Chain amplification associated with exponential radiance

Abstract

Disclosed herein are compositions for chain amplification coupled with exponential radiance for signal amplification. Further disclosed herein are kits for chain amplification coupled with exponential radiance for signal amplification. Further disclosed herein are methods for chain amplification coupled with exponential radiance for signal amplification.

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 63 / 192,554, filed May 24, 2021. The contents of the above-referenced applications are incorporated by reference in their entireties.

[0002] [Statement regarding federally sponsored research] This invention was made with Government support under Grant No. HD075605 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] The present disclosure provides methods, compositions, and kits for scalable signal amplification of amplicons that are applicable to multiplexed imaging for profiling biological samples. [Background technology]

[0004] Transcriptional profiling of cells is useful for many purposes. Microscopic imaging that resolves multiple mRNAs within a single cell can provide information on transcript abundance and localization, which is important for understanding the molecular basis of cellular identity and developing treatments for disease. Molecular profiling, such as transcriptome profiling, of biological samples is useful for a variety of purposes. For example, evaluating the expression levels of genes can detect and identify abnormal growth conditions, such as cancer.

[0005] Techniques such as qPCR and microarrays are useful but do not reach single molecule sensitivity. Next generation sequencing, on the other hand, involves sample amplification and reverse transcription of mRNA, which can introduce bias and inaccuracies in quantification. Furthermore, sample preparation and sequencing are time consuming and expensive. Although imaging has been used to quantify mRNA transcription, it has been limited to a few hundred genes. Many scientific questions would be easier to understand if thousands of genes, or even the entire transcriptome, could be quantified.

[0006] What is needed are better methods and systems to perform imaging-based transcriptome profiling in a time-efficient manner with high precision and single-molecule sensitivity. [Prior art documents] [Non-patent literature]

[0007] The following references are incorporated in their entirety: [Non-Patent Document 1] Agouridas, V., El Mahdi, O., Diemer, V., Cargoet, M., Monbaliu, JM, & Melnyk, O. (2019). Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations. Chemical Reviews, 119(12). https: / / doi.org / 10.1021 / acs.chemrev.8b00712 [Non-Patent Document 2] Bauer, RJ, Zhelkovsky, A., Bilotti, K., Crowell, LE, Evans, TC, Jr, McReynolds, LA, & Lohman, GJS (2017). Comparative analysis of the end-joining activity of several DNA ligases. PloS One, 12(12), e0190062. [Non-Patent Document 3] Bharti, A., Rashmi, T., Pankaj, A., & Rohit, B. (2017). Chemical Crosslinking: Role in Protein and Peptide Science. Current Protein & Peptide Science, 18(9), 946-955. [Non-Patent Document 4] Bi, X., & Liang, A. (2016). Emerging Concepts in Analysis and Applications of Hydrogels. In Emerging Concepts in Analysis and Applications of Hydrogels. IntechOpen. [Non-Patent Document 5] Braasch, DA, & Corey, DR (2001). Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chemistry & Biology, 8(1). https: / / doi.org / 10.1016 / s1074-5521(00)00058-2 [Non-Patent Document 6] Chen, F., Tillberg, PW, & Boyden, ES (2015). Expansion microscopy. Science, 347(6221), 543-548. [Non-Patent Document 7] Chen , F. , Wassie , AT , Cote , AJ , Sinha , A. , Alon , S. , Asano , S. , Daugharthy , ER , Chang , JB , Marblestone , A. , Church , GM , Raj , A. , & Boyden , ES (2016). Nanoscale imaging of RNA with expansion microscopy. Nature Methods, 13(8).

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[0008] The present disclosure provides methods, compositions and kits for accurate and deterministic signal amplification, linked amplification tethered with exponential radiance (LANTERN). LANTERN allows for scalable signal amplification of amplicons that can be applied to multiplexed imaging for profiling biological samples. The present disclosure describes compositions and kits, as well as the making and use of the same, and other solutions to problems in related fields. [Means for solving the problem]

[0009] In some embodiments, a composition for amplification coupled with exponential radiance is provided that includes a plurality of probes, the composition includes one or more primary probes capable of binding to one or more targets, where each primary probe includes one or more secondary probe binding sites and optionally one or more readout probe binding sites. In certain embodiments, the composition includes one or more secondary probes, each capable of binding to a primary probe, where each secondary probe includes one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition optionally includes one or more tertiary probes, each capable of binding to a secondary probe, where each tertiary probe includes one or more quaternary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition optionally includes one or more quaternary probes, each capable of binding to a tertiary probe, where each quaternary probe includes one or more readout probe binding sites. In certain embodiments, the composition includes one or more readout probes that can bind to and detect binding sites on one or more primary, secondary, tertiary, or quaternary probes. In certain embodiments, the composition comprises one or more molecules capable of stabilizing one or more primary, secondary, tertiary, or quaternary probes when or after the probes hybridize.

[0010] In some embodiments, a kit for chain amplification coupled with exponential radiance is provided, the kit comprising a plurality of probes, the composition being that each primary probe comprises one or more secondary probe binding sites and optionally one or more readout probe binding sites. In certain embodiments, the kit comprises one or more secondary probes, each capable of binding to a primary probe, where each secondary probe comprises one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the kit optionally comprises one or more tertiary probes, each capable of binding to a secondary probe, where each tertiary probe comprises one or more quaternary probe binding sites or one or more readout probe binding sites. In certain embodiments, the kit optionally comprises one or more quaternary probes, each capable of binding to a tertiary probe, where each quaternary probe comprises one or more readout probe binding sites. In certain embodiments, the kit comprises one or more readout probes, each capable of binding to a binding site on one or more primary, secondary, tertiary, or quaternary probes, and capable of detection. In certain embodiments, the kit comprises one or more molecules capable of stabilizing one or more primary, secondary, tertiary, or quaternary probes when or after the probes hybridize.

[0011] In some embodiments, a method of linkage amplification coupled with exponential radiance comprising ligation amplification fluorescence in situ hybridization is provided, the method comprising contacting a sample with one or more primary probes that bind to one or more targets. In certain embodiments, the method comprises hybridizing one or more secondary probes to the primary probe, where each secondary probe comprises one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the method optionally comprises hybridizing one or more tertiary probes to at least one secondary probe, where each tertiary probe comprises one or more quaternary probe binding sites or one or more readout probe binding sites. In some embodiments, the method optionally comprises hybridizing one or more quaternary probes to at least one tertiary probe, where each quaternary probe comprises one or more readout probe binding sites. In some embodiments, the method comprises stabilizing one or more primary, secondary, tertiary, or quaternary probes during or after steps (i)-(iv). In certain embodiments, the method includes hybridizing a detectable readout probe to one or more readout probe binding sites. In certain embodiments, the method includes imaging the cell after step (vi) so that the interaction of the primary probe to the nucleic acid is detected. In certain embodiments, the method optionally includes repeating the contacting and imaging steps, each time using a new number of detectably labeled readout probes, where at least one readout probe for one target is different from at least one other readout probe for the same target in its detectable moiety, such that the targets in the sample are described by barcodes and can be distinguished from one other target in the sample by the differences in their barcodes. In some embodiments, any of the previous embodiments are repeated individually or in any combination thereof.

[0012] In some embodiments, the method is used to generate probes for use in efficient and scalable signal amplification methods that can be applied to multiplexed imaging. In certain embodiments, the method is used to generate probes for use in sequential Fluorescence In Situ Hybridization (seqFISH) of RNA and DNA. In certain embodiments, the method is used to generate probes for use in immunofluorescence studies.

