates an exemplary embodiment in which each secondary amplifier probe can be bound by readout probes that can be used to detect the molecular target in the sample.
  • the readout probes can be fluorophore- conjugated ssDNA oligo sequences.
  • Figure 4A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. After binding, each amplifier probe was either cleaved and partially washed away or displaced from the primary probe so that another amplifier can bind to the primary probe. Multiple rounds of adding amplifier probes results in the presence of multiple amplifier probes near the primary probe and molecular target of the probes. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.
  • Figure 4B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary amplifier probes that are bound to the sample.
  • the secondary amplifier probes have a region reverse complementary to the initially bound amplifier probes.
  • the number of secondary amplifier probes that can bind to the sample depends on the number of initial amplifier probes present in the sample. In this exemplary embodiment, the number of secondary amplifiers that can bind to the initial amplifier probes is 6. Only a single step with 6 amplifiers is shown in this example. As described before the procedure with binding, cleavage or displacement and binding of additional amplifier probes can be repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample. For an exponential amplification in the subsequent steps of this example, each of the amplifier probes bound here has two binding sites for amplifier probes in the next amplification round.
  • Figure 4C illustrates an exemplary embodiment of binding exponential amplifier probes to the sample.
  • 12 tertiary amplifier probes are bound to the 6 amplifier probes that were bound to the sample in the previous step. In this example, no displacement or cleavage or the probes takes place.
  • Each amplifier is bound to the sample, and has two binding sites available for subsequent amplifier probe binding.
  • Figure 4D illustrates an exemplary embodiment of binding exponential amplifier probes to the sample.
  • This example depicts an exemplary design of an exponential amplification process following an amplification factor according to a Pell number series.
  • Figure 5 A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe.
  • a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary probe.
  • the primary probe further has a binding feature R+ for an amplifier probe, which can bind the primary probe through the reverse complementary feature R-. Dotted lines at the ends of the primary probe indicate that A pically, each primary’ probe contains multiple unique amplifier binding sites, which are omitted for clarity in this exemplary figure.
  • the first amplifier has a feature that allows binding to the sample (circle), and a cleavable feature (square), and two binding sites for the second amplifier (A+). In total, in the step of the method labelled here as round 1. there are a total of two binding features Anin the sample.
  • Figure 5B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked and cleaved first amplifier at feature A+. After crosslinking and cleaving the probe, only the cross-linkable portion of the first amplifier, which includes the binding sites for the second amplifier, remains in the sample. Note that the position of the crosslinked probes is shifted in the illustration for clarity' purposes. Elements that were present before this round are illustrated in light grey for clarity'.
  • the secondary amplifier has two binding features R+, which is designed to be identical to the binding feature R+ on the primary probe.
  • Figure 5C illustrates an exemplary embodiment of the next round in which the same amplifier is applied to the sample as in the initial round.
  • the amplifier binds the secondary' amplifier at feature R+ through its binding feature R-.
  • the first amplifier has two binding features A+.
  • the newly added number of binding features A+ in this round of amplification is 10.
  • the total number of available binding features A+ in the sample is now 12.
  • Figure 5D illustrates an exemplary' embodiment of another round of amplification with secondary amplifier probes, which bind to the A+ sites as described in figure 5B.
  • Figure 5E illustrates an exemplary embodiment of the next round of amplification, in which the first amplifier is added to the sample again. Binding is similar as described in figure 5C. The newly added number of binding features A+ in this round of amplification is 58. The total number of available binding features A+ in the sample is now 70. The process can be repeated through additional rounds until a sufficient amount of features A+ are bound in the sample.
  • feature A+ can be bound by fluorophore-conjugated readout probes. Alternatively, it is also possible to bind the readout probe to any other amplifier probe element shown in this example.
  • Figure 6A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe.
  • a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary 7 probe.
  • the primary probe further has a binding feature R+ for an amplifier probe, which can bind the primary probe through the reverse complementary feature R-. Dotted lines at the ends of the primary probe indicate that typically, each primary probe contains multiple unique amplifier binding sites, which are omitted for clarity in this exemplary' figure.
  • the first amplifier has a feature that allow s binding to the sample (circle), and two binding sites for the second amplifier (A+). In total, in the step of the method labelled here as round 1. there are a total of two binding features A+ in the sample.
  • Figure 6B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked first amplifier at feature A+.
  • the secondary' amplifier has two binding features R+.
  • w hich can be identical to the binding feature R+ of the primary probe, for an amplifier probe.
  • no cleavage of displacement of amplifier probes takes place.
  • Figure 6C illustrates an exemplary embodiment of four amplifier probes, which can be identical to the amplifier probes used in round 1. binding at feature R- to the crosslinked first amplifier at feature R+.
  • the amplifier has two binding features A+.
  • FIG. 7A illustrates an exemplary embodiment of a primary' probe bound with an amplifier probe. Details on an exemplary design of an amplifier probe are given. Only one amplifier is shown here, with potentially multiple of the same or different amplifiers binding to primary probes.
  • the amplifier probe consists of a primary probe binding site (R-), w hich is a reverse complement to an amplifier probe binding site on the primary probe (R+).
  • R- primary probe binding site
  • w hich is a reverse complement to an amplifier probe binding site on the primary probe (R+).
  • a feature that allow s cleavage or probe displacement (rectangle) is part of the amplifier and allows freeing up the primary probe site after crosslinking to complete one cycle of amplification.
  • the cleavage site can also be placed within the primary probe binding site R- or other features of the amplifier probe adjacent to the primary' probe binding site R-.
  • R- can be a toehold sequence for a displacer strand that is reverse complementary’ to the amplifier probe.
  • Other features of the amplifier adjacent to the primary probe binding site R- can be an additional toehold feature for effective displacer strand binding.
  • the amplifier further features an example binding site A+, which is bound by other amplifier probes or by readout probes that allow' detection of the signal, for example by conjugating fluorescent dyes to the readout probes.
  • Figure 7B illustrates an exemplary embodiment of an amplifier probe similar to the one described in example Figure 7A, but it shows two binding sites A+ instead of one for designs that involve, for example, stronger linear amplification or non-linear amplification schemes.
  • This example depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary’ probe.
  • Figure 8A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary' probe.
  • the primary' probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-, R2-, R3- and R4-.
  • the dotted rectangle indicates part of the primary probe that follows panels Figures 8 B-D.
  • the white circle at the end of the amplifier probes illustrates a feature that can be bound, for example crosslinked by UV-light, to the sample.
  • Figure 8B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+/R3-.
  • the example amplifier has a toehold sequence binding site T+ and an amplifier binding site A3+.
  • the crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • Figure 8C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe.
  • binding is aided by a toehold feature T+ on the amplifier probe, which allows binding of the displacement probe R3+T- to be favoured compared to the binding of the amplifier at binding site R3- to the primary probe at R3+.
  • Figure 8D illustrates an exemplary embodiment of a displacement probe remaining attached to the amplifier probe after unbound displacer probes are washed or otherwise removed from the sample. During this step, the previously bound amplifier probe cannot bind to the primary probe at R3+/R3- again because the bound displacement probe is blocking the binding site. The R3+ site of the primary probe therefore remains available for other amplifier probes in subsequent binding round.
  • Figure 8E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (crossed-out circle).
  • This example depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe.
  • Figure 9A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe.
  • the primary probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-. R2-, R3- and R4-.
  • the dotted rectangle indicates part of the primary probe that follow s panels Figures 9 B-D.
  • the white circle at the end of the amplifier probes illustrates a feature that can be bound, for example crosslinked by UV-light, to the sample.
  • Figure 9B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary' probe at site R3+/R3-.
  • the example amplifier has two d amplifier binding sites A3+.
  • the crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • Figure 9C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe.
  • binding is aided by a toehold feature A3(l-n)-, which is the first n nucleotides of the A3- sequence that can bind to the A3+ sequence on the amplifier probe.
  • the length is for example 10 nt, so that the displacement probe in this example has a total length of 25nt.
  • the toehold sequence allows binding of the displacement probe R3+ A3(l-n)- to be favoured compared to the binding of the amplifier at binding site R3- to the primary probe at R3+.
  • the R3+ A3(l-n)- displacer allows amplifier probes with two feature sites A3+ to be used flexibly also for exponential amplification rounds without displacement.
  • Figure 9D illustrates an exemplary embodiment of a displacement probe remaining attached to the amplifier probe after unbound displacer probes are washed or otherwise removed from the sample. During this step, the previously bound amplifier probe cannot bind to the primary probe at R3+/R3- again because the bound displacement probe is blocking the binding site. The R3+ site of the primary probe therefore remains available for other amplifier probes in subsequent binding round.
  • Figure 9E illustrates an exemplary 7 embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (crossed-out circle).
  • This example provides an example design of a protocol for the displacement of probes using photo-crosslinking and amplifier cleavage for unbinding from the primary probe.