[0013] In contrast to other methods such as hybridization chain reaction (HCR), which are only capable of amplifying a few amplicons at a time and are prohibitively time-consuming when imaging tens or hundreds of species from a single sample, the methods disclosed herein for primary, secondary, tertiary, quaternary, and readout probes are significantly simpler and less costly to synthesize than other chemical modifications and are readily compatible with existing enzymatic probe synthesis protocols. [Brief description of the drawings]

[0014] [Figure 1]Left: Overview of the LANTERN scheme showing two different target sequences as an example. Different colors represent the orthogonal sequences used to amplify the different targets (denoted as "A" and "B") and the orthogonal readout probe binding site (denoted as "C"). One round of amplification is shown, corresponding to a 2*2*4=16-fold amplification compared to direct primary probe readout. Right: Top: Widefield RNA FISH for Eef2 in NIH3T3 cells with two probe sets of different colors, magenta and cyan. (A) Conventional smFISH, exposure time 100 ms. (B) Six rounds of LANTERN, exposure time 1 ms. Bottom: Confocal RNA LANTERN for Eef2 in NIH3T3 cells (6 rounds). (C) First hybridization (D) Rehybridization #12, corresponding to about 10 hours of real time, showing the stability of the amplified signal for repeated formamide stripping and rehybridization. Unless otherwise noted, all images were taken with a 63× oil immersion objective. [Diagram 2] Left: Pairwise colocalization heatmaps in a pooled test of 60 LANTERN amplifiers. 2D colocalization was estimated by searching in maximum projection images for local maxima in one image within a 3 pixel box surrounding the local maxima in the second image. Off-diagonal squares corresponding to adjacent amplifiers (e.g. 1 and 2, 3 and 4) show high colocalization because pairs of amplifiers targeted the same gene for this experiment. Center: Scatter plot showing the correlation between the number of dots detected by smFISH (horizontal axis) and LANTERN (vertical axis) in the same gene. Each gene was assigned two amplifiers. Right: Scatter plot showing the correlation between the number of dots detected by the first amplifier (horizontal axis) and the number of dots detected by the second amplifier (vertical axis) for the same gene. [Diagram 3]Widefield images of RNA FISH of Eif4g1 in NIH3T3 cells showing colocalization between two orthogonal amplified sequences (A and B) and conventional smFISH (C) on the same gene. Exposure times were 40 ms (A and B) and 400 ms (C). (D) shows all three channels simultaneously. DAPI nuclear stain is shown in grey. [Figure 4] Confocal images of telomeric DNA FISH in NIH3T3 cells (cyan). Left: non-amplified telomeric DNA FISH, exposure time 500 ms. Right: LANTERN amplified telomeric DNA FISH, exposure time 500 ms. Images are shown with uniform contrast. DAPI nuclear stain is shown in grey. [Diagram 5] Widefield images of NIH3T3 cells stained with DNA-labeled antibodies before (A and C) and after (B and D) LANTERN amplification. A and B were stained with anti-lamin B and a DNA-binding secondary antibody, showing nuclear lamina staining. Exposure times 200 ms (A) and 10 ms (B). C and D were stained with anti-TIMM44 and a DNA-binding secondary antibody, showing mitochondrial staining. Exposure times 200 ms (C) and 20 ms (D). [Figure 6] A-C: Confocal images of RNA FISH for Eef2 in a human breast cancer biopsy sample. (A) Before clearing, showing high background, exposure 1 s. (B) After clearing, showing reduced background, exposure 1 s. (C) After clearing and LANTERN amplification, showing brighter and sharper dots, exposure 100 ms. (D-E) Confocal images of RNA FISH for Eef2 in adult mouse brain sections. D: smFISH signal (cyan), exposure 4 s. (E) Different brain sections after clearing and LANTERN amplification (cyan), exposure 50 ms. (F-G) Confocal images of RNA FISH for Notch1 (cyan and magenta) and Lfng (green) introns in whole mount chicken embryos. (F) Unamplified signal, barely visible at this threshold, exposure 1 s. (G) Signal after LANTERN, exposure 1 s. All samples from the same species are shown at the same contrast level, and the nuclear DAPI stain is grey in the lower panel. [Figure 7]A representative amplifier that amplifies and highly colocalizes fluorescent signals. [Figure 8] LANTERN is very specific. Most of the amplified signal comes from correctly ligated padlock probes. The images are displayed with the same contrast and show higher intensity fluorescent spots from LANTERN compared to non-amplified fluorescent spots. [Figure 9] LANTERN overcomes autofluorescence and lipofuscin in Alzheimer's human brain sections. (A) LANTERN amplified fluorescent spots of gene Eef2 are retained when thresholding lipofuscin intensity counts, showing that the amplified fluorescent spots are much brighter. (B) Compared to the non-amplified, single molecule FISH fluorescent spots, thresholding removes the transcription spots, but lipofuscin still remains strong. Images are displayed with the same contrast to show the brightness of the signal after LANTERN amplification. [Figure 10] LANTERN amplifies the fluorescent signal in mouse brain tissue sections that were perfused and fixed overnight with paraformaldehyde. The contrast in the top panel is 10x lower than the middle panel, which has the same contrast as the bottom panel. This shows that the fluorescent signal in the same single cell is amplified after six cycles of LANTERN. [Figure 11]Quantitative assessment of LANTERN in a highly multiplexed seqFISH+ experiment. (A) Top panel: An example of "pseudocolor" hybridization in a pre-LANTERN seqFISH+ experiment with 5 s exposure times for all fluorescent channels. Bottom panel: LANTERN amplified images with 200 ms exposure in the 647 nm channel, 300 ms exposure in the 561 nm channel, and 400 ms exposure in the 488 nm channel. Images of the same fluorescent channels are displayed with the same contrast. (B) LANTERN fold amplification in each fluorescent channel. The 647 nm, 561 nm, and 488 nm fluorescent channels showed 76.03-, 59.49-, and 59.82-fold amplification, respectively, after 10 cycles of LANTERN compared to smFISH. (C) Comparison of a seqFISH+ experiment with 3,000 genes profiled with LANTERN to bulk RNA-seq measurements. 45 pairs of unique LANTERN amplifiers are used in this experiment. The results show good Pearson correlation coefficients for RNA-seq measurements. [Figure 12] An alternative scheme for LANTERN. (A) A pair of split amplifiers can be used to hybridize to the padlock primary probe in this case and the reverse padlock primary probe in the other case. The split amplifiers are then ligated using an enzymatic ligase, but incorrectly bound split amplifiers are not ligated and are washed away with a high concentration formamide wash. A pair of split tertiary amplifiers is then hybridized to the ligated secondary amplifiers, followed by enzymatic ligation and a harsh formamide wash. This cycle of split amplifier hybridization, enzymatic ligation, and harsh formamide washes is repeated until the desired amplification factor is achieved. Finally, the amplified scaffolds can be detected with a fluorescent dye-conjugated readout oligonucleotide. (B) An optional design of the split amplifier that includes an additional sequence that can be further ligated with a ligation enzyme via a splint sequence to stabilize correctly bound amplifiers. (C) Amplified sample of Eef2 transcript detected with the split amplifier in the 647 nm fluorescence channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The following description is presented to enable those skilled in the art to make and use the disclosed subject matter, and to incorporate it into the context of application. As with the various applications in various applications, various modifications will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to a wide range of embodiments. Thus, the present disclosure is not intended to be limited to the embodiments presented, but should be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0016] [Definition] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the art.

[0017] As used herein, the terms "approximately" or "about" in reference to numerical values ​​are generally deemed to include numerical values ​​that fall within 5%, 10%, 15%, or 20% in either direction (greater or lesser) of the numerical value unless otherwise specified or apparent from the context (except where such numerical value is less than 0% or greater than 100% of its possible value).

[0018] As used herein, the term "LANTERN" is an acronym that stands for linked amplification tethered with exponential radiance.

[0019] The term "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 have a variety of lengths. In certain embodiments, oligonucleotides can range from about 2 to about 1000 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can range from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, and about 20 to about 30 nucleotides in length. In some embodiments, oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, oligonucleotides are at least 4 nucleotides in length. In some embodiments, oligonucleotides are at least 5 nucleotides in length. In some embodiments, oligonucleotides are at least 6 nucleotides in length. In some embodiments, oligonucleotides are at least 7 nucleotides in length. In some embodiments, oligonucleotides are at least 8 nucleotides in length. In some embodiments, oligonucleotides are at least 9 nucleotides in length. In some embodiments, oligonucleotides are at least 10 nucleotides in length. In some embodiments, 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 at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands at least 21 nucleotides in length.

[0020] As used herein, the term "probe" or "probes" refers to any synthetic or naturally occurring molecule that can directly or indirectly bind to a molecular target (e.g., an mRNA sample, a DNA molecule, a protein molecule, an RNA and DNA isoform molecule, a single nucleotide polymorphism molecule, etc.). For example, a probe can include a nucleic acid molecule, an oligonucleotide, a protein (e.g., an antibody or an antigen binding sequence), or a combination thereof. For example, a protein probe can be conjugated to one or more nucleic acid molecules to form a chimeric probe. As disclosed herein, in some embodiments, the probe itself can generate a detectable signal. In some embodiments, the probe is conjugated, directly or indirectly through an intermediate molecule, to a signal moiety (e.g., a dye or fluorophore) that can generate a detectable signal.

[0021] As used herein, the term "binding site" refers to a site on a probe at which another molecule can bind to the probe. In certain embodiments, the binding site on a probe binds to another molecule through non-covalent interactions.

[0022] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample includes a biological tissue or fluid. In some embodiments, the biological sample is or includes the following: bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymphatic fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation such as ductal lavage cytology or bronchoalveolar lavage, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluids, secretions, and / or excretions, and / or cells thereof. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, excreta, etc.), and the like. In some embodiments, as the context will make clear, the term "sample" refers to a preparation obtained by processing (e.g., removing one or more components and / or adding one or more agents) a primary sample. For example, filtration through a semi-permeable membrane. Such a "processed sample" may consist, for example, of nucleic acids or proteins extracted from the sample, or nucleic acids or proteins obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components. In some embodiments, the term "sample" refers to nucleic acids such as DNA, RNA, transcripts, or chromosomes. In some embodiments, the term "sample" refers to nucleic acids extracted from cells.

[0023] As used herein, the term "substantially" refers to the qualitative state of exhibiting the entire or near extent of a characteristic or property of interest. One of ordinary skill in the art of biology will understand that biological and chemical phenomena rarely or only rarely proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and / or chemical phenomena.

[0024] As disclosed herein, the term "label" generally refers to a molecule that can recognize and bind to a specific target site within a molecular target within a cell. For example, the label can include an oligonucleotide that can bind to the molecular target within a cell. The oligonucleotide can be linked to a moiety that has affinity for the molecular target. The oligonucleotide can be linked to a first moiety that can be covalently linked to the molecular target. In certain embodiments, the molecular target includes a second moiety that can form a covalent linkage with the label. In certain embodiments, the label includes a nucleic acid sequence that can provide identification of a cell that contains or has contained the molecular target. In certain embodiments, a plurality of cells are labeled, and each cell of the plurality of cells has a unique label relative to other labeled cells.

[0025] As disclosed herein, the term "barcode" generally refers to the nucleotide sequence of the label produced by the methods described herein. The barcode sequence is typically of sufficient length and uniqueness to identify a single cell that contains the molecular target.

[0026] As disclosed herein, the term "linked" refers to a covalent bond or a non-covalent interaction between two molecules. In particular, one type of non-covalent interaction is hybridization.

[0027] As disclosed herein, the term "cis-ligated" generally refers to the ligation of oligonucleotides 5'-3' on the same oligonucleotide. In certain embodiments, "cis-ligated" refers to ligating the ends of an oligonucleotide together. In certain embodiments, "cis-ligated" refers to ligating one end of an oligonucleotide to a nucleotide anywhere before the other end of the oligonucleotide.

[0028] As disclosed herein, the term "trans-ligated" generally refers to the ligation of 5'-3' oligonucleotides on different oligonucleotides. In certain embodiments, "trans-ligated" refers to ligating together an end of one oligonucleotide and an end of another oligonucleotide. In certain embodiments, "trans-ligated" refers to one end of an oligonucleotide being ligated to any nucleotide on a different oligonucleotide.

[0029] As disclosed herein, the term "splint probe" or "splint" or "splint sequence" refers to a probe that is complementary to another probe that hybridizes or binds to the complementary probe, and the probes are not covalently linked to each other. In certain embodiments, the term "splint probe" uses the definition and technique of Lohman et al. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase. Non-patent literature Nucleic Acid Research, 2014, vol.42 No.3 1831-1844 is incorporated by reference in its entirety.

[0030] [Embodiment] The present disclosure describes embodiments relating to a chain amplification composition that combines exponential radiance and includes multiple probes.

[0031] The present disclosure describes embodiments of kits for chain amplification coupled with exponential radiance that include multiple probes.

[0032] The present disclosure describes embodiments relating to methods of chain amplification involving multiple probes and coupled exponential radiance.

[0033] This disclosure describes an efficient and scalable signal amplification method that can be applied to multiplexed imaging such as RNA and DNA seqFISH and immunofluorescence (Figure 1, Figures 3-5). In some embodiments, amplification of tens or hundreds of orthogonal amplicons can be performed at once.

[0034] In contrast to other methods, such as hybridization chain reaction (HCR), which are only capable of amplifying a few amplicons at a time and are extremely time-consuming when imaging tens or hundreds of species from a single sample, the primary, secondary, tertiary, quaternary, and readout probes are significantly simpler and less costly to synthesize than other chemical modifications and are readily compatible with existing enzymatic probe synthesis protocols.

[0035] In some embodiments, a composition for chain amplification fluorescence in situ hybridization is provided that includes a plurality of probes, the composition includes one or more primary probes capable of binding to one or more targets, each primary probe including one or more secondary probe binding sites and optionally one or more readout probe binding sites. In certain embodiments, the composition includes one or more secondary probes, each capable of binding to a primary probe, each secondary probe including one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition optionally includes one or more tertiary probes, each capable of binding to a secondary probe, each tertiary probe including one or more quaternary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition optionally includes one or more quaternary probes, each capable of binding to a tertiary probe, each quaternary probe including one or more readout probe binding sites. In certain embodiments, the composition includes one or more readout probes capable of binding to and detecting binding sites on one or more primary, secondary, tertiary, or quaternary probes. In certain embodiments, the composition comprises one or more molecules capable of stabilizing one or more primary, secondary, tertiary, or quaternary probes when or after the probes hybridize.

[0036] In some embodiments, a kit for chain amplification coupled to exponential radiance comprising a plurality of probes is provided, the composition comprising: wherein each primary probe comprises one or more secondary probe binding sites and optionally one or more readout probe binding sites. In certain embodiments, the composition comprises one or more secondary probes, each capable of binding to a primary probe, each secondary probe comprising one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition optionally comprises one or more tertiary probes, each capable of binding to a secondary probe, each tertiary probe comprising one or more quaternary probe binding sites or one or more readout probe binding sites. In certain embodiments, the composition comprises one or more readout probes capable of binding to and detecting binding sites on one or more primary, secondary, tertiary, or quaternary probes. In certain embodiments, the kit comprises one or more molecules capable of stabilizing one or more primary, secondary, tertiary, or quaternary probes when or after the probes hybridize or after the probes hybridize.