  • Figure 10A illustrates an exemplary embodiment of a primary 7 probe binding to a target molecule and amplifier probes binding to the primary probe similar to Figure 9A.
  • the primary probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-. R2-, R3- and R4-.
  • the white circle at the end of the amplifier probes illustrate a feature that can be bound, for example crosslinked by UV -light, to the sample.
  • Figure 10B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+/R3-.
  • the crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.
  • the white rectangle illustrates an example cleavable feature of the amplifier probe.
  • Figure 10C illustrates an exemplary embodiment of the amplifier probe that was cleaved at the cleavable feature.
  • the portion of the amplifier that includes binding feature A+ remains in proximity to the primary probe, as it was linked to the sample through the linkable feature illustrated by the crossed-out circle.
  • Figure 10D illustrates an exemplary embodiment of the remaining portion of the cleaved amplifier which remains bound to example binding site R3+ through binding feature R3-. This portion of the cleaved amplifier can be removed, for example, through wash conditions suitable for the short remaining fragment.
  • Figure 10E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle), which has become available through the steps described in Figures 10B-10D.
  • This example depicts exemplary images of a linear amplification process of the fluorescence signal intensity using diazirine modified oligonucleotides of a single gene in a cell culture sample.
  • NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 4% PFA in lx PBS at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples were dried with nitrogen gas and a custom flow cell was attached to the glass coverslip. After rinsing with lx PBS, 7.5 mM BS-PEG5 in lx PBS was added for 30 min at room temperature. Samples were incubated with 100 mM /V-(Propionyloxy) succinimide in lx PBS two times for 30 min at room temperature.
  • Samples were rinsed with 40% wash buffer (40% formamide, 2x SSC, 0.1% Triton-XlOO) three times.
  • Primary probes were hybridized with a 40% hybridization buffer (40% formamide, 0.1 mg/mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate) containing 5 nM/oligo at 37 C for 12 hours.
  • the sample was rinsed with a 40% wash buffer 3 times and incubated at 37 C once. Subsequently, the sample was rinsed with 2X SSC solution.
  • 100 nM/oligo amplifier probes were hybridized to the sample in a 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO) at room temp for 30 min.
  • the sample was rinsed with 10% wash buffer (Wash buffer: 10% formamide, 2X SSC, 0.1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 45 min.
  • samples were rinsed with 2x SSC and incubated with 1 uM of displacer strand in a 15% hybridization buffer at room temperature for 15 min.
  • sample Post displacement, the sample was rinsed with a 20% wash buffer (20% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated multiple times to obtain sufficient signal amplification. Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM/oligo readouts at room temperature for 15 min. Samples were stained with 3 ug/mL DAPI in 2X SSC for 1 min, then rinsed with 2X SSC twice.
  • a 20% wash buffer (20% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated multiple times to obtain sufficient signal amplification. Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM/oligo readouts at room temperature for 15 min. Samples were stained with 3 ug/mL DAPI in 2X SSC for 1 min, then rinsed with 2X SSC twice.
  • Anti-bleaching buffer 100 mM Tris-HCl pH 8, 4x SSC, 2 mM Trolox, 20% (w/v) D- Glucose, 1 :100 Glucose Oxidase (200 U/mL), 1: 1000 Catalase) w as applied and samples were imaged using a 63x, 1.4 NA Leica objective and a spinning disk confocal microscope (Andor Dragonfly).
  • Figure 11 A illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification.
  • Figure 1 IB illustrates an exemplary embodiment of a fluorescent intensity signal increase after five consecutive rounds of linear amplification.
  • Figure 11C illustrates an exemplary embodiment of a fluorescent intensity signal increase after eleven consecutive rounds of linear amplification.
  • FIG. 12 depicts exemplary images of a linear amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample.
  • the scale bar is 20 microns.
  • Approximately 100000 NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 4% PFA in lx PBS at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples w ere dried with nitrogen gas and a custom flow cell was attached to the glass coverslip.
  • the sample was rinsed with a 40% wash buffer 3 times and incubated at 37 C once. Subsequently, the sample was rinsed with 2X SSC solution. Then, 100 nM/oligo amplifier probes were hybridized to the sample in a 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO) at room temp for 30 min. The sample was rinsed with 10% wash buffer (Wash buffer: 10% formamide, 2X SSC, 0.1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 45 min.
  • 10% hybridization buffer 10% formamide, 2X SSC, 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO
  • samples were rinsed with 2x SSC and incubated with 1 uM of displacer strand in a 15% hybridization buffer at room temperature for 15 min.
  • Post displacement the sample was rinsed with a 20% wash buffer (20% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated multiple times to obtain sufficient signal amplification.
  • Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM/oligo readouts at room temperature for 15 min. Samples were stained with 3 ug/mL DAPI in 2X SSC for 1 min, then rinsed with 2X SSC twice.
  • Anti-bleaching buffer 100 mM Tris-HCl pH 8, 4x SSC, 2 mM Trolox, 20% (w/v) D- Glucose, 1 :100 Glucose Oxidase (200 U/mL), 1: 1000 Catalase
  • Figure 12 illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.
  • Figure 13 illustrates an exemplary embodiment of a fluorescent intensity distribution change over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.
  • This example depicts exemplary images of a linear and exponential amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample.
  • NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 3% glyoxal, 0.8% acetic acid. 150 mM NaCl. and 45 mM NaOH at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples were dried with nitrogen gas and a custom flow cell was attached to the glass coverslip. After rinsing with lx PBS, the sample was incubated with 0.1% NaBH4 for 15 minutes, and 7.5 mM BS-PEG5 in lx PBS was added for 15 min twice at room temperature.
  • Samples were incubated with 100 mM N- (Propionyloxy) succinimide in lx PBS two times for 15 min at room temperature. Samples were rinsed with 50% wash buffer (50% formamide, 2x SSC, 0.1% Triton-XlOO) three times. Primary probes were hybridized with a 50% hybridization buffer (50% formamide, 0. 1 mg/mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate) containing 5 nM/oligo at 37 C for 16 hours. After primary probe hybridization, the sample was rinsed with a 55% wash buffer 3 times and incubated at 37 C once. Subsequently, the sample was rinsed with 4x SSC solution.
  • 50% wash buffer 50% formamide, 2x SSC, 0.1% Triton-XlOO
  • Primary probes were hybridized with a 50% hybridization buffer (50% formamide, 0. 1 mg/mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate)
  • FIG. 14A illustrates an exemplary embodiment of a fluorescent intensity signal increase. Labelling above exemplary images shows number of total amplification rounds.
  • Example image contrast is adjusted to illustrate the similarity of fluorescent signal features such as signal dot size and signal dot number in different amplification rounds. Each dot correspond to a signal from a single molecule of nucleic acid.
  • Figure 14B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.
  • Figure 15 illustrates additional designs for crosslinking amplification.
  • Figure 15 A illustrates schematics of a sacrificial layer design for amplification.
  • Primary probes were hybridized on a RNA molecule, followed by secondary and tertiary amplifier hybridization. In this scenario, the secondary amplifiers do not have crosslinking groups, but the tertiary amplifiers have crosslinking groups.
  • a stringent stripping step was performed (60% formamide wash), which removed the secondary 7 amplifiers.
  • Other probes that were not crosslinked were also be removed- such as the primary probes. The RNA could also be degraded without affecting the subsequent results.
  • FIG. 15B illustrates quantification of signal of individual amplified dot in cells implementing the scheme shown in Figure 15 A. After 3 rounds of secondary and tertiary hybridization, crosslinking and washing, a mean of 16.7-fold amplification was achieved.
  • FIG. 15C illustrates a bridge adapter design that generates signal only when two amplified balls are physically proximal to each other.
  • the highlighted bridge probe with a thicken line shows the bridge adapter that bind across two amplified balls.
  • the thicken line represents a readout probe binding site.
  • the amplification was performed with or without a sacrificial amplifier layer.
  • the primary probes are designed to hybridize on adjacent regions on the target analytes, the RNA illustrated.
  • Each of the primary probes contained amplifier binding sites such that two amplified balls result from adjacent pairs of amplifiers.
  • Figure 15D illustrates images of cells amplified using the scheme shown in Figure 20C. 24 pairs of primary probes were used to target Eef2 mRNA in mammalian cell culture (NIH 3T3). Each pair contained primary probes that were hybridized on adjacent regions on the mRNA that are spaced one nucleotide apart. Each primary probe contained 2 amplifier binding sites for a total of 4 sites. Three of the sites were amplified.
  • Amplification was performed using the sacrificial secondary layers which are removed by washes while the tertiary amplifiers are crosslinked to the cell by Click reactions.
  • Many dots colocalize between amplifiers 1-3 indicating accurate amplification and detection of single molecules of mRNAs in cells.
  • non-specifically amplified dots in each of the amplified channels are not observed in the bridge adapter channel (right panel).
  • the lower right comers of each image show zoomed-in images shown in the white box.
  • the arrows in the insets on the lower right show regions where nonspecific dots appear in the individual amplified channel, but not in the bridge channel.