[0037] In some embodiments, a method for chain amplification coupled with exponential radiance is provided, including a method for chain amplification coupled with exponential radiance, the method comprising contacting a sample with one or more primary probes that bind to one or more targets, each primary probe hybridizing to the target. In certain embodiments, the method comprises hybridizing one or more secondary probes to the primary probe, each secondary probe comprising one or more tertiary probe binding sites or one or more readout probe binding sites. In certain embodiments, the method optionally comprises hybridizing one or more tertiary probes to at least one secondary probe, each tertiary probe comprising one or more quaternary probe binding sites or one or more readout probe binding sites. In some embodiments, the method optionally comprises hybridizing one or more quaternary probes to at least one tertiary probe, each quaternary probe comprising one or more readout probe binding sites. In some embodiments, the method comprises stabilizing one or more primary, secondary, tertiary, or quaternary probes during or after steps (i)-(iv). In certain embodiments, the method comprises hybridizing detectable readout probes to one or more readout probe binding sites. The method comprises imaging the cell so that the interaction of the primary probe with the nucleic acid is detected. The method optionally repeats the contacting and imaging steps, each time using a new plurality of detectably labeled readout probes, where at least one readout probe for one target is different from at least one other readout probe for the same target of its detectable moiety. In some embodiments, any of the previous embodiments are repeated individually or in any combination thereof.

[0038] [sample] In some embodiments, the method includes analyzing a sample, the sample being comprised of bacterial cells, archaeal cells, eukaryotic cells, or a combination thereof. In certain embodiments, the sample includes a tissue, a cell, or an extract from a cell. In certain embodiments, the sample includes a biofilm. In certain embodiments, the sample includes cells obtained from a patient.

[0039] [target] In some embodiments, the target is selected from a transcript, RNA, a DNA locus, a chromosome, DNA, a protein, a lipid, a glycan, a cellular target, an organelle, and any combination thereof. In some embodiments, the transcript, RNA, a DNA locus, a chromosome, DNA, a protein, a lipid, a glycan, a cellular target, an organelle, and any combination thereof is attached to an oligonucleotide.

[0040] [Primary, secondary, tertiary, and quaternary probes] In some embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least one readout probe binding site. In certain embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least two readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least three readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least four readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least five readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least six readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least seven readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probe comprises at least eight readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probes comprise at least 9 readout probe binding sites. In some embodiments, in any of the preceding embodiments, the primary, secondary, tertiary, or quaternary probes comprise at least 10 readout probe binding sites.

[0041] In some embodiments, the primary probe of any of the previous embodiments comprises a nucleic acid sequence complementary to a target nucleic acid sequence.

[0042] In some embodiments, sequences complementary to a target nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, sequence complementarity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0043] In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length.In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the primary probe of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0044] In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length.In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the secondary probe of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0045] In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length.In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the tertiary probe of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0046] In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length.In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the quaternary probe of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0047] In some embodiments, the secondary probe complements a secondary probe binding site on a primary probe. In some embodiments, the tertiary probe complements a secondary probe binding site on a secondary probe. In some embodiments, the quaternary probe complements a tertiary probe binding site on a tertiary probe. In some embodiments, the probe complement comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity.

[0048] In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 100 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 10 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 20 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 30 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 40 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 50 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 60 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 70 nucleotides. In some embodiments, the length of the primary, secondary, tertiary, and quaternary probe binding sites ranges from 5 to 80 nucleotides. In some embodiments, the lengths of the primary, secondary, tertiary, and quaternary probe binding sites range from 5 to 90 nucleotides.

[0049] In some embodiments, a composition or kit according to any of the previous embodiments comprises two or more primary probes capable of binding to two or more targets.

[0050] In some embodiments, a composition or kit according to any of the preceding embodiments comprises two or more secondary probes capable of binding to a primary probe, each secondary probe comprising two or more tertiary probe binding sites or two or more readout probe binding sites.

[0051] In some embodiments, a composition or kit according to any of the preceding embodiments comprises two or more tertiary probes, each capable of binding to two or more tertiary probe binding sites, and each tertiary probe comprises one or more readout probe binding sites.

[0052] In some embodiments, the kit of any of the previous embodiments comprises a DNA ligase.

[0053] In some embodiments, the method of any of the previous embodiments includes contacting the sample with two or more primary probes that bind to one or more targets, where the two or more primary probes hybridize to the targets.

[0054] In some embodiments, the method of any of the previous embodiments includes hybridizing a secondary probe to the ligated primary probe, where the secondary probe includes two or more tertiary probe binding sites or two or more readout probe binding sites.

[0055] In some embodiments, the method of any of the previous embodiments comprises hybridizing a tertiary probe to at least two secondary probes, and wherein each tertiary probe comprises two or more readout probe binding sites.

[0056] In some embodiments, the method of any preceding claim comprises hybridizing a detectable readout probe to two or more readout probe binding sites.

[0057] In some embodiments, the method of any of the preceding embodiments further comprises repeating the contacting and imaging steps, each time with a new plurality of detectably labeled readout probes, each of which has at least one readout probe for one target that differs from at least one readout probe for the same target in a previous plurality at least in their detectable moieties.

[0058] [Amplifier Fragment] In some embodiments, each secondary, each tertiary, and / or each quaternary probe includes at least two amplifier fragments.

[0059] In some embodiments, a secondary, secondary and tertiary, secondary, tertiary and quaternary, secondary and quaternary, tertiary, tertiary and quaternary, or quaternary probe comprises at least two amplifier fragments.

[0060] In certain embodiments, the amplifier fragment of the secondary probe comprises at least a first amplifier fragment, where the first amplifier fragment comprises a region of complementarity to the primary probe, and the region of complementarity hybridizes to the primary probe. In certain embodiments, the amplifier fragment of the secondary probe comprises at least a second amplifier fragment, where the second amplifier fragment comprises a region of complementarity to the primary probe, and the region of complementarity hybridizes to the primary probe.

[0061] In some embodiments, the amplifier fragment of the tertiary probe comprises at least a first amplifier fragment, where the first amplifier fragment comprises a region of complementarity to the secondary probe or a first or second amplifier fragment of the secondary probe, and the region of complementarity hybridizes to the secondary probe or the first or second amplifier fragment of the secondary probe. In certain embodiments, the amplifier fragment of the tertiary probe comprises at least a second amplifier fragment, where the second amplifier fragment comprises a region of complementarity to the secondary probe or a first or second amplifier fragment of the secondary probe, and the region of complementarity hybridizes to the secondary probe or the first or second amplifier fragment of the secondary probe.

[0062] In some embodiments, the amplifier fragment of the quaternary probe comprises at least a first amplifier fragment, where the first amplifier fragment comprises a region of complementarity to the tertiary probe or a first or second amplifier fragment of the tertiary probe, and the region of complementarity hybridizes to the tertiary probe or the first or second amplifier fragment of the tertiary probe. In certain embodiments, the amplifier fragment of the quaternary probe comprises a second amplifier fragment, where the second amplifier fragment comprises a region of complementarity to the tertiary probe or a first or second amplifier fragment of the tertiary probe, and the region of complementarity hybridizes to the tertiary probe or the first or second amplifier fragment of the tertiary probe.

[0063] In some embodiments, the composition, kit, or method of any of the previous embodiments includes a ligase ligating the first or second fragment of any secondary, tertiary, or quaternary amplifier fragment.