  • Nonspecific amplification occurred because amplifiers, either secondary or tertiary probes, can stick to the cell non-specifically rather than the real analyte targets.
  • These nonspecific events can be further amplified to give signal comparable to the real signals. However, because the nonspecific events are random, they are unlikely to occur at the same location for two different amplifier sequences, such as amplifier 2 and amplifier 3.
  • bridge adapters that require two or more amplifier balls to be present in close proximity serves as a co-incidence detector that rejects nonspecific binding and detect only target analytes where the two primary' probes and the amplified products occur in close physical proximity.
  • This approach when applied to different analytes present at close proximities, such as RNA-DNA.
  • RNA-protein, DNA-protein. protein-protein or other molecules enabled detection of molecular interactions using amplification and bridge readouts.
  • FIG. 1 depicts exemplary images of a linear and exponential amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample.
  • Cells were prepared as described in example 14.
  • Figure 16A illustrates an exemplary embodiment of a fluorescent intensity signal increase for a small part of a recorded image to visualize individual fluorescence intensity peaks. Each dot corresponds to a signal from a single molecule of nucleic acid. Labelling above exemplary images shows number of total amplification rounds. Contrast is matched for all example images, with a 1 Ox shorter exposure time for the rightmost two images to illustrate the increase in signal intensity.
  • Figure 16B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides.
  • peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.
  • Examples 17-20 describe RNA signal stabilization and amplification via clickchemistry.
  • the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using, for example, Alkyne- PEG4-NHS esters.
  • esterification besides adding the functional group to the cells via esterification, this could also be accomplished via a thiol or mercaptan nucleophile to make corresponding thioesters.
  • alkyne groups were attached to the cell where 3'-azide modified DNA oligos can crosslink to the cell via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions, preserving the spatial information and amplifying the signal of the unstable RNA, as shown in Figure 17A.
  • CuAAC copper(I)-catalyzed azide-alkyne cycloaddition
  • crosslinking can also be achieved using other established click-chemistry methods, including but not limited to strain-promoted azide-alkyne cycloadditions (SPAACs); tetrazine-trans-cyclooctene ligations (TCO-Tz; or inverse-electron demand Diels-Alder reactions); thiol-ene reactions; thiol-yne reactions; oxime ligations; hydrazone ligations; Diels-Alder reactions; and inverse-electrondemand Diels-Alder reactions (lEDDAs).
  • probes can be crosslinked to the cell via hydrazide treatment with carbonyls to form, for example, stable hydrazones.
  • the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using Alkyne-PEG4-NHS esters. After each round of amplifier hybridization, sample was washed with 20% formamide wash buffer (20% Formamide, 0.1% Triton X-100 in 4x SSC) before clicking to the cell to remove redundant amplifiers.
  • RNA signal can be preserved and amplified with this amplification scheme.
  • Signal could reach 5000 counts with 500ms exposure at 10% laser power from a standard confocal microscope (Andor Dragonfly) while maintaining good colocalization with the smFISH signal.
  • RNA binding site on primary probe was designed to be 30nt, amplifier binding sites were designed as 13nt/15nt at both ends of the primary probe.
  • excess probes were removed using a 30% formamide wash buffer (30% Formamide, 0.1% Triton X-100 in 4x SSC).
  • the primary probes were then anchored to the cells at the 3' end via CuAAC. Amplification of the signal was achieved with branch amplifiers, which contain a 13nt/15nt probe binding site at the 5’ end and two 13nt/15nt amplification sites at the 3' end. To minimize noise, a 10% formamide wash was applied following each round of amplifier hybridization.
  • the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using Alkyne-PEG4-NHS esters.
  • the amplifiers were subsequently crosslinked to the cells through the CuAAC reaction to establish the branching structure. Redundant amplifiers were stripped off using a 60% formamide wash buffer post-crosslinking. For rapid amplification with the short branch amplifiers, each cycle of amplifier hybridization can be reduced from 1 hour to 30 mins.
  • the amplification scheme is illustrated in Figure 19A.

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Abstract

La présente invention concerne des procédés d'amplification évolutive du signal d'un analyte dans un échantillon pour la localisation spatiale de l'analyte dans un échantillon biologique. La présente invention concerne également des procédés, et leur utilisation, ainsi que d'autres solutions à des problèmes dans le domaine concerné.
PCT/US2024/036455 2023-06-30 2024-07-01 Procédés et compositions d'amplification du signal pour la détection de cibles moléculaires par dépôt itératif de sondes Pending WO2025007154A1 (fr)

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