[0064] In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length.In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the amplifier fragment of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0065] In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 5 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 6 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 7 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 8 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 9 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 10 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 11 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 12 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 13 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 14 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 15 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 16 nucleotides in length.In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 17 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 18 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 19 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 20 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is at least 21 nucleotides in length. In some embodiments, the region of complementarity according to any of the previous embodiments comprises a region of the oligonucleotide according to any of the previous embodiments that is less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0066] In some embodiments, the region of complementarity of any of the previous embodiments comprises sequence complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0067] [Sprint Array] In some embodiments, the composition, kit, or method of any embodiment comprises a splint sequence.

[0068] In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length.In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide that is at least 20 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide that is at least 21 nucleotides in length. In some embodiments, each splint sequence of any of the previous embodiments comprises an oligonucleotide that is less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0069] In some embodiments, the splint sequence hybridizes to a first amplifier fragment, a second amplifier fragment, or both amplifier fragments of a secondary, tertiary, or quaternary amplifier fragment.

[0070] In certain embodiments, the splint sequence comprises at least two splint sequence fragments.

[0071] In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 6 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 7 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 8 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 9 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 10 ...1 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length.In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide at least 21 nucleotides in length. In some embodiments, the splint sequence fragment of any of the previous embodiments comprises an oligonucleotide less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0072] In some embodiments, the splint sequence fragments are ligated.

[0073] [Probe stabilization] In some embodiments, the method comprises stabilizing a primary, secondary, tertiary, or quaternary probe. In some embodiments, the method comprises stabilizing a primary probe. In some embodiments, the method comprises stabilizing a secondary probe. In some embodiments, the method comprises stabilizing a tertiary probe. In some embodiments, the method comprises stabilizing a quaternary probe.

[0074] In some embodiments, the probes are stabilized by a method selected from enzymatic ligation, chemical ligation, UV crosslinking with or without oligo splint probes, hybridization of splint probes, matrix-mediated crosslinking, and chemical crosslinking, or any combination thereof. In certain embodiments, the enzyme used for enzymatic ligation is selected from T4 ligase, T7 ligase, Quick ligase, T3 ligase, and Ampligase. In certain embodiments, the chemical ligation is selected from amine-phosphate, diamine, and thiol ligation. In certain embodiments, the matrix-mediated crosslinking comprises a hydrogel made from polyacrylamide or agarose. In certain embodiments, the chemical crosslinking for stabilization is selected from reversible crosslinkers such as paraformaldehyde, glutaraldehyde, and DSP (dithiobis succinimidyl propionate). In certain embodiments, the splint probe comprises a locked nucleic acid (LNA) or a peptide nucleic acid (PNA).

[0075] In some embodiments, the primary, secondary, tertiary, or quaternary probes described in any of the previous embodiments are cis-ligated through one or more additional molecules, such as oligonucleotide probes, LNA or PNA, or proteins, molecular complexes, or small chemical molecules.

[0076] In some embodiments, the primary, secondary, tertiary, or quaternary probes according to any of the previous embodiments are UV cis-ligated directly or indirectly via an intermediate molecule.

[0077] In some embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are cis-ligated to a cell or sample matrix, either directly or via an intermediate molecule. In certain embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are cis-ligated with a chemical crosslinker, including paraformaldehyde, glutaraldehyde, or a reversible crosslinker. In certain embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are cis-ligated with a matrix, including a native tissue matrix, a tissue matrix, or an exogenous matrix. In certain embodiments, the exogenous matrix comprises a hydrogel.

[0078] In some embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are stabilized prior to subsequent probe hybridization. In certain embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are stabilized prior to a stripping step.

[0079] In some embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are ligated using a ligase. In certain embodiments, the probes are ligated either in cis or in trans. In some embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are cis-ligated. In some embodiments, the primary, secondary, tertiary, or quaternary probes of any of the previous embodiments are trans-ligated.

[0080] In some embodiments, the primary, secondary, tertiary, or quaternary probes according to any of the previous embodiments are cis-ligated by acrylamide polymerization.

[0081] In some embodiments, the primary, secondary, tertiary, or quaternary probes according to any of the previous embodiments are cis-ligated by click chemistry.

[0082] In some embodiments, the primary, secondary, tertiary, or quaternary probes according to any of the previous embodiments are cis-ligated by reactive groups, where the reactive groups form a reactive pair selected from an alkene, an alkyne, an azide, an amide, an amine, a nitrone, a phosphate, a tetrazine, and a tetrazole.

[0083] In some embodiments, the method of any of the previous embodiments uses ligated or cross-linked primary probes. In some embodiments, the method of any of the previous embodiments uses ligated or cross-linked primary probes either in cis or in trans.

[0084] In some embodiments, the primary, secondary, or tertiary probes of any of the previous embodiments are ligated or cross-linked.

[0085] In some embodiments, the primary, secondary, or tertiary probes of any of the previous embodiments are cis-ligated.

[0086] In some embodiments, the enzyme according to any of the previous embodiments comprises a DNA or RNA ligase, or a DNA or RNA polymerase, and or any combination of the above.

[0087] In some embodiments, the kit of any of the previous embodiments comprises a DNA ligase.

[0088] [Fluorophore] In some embodiments, the composition, kit, or method of any embodiment comprises a detectable moiety. In some embodiments, any one of the compositions, kits, or methods described in any of the previous embodiments comprises at least two different detectable moieties. In certain embodiments, the detectable moieties are the same.

[0089] In some embodiments, the detectable moiety is any fluorophore deemed appropriate by one of skill in the art.

[0090] In some embodiments, the detectable moiety includes, but is not limited to, fluorescein, rhodamine, Alexa Fluors, DyLight Fluors, ATTO Dyes, or analogs or derivatives thereof. In certain embodiments, the detectable moiety includes, but is not limited to, fluorescein and chemical derivatives of fluorescein, eosin, carboxyfluorescein, fluorescein isothiocyanate (FITC), fluorescein amidite (FAM), erythrosine, rose bengal, fluorescein secreted by Pseudomonas aeruginosa, methylene blue, laser dyes, rhodamine dyes (e.g., rhodamine, rhodamine 6G, rhodamine B, rhodamine 123, auramine O, sulforhodamine 101, sulforhodamine B, Texas Red).

[0091] In some embodiments, the detectable moiety includes the following: ATTO dyes, acridine dyes (e.g., acridine orange, acridine yellow), Alexa Fluor, 7-aminoactinomycin D, 8-anilinonaphthalene-1-sulfonate, auramine rhodamine stain, benzanthrone, 5,12-bis(phenylethynyl)naphthacene, 9,10-bis(phenylethynyl)anthracene, black light paint, brainbow, calcein, carboxyfluorescein, carboxyfluorescein diacetate succinimidyl ester, carboxyfluorescein succinimidyl ester, 1-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-diphenylanthracene, coumarin, cyanine dyes (e.g., cyanines such as Cy3 and Cy5, DiOC6, SYBR Green I), DAPI, dark quenchers, DyLight 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, merbromin), Fluoro-Jade stain, Fura-2, Fura-2-acetoxymethyl ester;These include, but are not limited to, green fluorescent protein, Hoechst stain, Indian-yellow, Indo-1, Lucifer yellow, luciferin, merocyanine, fluorescent brighteners, oxazine dyes (e.g., cresyl violet, Nile blue, Nile red), perylene, phenanthridine dyes (ethidium bromide, propidium iodide), phloxine, phycobilin, phycoerythrin, phycoerythrobilin, pyranine, rhodamine, rhodamine 123, rhodamine 6G, ribogreen, RoGFP, rubrene, SYBR Green I, (E)-stilbene, (Z)-stilbene, sulforhodamine 101, sulforhodamine B, Synapto-pHluorin, tetraphenylbutadiene, tetrasodium tris(batophenanthroline disulfonate)ruthenium(II), Texas Red, TSQ, Umbelliferone, or yellow fluorescent protein;

[0092] In some embodiments, the detectable moiety includes, but is not limited to, the 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. Individual members of the family are roughly numbered according to their excitation maxima (in nm). Certain Alexa Fluor dyes are synthesized by sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. In some embodiments, sulfonation renders the Alexa Fluor dyes negatively charged and hydrophilic. In some embodiments, the Alexa Fluor dyes are more stable, brighter, and less pH sensitive than common dyes of comparable excitation and emission (e.g., fluorescein, rhodamine), as well as some newer cyanine systems. 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.

[0093] In some embodiments, the detectable moiety comprises one or more fluorescent dyes of the DyLight Fluor family (Dyomics and Thermo Fisher Scientific). Exemplary DyLight Fluor family dyes include, but are not limited to, DyLight-350, DyLight-405, DyLight-488, DyLight-549, DyLight-594, DyLight-633, DyLight-649, DyLight-680, DyLight-750, or DyLight-800.

[0094] In some embodiments, the detectable moiety comprises a nanomaterial. In some embodiments, the fluorophore is a nanoparticle. In some embodiments, the detectable moiety is or comprises a quantum dot. In some embodiments, the fluorophore is a quantum dot. In some embodiments, the detectable moiety comprises a quantum dot. In some embodiments, the detectable moiety is or comprises a gold nanoparticle. In some embodiments, the detectable moiety is a gold nanoparticle. In some embodiments, the detectable moiety comprises a gold nanoparticle.

[0095] [Read Probe] In some embodiments, one or more of the readout probes of any of the previous embodiments comprises an oligonucleotide or an antibody having a detectable moiety.

[0096] In some embodiments, one or more readout probes of any of the previous embodiments comprise oligonucleotides having the same sequence.

[0097] In some embodiments, one or more readout probes of any of the previous embodiments comprise oligonucleotides having different sequences.

[0098] In some embodiments, one or more readout probes of any of the previous embodiments comprise an oligonucleotide at least 17 nucleotides in length.

[0099] In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 5 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 10 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 11 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 12 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 13 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 14 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 15 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 16 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 17 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 18 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 19 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide at least 20 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide that is at least 21 nucleotides in length. In some embodiments, the readout probe of any of the previous embodiments comprises an oligonucleotide that is less than 30, 50, 100, 200, 250, 500, 750, or 1000 nucleotides in length.

[0100] In some embodiments, the readout probe complements a readout probe binding site on a primary probe. In some embodiments, the readout probe complements a readout probe binding site on a secondary probe. In some embodiments, the readout probe complements a readout probe binding site on a tertiary probe. In some embodiments, the readout probe complements a readout probe binding site on a quaternary probe. In some embodiments, the readout probe is complementary to a splint sequence fragment. In some embodiments, the probe complement comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity.

[0101] In some embodiments, the length of the readout probe binding site is in the range of 5 to 100 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 10 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 20 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 30 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 40 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 50 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 60 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 70 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 80 nucleotides. In some embodiments, the length of the readout probe binding site is in the range of 5 to 90 nucleotides.

[0102] In some embodiments, a composition or kit according to any of the previous embodiments comprises two or more readout probes capable of binding to and detecting at least one of the one or more readout probe binding sites.

[0103] In some embodiments, a composition or kit according to any of the previous embodiments comprises two or more readout probes capable of binding to and detecting at least one of the one or more readout probe binding sites.

[0104] [Sample imaging] In some embodiments, the method comprises imaging the probe or barcode. In some embodiments, the method comprises imaging the target probe or barcode. As will be appreciated by those skilled in the art, different techniques can be used for the imaging step.

[0105] In some embodiments, imaging methods include, but are not limited to, epifluorescence microscopy, confocal microscopy, different types of super-resolution microscopy (PALM / STORM, SSIM / GSD / STED), and light sheet microscopy (SPIM, etc.).

[0106] In some embodiments, the imaging method including the exemplary super-resolution technique includes the following: 5These include, but are not limited to, M and 4Pi microscopy, Stimulated Emission Depletion microscopy (STEDM), Ground State Depletion microscopy (GSDM), 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.Examples include the following non-patent documents: Chi, 2009, "Super-resolution microscopy: breaking the limits," Nature Methods 6(1): 15-18; Blow, 2008, "New ways to see a smaller world," Nature 456:825-828; Hell, et al, 2007, "Far-Field Optical Nanoscopy," Science 316: 1153; R. Heintzmann and G. Ficz, 2006, "Breaking the resolution limit in light microscopy," Briefings in Functional Genomics and Proteomics 5(4):289-301; Garini et al., 2005, "From micro to nano: recent advances in high-resolution microscopy," Current Opinion in Biotechnology 16:3-12; and Bewersdorf et al, 2006, "Comparison of I. 5 M and 4Pi-microscopy," 222(2): 105-1 17; and the non-patent publication Wells, 2004, "Man the Nanoscopes," JCB 164(3):337-340.

[0107] In some embodiments, electron microscopes (EM) are used for imaging.

[0108] In some embodiments, the imaging step detects the target. In some embodiments, the imaging step localizes the target. In some embodiments, the imaging step provides three-dimensional spatial information of the target. In some embodiments, the imaging step quantifies the target. By using multiple contacting and imaging steps, the provided methods can provide spatial and / or quantitative information of multiple targets with surprisingly high throughput. For example, when using detectable F different labels, spatial and / or quantitative information of up to FN targets can be obtained through N contacting and imaging steps.

[0109] Certain techniques for imaging are known in the art. See, for example, International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014, entitled "MULTIPLEX LABELING OF MOLECULES BY SEQUENTIAL HYBRIDIZATION BARCODING," the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0110] In some embodiments, the method includes analyzing cell size and shape, markers, immunofluorescence, or any combination thereof.

[0111] [Remove probe] In some embodiments, the method according to any of the previous embodiments comprises washing the sample after each step. In certain embodiments, the sample is washed with a buffer that removes non-specific hybridization reactions. In certain embodiments, formamide is used in the washing step. In certain embodiments, the washing buffer is stringent. In certain embodiments, the washing buffer comprises 10% formamide, 2xSSC, and 0.1% Triton X-100s.

[0112] In some embodiments, the method comprises removing one or more probes after one or more imaging steps. In some embodiments, removing the probes comprises contacting the plurality of readout probes with an enzyme that digests the probes. In some embodiments, removing comprises contacting the plurality of probes with DNase, contacting the plurality of probes with RNase, photobleaching, strand displacement, formamide wash, thermal denaturation, chemical denaturation, cleavage, or a combination thereof. In some embodiments, removing comprises photobleaching to remove the probes.

[0113] In some embodiments, the method further comprises removing the readout probe after one or more imaging steps. In some embodiments, the removing step comprises contacting the plurality of readout probes with an enzyme that digests the readout probe. In some embodiments, the method comprises removing the readout probe using a stripping reagent, a wash buffer, photobleaching, chemical bleaching, and any combination thereof. In some embodiments, the method comprises contacting the plurality of target readout probes with DNase, contacting the plurality of target probes with RNase, photobleaching, strand displacement, formamide wash, heat denaturation, or a combination thereof. In some embodiments, the target readout probe is removed by photobleaching.

[0114] In some embodiments, the method comprises clearing the sample. In some embodiments, the sample is cleared by CLARITY. In some embodiments, the sample is cleared by post-hydrogel embedding.

[0115] Certain techniques for removing probes are known in the art, see, e.g., International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014, entitled "MULTIPLEX LABELING OF MOLECULES BY SEQUENTIAL HYBRIDIZATION BARCODING," the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0116] The following non-limiting methods are provided to further illustrate the embodiments of the invention disclosed herein. Those skilled in the art will understand that the techniques disclosed in the following examples represent approaches that have been found to work well in the implementation of some embodiments of the invention, and therefore are considered to constitute examples of its implementation. However, those skilled in the art should understand in light of this disclosure that many changes can be made to the specific embodiments disclosed, while still obtaining the same or similar results, without departing from the spirit and scope of the invention.

[0117] [method] [Probe design and synthesis] Primary probes specific for gene or antibody barcodes are designed to target the RNA or DNA sequence of interest, as is common in FISH techniques. Typically, a complementary region of 25-35 nucleotides is used. In RNA FISH, 10-30 of these are designed for each transcript.

[0118] Instead of a direct readout probe site, the primary probe has 5' and 3' consensus sequences that can be used to hybridize sites 1-4 in the first round of amplification padlock probes (hereafter referred to as 'A' sequences), as well as a splint for ligation.

[0119] Three pools of 5'-phosphorylated amplification probes are synthesized: the first pool (secondary probes) bind to the "A" sequence on the primary probe and display multiple repeats of the "B" sequence; the second pool (tertiary probes) align to the "B" sequence and display multiple repeats of the "A" sequence; and the third pool (readout probes) bind to either the "A" or "B" sequence and display binding sites for a fluorescently conjugated readout probe.

[0120] [Amplification protocol] Optionally, samples may be precleared with 8% SDS or Triton X-100 for 30 min at RT, or ethanol or methanol at -20°C for 1-24 h.

[0121] The primary probe or antibody is incubated with a typical sample in smFISH, for example in a standard hybridization or standard immunofluorescence blocking buffer.

[0122] If the primary probes are 5'-phosphorylated, they are ligated by a ligase.

[0123] If the primary probe has a 5' acryldite group, the sample is embedded in a polyacrylamide gel. After polymerization, the sample is digested / cleared, for example, with proteinase K in 1% SDS / 50 mM Tris HCl / 2 mM CaCl2 at 37°C for 1-24 hours.

[0124] The sample undergoes repeated cycles of hybridization, washing, stabilization, and ligation with secondary and tertiary probes and washing, and is then read with the readout oligo.

[0125] Amplified samples can be optionally post-fixed, a step that may ensure the stability of the amplification over many imaging rounds of formamide stripping and rehybridization.

[0126] [Imaging] Chemically conjugated fluorescent probes are hybridized directly or using a short bridge to a unique 10-17 nucleotide sequence on each readout adapter at RT-37°C in an appropriate buffer (e.g., 10% ethylene carbonate, 4xSSC, 10% 6.5-10 kDa dextran sulfate for 5-40 min, followed by a mild wash such as 10% formamide, 2xSSC, 0.1% Triton X-100, then a nuclear stain such as DAPI).

[0127] As with regular smFISH, imaging is performed in an anti-bleaching buffer system containing 4xSSC, 25 mM Tris HCl, glucose oxidase, catalase, and Trolox, using optimal filter sets and laser illumination. In confocal or widefield microscopes equipped with CMOS cameras, laser powers of 50-500 mW and objectives between 20-100x are typically used. Importantly, amplification allows for a significant reduction in exposure time per Z slice, down to 10 ms in spinning disk confocal microscopes, which can greatly reduce background signal. Alternatively, widefield setups can be used to image smFISH in tissue samples, which typically requires confocal imaging.

[0128] The fluorescent readout probe is stripped by up to 60% formamide wash with or without strand displacement using a 5-10 nucleotide toehold. Strand displacement may help to completely extinguish the fluorescent signal from higher GC content readout sequences. EXAMPLES

[0129] Example 1 The methods disclosed herein target nucleic acids or proteins of interest in situ or in vitro using DNA primary probes or DNA-binding antibodies, which can be ligated to circular single-stranded DNA (ssDNA) or cross-linked to polyacrylamide gels and contain secondary probe binding sites.

[0130] As an example, the primary binding site (Figure 1) is hybridized to a ssDNA primary probe, which may comprise a locked nucleic acid (LNA) or other modified oligonucleotide for higher affinity.

[0131] The 5' end of the primary probe is modified with either phosphate, which allows for covalent circularization by DNA ligase, or acrylamide, which allows for free radical polymerization into polyacrylamide crosslinks. This ligation / polymerization step stabilizes the primary probe bond to the targeted nucleotide during subsequent hybridization and washing cycles.

[0132] Ligating probes with enzymes has several advantages over designing probes for ligation using click chemistry. Enzymatic ligation provides better specificity because the ends of the probe must be adjacent in order to ligate. In contrast, click chemistry often ligates nonadjacent ends of the probe. Using enzymatic ligation ensures that amplification is highly specific. In addition, the use of enzymes allows for inexpensive mass production of oligonucleotides for experiments. Individual click chemistry probes are often purchased for approximately $1000 per probe.

[0133] Secondary probes with 5'-phosphorylated modifications and padlocks are then hybridized to one or more secondary binding sites in the primary probe and subsequently ligated with DNA ligase. These secondary probes contain two or more tertiary probe binding sites. After hybridization and ligation of the secondary probes, tertiary probes with 5'-phosphorylated modifications and padlocks are hybridized to two or more tertiary binding sites in the secondary probe and subsequently ligated with DNA ligase. These tertiary probes contain two or more secondary probe binding sites.

[0134] Importantly, the above steps can be repeated, because the secondary and tertiary probes have more than one secondary or tertiary probe binding site, allowing for exponential amplification of the secondary and tertiary probes. For example, if secondary and tertiary probes, each with two probe binding sites, undergo two rounds of hybridization and ligation, one secondary probe binding site will be used for two amplifications. 4 = 16 secondary probe binding sites. Additional steps of hybridization and ligation of secondary and tertiary probes can theoretically increase the number of readout probe binding sites by several thousand-fold, and in practice several hundred-fold has been achieved (Figure 1 and Figure 6E).

[0135] After the desired number of iterations, a padlock-type probe with phosphorylation at the 5' end, containing multiple readout probe binding sites, is hybridized and ligated in the same way. The readout binding sites are then visualized by a 17-nt or shorter readout probe conjugated to a fluorophore, resulting in an exponentially amplified signal compared to that obtained directly from the primary probe. The fluorescent signal from the readout probe can be stripped using up to 60% formamide solution without affecting the primary probe and padlock structure (Figure 1).

[0136] By preparing orthogonal sets of secondary, tertiary, and readout probe sequences, signal amplification can be performed for many nucleic acid or protein species of interest in both cell, tissue, whole tissue specimen samples, or extracted in vitro samples (Figure 2). This amplification process for many targets can be accomplished simultaneously, because the sequences are orthogonal and do not cross-hybridize with each other (Figure 2). Multiplexing amplification can be performed either in nonbarcoded seqFISH, where individual targets are imaged one at a time sequentially, or in barcoded seqFISH, where the color or pseudocolor assigned to each target is varied over multiple rounds of barcode hybridization. For example, 20 pseudocolors are imaged four times each (20 4 =160,000), more than 20,000 genes can be detected by barcoding.

[0137] We found good agreement between dots detected by LANTERN and conventional smFISH, and between two different LANTERN amplifiers (Figure 2, center and right, and Figure 3). In addition to RNA FISH, LANTERN can be used to amplify genomic DNA FISH signals (Figure 4) and protein signals from DNA-bound antibodies (Figure 5). Furthermore, we applied LANTERN to amplify RNA FISH signals in multiple tissues and cells, including mouse brain, human breast cancer biopsies, and whole tissue specimens chicken embryos (Figure 6). LANTERN is highly compatible with polyacrylamide hydrogel embedding protocols, which can be performed before or after embedding and clearing of samples.

[0138] Our method has the following advantages over existing amplification methods: Signals amplified by LANTERN can be stably visualized even after multiple hybridization and stripping of the readout probe because the primary, secondary, and tertiary probes are physically intertwined (Figure 1B). In contrast, signals from other amplification methods, such as branched DNA amplification, may decrease after several hybridization and stripping of the readout probe.

[0139] The level of amplification is determined by the number of repeats and the number of binding sites on the probe in successive rounds, unlike methods such as rolling circle amplification (RCA) and hybridization chain reaction (HCR), which are stochastic in nature.

[0140] Presumably due to the rigid nature of the dsDNA nanostructures, the amplified signal is highly stable over several rounds (Fig. 1). After correcting for imperfect stage movements by image alignment, we found that the centers of highly amplified RNA FISH dots could be located using Gaussian fitting with an average root-mean-square accuracy of ∼3 nm in X and Y directions over 12 rehybridizations, i.e., over a period of approximately 10 h in real time.

Claims

1. A composition comprising a plurality of probes for chain amplification coupled with exponential radiance, the composition comprising: (i) one or more primary probes, each capable of binding to one or more targets, the primary probes comprising one or more secondary probe binding sites and, optionally, one or more readout probe binding sites; (ii) one or more secondary probes, each capable of binding to said primary probe, said secondary probes comprising one or more tertiary probe binding sites or one or more readout probe binding sites; (iii) optionally, one or more tertiary probes, each capable of binding to said secondary probe, said tertiary probes comprising one or more quaternary probe binding sites or one or more readout probe binding sites; (iv) optionally, one or more quaternary probes, each capable of binding to said tertiary probe, said quaternary probes comprising one or more readout probe binding sites; (v) one or more readout probes capable of binding to and being detected by a readout probe binding site on one or more primary, secondary, tertiary, or quaternary probes; and (vi) one or more molecules that can subsequently stabilize one or more primary, secondary, tertiary, or quaternary probes when the probes hybridize; and A composition comprising:

2. A method for chain amplification coupled with exponential radiance, comprising the steps of: (i) contacting the sample with one or more primary probes that bind to one or more targets, such that each primary probe hybridizes to a target; (ii) hybridizing one or more secondary probes to the primary probes, each secondary probe comprising one or more tertiary probe binding sites or one or more readout binding sites; (iii) optionally hybridizing one or more tertiary probes to at least one secondary probe, each tertiary probe comprising one or more quaternary probe binding sites or one or more readout binding sites; (iv) optionally hybridizing one or more quaternary probes to at least one tertiary probe, each quaternary probe comprising one or more readout binding sites; and (v) stabilizing one or more of the primary, secondary, tertiary, or quaternary probes during or after steps (i)-(iv); (vi) hybridizing a detectable readout probe to the one or more readout probe binding sites; (vii) imaging the cells after step (vi) to detect the interaction of the primary probes with the nucleic acid; and (viii) optionally repeating the contacting and imaging steps each time with a new number of detectably labeled readout probes, at least one readout probe for one target differing in its detectable moiety from at least one other readout probe for the same target, such that targets in the sample are described by barcodes and are distinguishable from other targets in the sample by differences in their barcodes; A method comprising:

3. 3. The method of claim 2, wherein any of steps (i) to (vii) are repeated individually or in any combination thereof.

4. 3. The method of claim 2, comprising stabilizing the primary, secondary, tertiary, or quaternary probe.

5. 3. The method of claim 2, wherein the secondary, secondary and tertiary, secondary and tertiary and quaternary, secondary and quaternary, tertiary, tertiary and quaternary, or quaternary probe comprises at least two amplifier fragments.

6. 6. The method of claim 5, wherein the amplifier fragment of the secondary probe comprises at least: (a) a first amplifier fragment, the first amplifier fragment including a region of complementarity to the primary probe, the region of complementarity hybridizing to the primary probe; and (b) a second amplifier fragment, the second amplifier fragment comprising a region of complementarity to the primary probe, and the region of complementarity hybridizes to the primary probe; and A method comprising:

7. 6. The method of claim 5, wherein the amplifier fragment of the tertiary probe comprises at least: (a) a first amplifier fragment, the first amplifier fragment including a region of complementarity to a secondary probe or the first or second amplifier fragment of the secondary probe, and the region of complementarity hybridizes to the secondary probe or the first or second amplifier fragment of the secondary probe; and (b) a second amplifier fragment, the second amplifier fragment comprising a region of complementarity to the secondary probe or the first or second amplifier fragment of a secondary probe, and the region of complementarity hybridizes to the secondary probe or the first or second amplifier fragment of a secondary probe; A method comprising:

8. 6. The method of claim 5, wherein the amplifier fragment of the quaternary probe comprises at least: (a) a first amplifier fragment, the first amplifier fragment including a region of complementarity to a tertiary probe or the first or second amplifier fragment of the tertiary probe, and the region of complementarity hybridizes to the tertiary probe or the first or second amplifier fragment of the tertiary probe; and (b) a second amplifier fragment, the second amplifier fragment including a region of complementarity to the tertiary probe or the first or second amplifier fragment of the tertiary probe, and the region of complementarity hybridizes to the tertiary probe or the first or second amplifier fragment of the tertiary probe; A method comprising:

9. 3. The method of claim 2, A ligase ligates said first or second fragment to any secondary, tertiary, or quaternary amplifier fragment.

10. 3. The method of claim 2, the splint sequence hybridizes to the first amplifier fragment, the second amplifier fragment, or both of the secondary, tertiary, or quaternary amplifier fragments; and A method, wherein the splint sequence comprises at least two splint sequence fragments.

11. The method of claim 2, wherein the splint sequence fragments are ligated.

12. 3. The method of claim 2, wherein a readout probe hybridizes to either the first or second amplifier fragment of the secondary, tertiary, or quaternary probe.

13. The method of claim 2, wherein the readout probe hybridizes to a splint sequence fragment.

14. The method of claim 2, wherein the stabilization is selected from the group consisting of enzymatic ligation, chemical ligation, UV cross-linking with or without an oligo splint probe, hybridization of a splint probe, cross-linking via a matrix, and chemical cross-linking, and any combination thereof.

15. 3. The method of claim 2, wherein the target is selected from a transcript, RNA, a DNA locus, a chromosome, DNA, a protein, a lipid, a glycan, a cellular target, an organelle, and any combination thereof.

16. 16. The target of claim 15, wherein the target is bound to one or more oligonucleotide sequences.

17. The method of claim 2, wherein the primary, secondary, tertiary, or quaternary probes are stabilized by cis-ligation.

18. 3. The method of claim 2, wherein the primary, secondary, tertiary, or quaternary probes are stabilized by cis-ligation while the probes are in contact or hybridized.

19. 3. The method of claim 2, wherein each primary probe comprises a nucleic acid sequence complementary to a target nucleic acid sequence.

20. 3. The method of claim 2, wherein the primary, secondary, tertiary, or quaternary probes are cis-ligated by a reactive group on the probe, the reactive group being a reactive pair selected from an alkene, an alkyne, an azide, an amide, an amine, a nitrone, a phosphate, a tetrazine, and a tetrazole.

21. The method of claim 2 , wherein one or more of the readout probes comprises an oligonucleotide or an antibody having a detectable moiety.

22. The method of claim 2 , wherein the readout probes comprise oligonucleotides having identical sequences.

23. The method of claim 2 , wherein the readout probes comprise oligonucleotides having different sequences.

24. 3. The method of claim 2, further comprising repeating the contacting and imaging steps each time using a new plurality of detectably labeled readout probes, each new plurality of at least one readout probe for a target being different from at least one readout probe for the same target in a previous plurality, where they differ in at least a detectable moiety.

25. 3. The method of claim 2, wherein the primary, secondary, or tertiary probes are ligated or crosslinked in cis or trans.

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