Compositions and methods for molecular barcoding

Abstract

Provided herein are nucleic acid oligonucleotides (oligos), such as hairpin tag nucleic acid molecules and DNA / RNA hybrid hairpin tag nucleic acid molecules, as well as capture oligo (CO) nucleic acid molecules and DNA / RNA chimeric CO nucleic acid molecules; spatially encoded capture arrays; the use of the oligos in encoding and decoding features; and the use of the oligos and / or arrays with other provided descriptions, including in methods and workflows for spatial encoding and quantification of targets, such as biomarkers, in biological samples. TIFF2026508331000004.tif82134

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of earlier filing of U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listing A computer-readable text file entitled "O046-0080PCT.xml", created on or about February 28, 2024, with a file size of 63,808 bytes, contains the sequence listing of the present application, and is incorporated herein by reference in its entirety.

[0003] Field of Disclosure The present disclosure relates generally to methods and systems for analyzing targets such as biological samples and molecules, and more particularly to molecular barcode and spatially encoded analysis. [Background technology]

[0004] Background to the disclosure Determining the identity and / or location of target molecules (such as proteins or nucleic acids) in a sample can be essential for clinical applications, diagnostics, and biomedical research. In situ hybridization (ISH), immunohistochemistry, laser capture microdissection, and similar techniques allow visualization of the location of target molecules within a sample, such as a biological sample.

[0005] The identity of target molecules can also be determined using methods that label the target molecules and track them through the amplification and / or sequencing process (such as stochastic barcoding). However, there remains an ongoing need for methods and systems that reliably correlate the identity of target molecules with their location within a sample, such as a substantially two-dimensional (2D) biological sample.

[0006] Spatial biology platforms have revolutionized biological research and are rapidly becoming essential for understanding and treating cancer. However, currently available commercial platforms, whether for proteomic profiling or transcriptomic analysis, are expensive and unable to scale for widespread adoption. Current imaging-based approaches used for proteomic profiling can provide sharp detail but are time-consuming and limited in the number of protein targets that can be analyzed on a single tissue slide. While imaging-free systems enable simple data acquisition via next-generation sequencing (NGS) readout, these platforms struggle to achieve subcellular resolution, have high costs per assay (due to the need to purchase specialized equipment), and require multiple manual intervention steps. Summary of the Invention

[0007] Disclosure Overview Described herein is a scalable, low-cost, image-free spatial platform with subcellular resolution suitable for use in, for example, basic research and clinical laboratory environments. The described barcoded tags and isothermal workflow enable information transfer to spatially encoded arrays in a single, hands-free step. The workflow is non-destructive to the source material (e.g., fresh-frozen or fixed tissue samples) and can function without specialized equipment to generate digital "images" of the presence and abundance of components (e.g., proteins) at subcellular resolution. The described platform enables coordinate spatial and structural analysis of, for example, cellular proteomic profiles for research deployment and clinical laboratories. Also provided are spatial capture arrays for spatial encoding of information recovered from binding probes (e.g., antibody probes) conjugated with the tags disclosed herein, as well as isothermal spatial encoding workflows that provide decoding of the recovered information, for example, by next generation sequencing, enabling digital display of protein profiles in biological samples.

[0008] Provided herein are DNA / RNA hybrid tags along with workflows for spatial encoding, identification, and / or quantification of biomarkers in biological samples.

[0009] A first embodiment is a DNA / RNA hybrid hairpin tag having a structure shown or described herein. By way of example, the DNA / RNA hybrid hairpin tag can include (in 5'->3' order): an attachment moiety, a stretch of RNA bases, a tag PCR handle, a tag ID barcode, a tag PCR handle complementary sequence, and a stretch of DNA bases complementary to at least a portion of the stretch of RNA bases. For example, an example DNA / RNA hybrid hairpin tag has the structure shown in FIG. 1A or has the sequence of tag v1, tag v2, tag v3, tag v5, or tag v6 (SEQ ID NOs: 3-7, respectively).

[0010] Also provided are released hairpin tags derived from the DNA / RNA hybrid hairpin tags described herein, including, for example, those shown in Figure 1B.

[0011] An additional embodiment is a tagging element (structure, component, etc.), which comprises an element having attached thereto (through an attachment moiety) a DNA / RNA hybrid hairpin tag as shown or described herein. In an example of this embodiment, the element comprises one or more of a biological molecule (such as a protein or nucleic acid), a cell or tissue, an affinity molecule (such as an antibody), a bead, or another addressable feature.

[0012] Yet another embodiment is a capture oligo (CO) having a structure shown or described herein. For example, an exemplary CO includes (in 5'->3' order): an attachment moiety, a first stretch of DNA bases, a tag ID barcode, and a second stretch of DNA bases complementary to at least a portion of the first stretch of DNA bases. For example, the CO may have the structure shown in Figure 2 or Figure 3.

[0013] Any of these capture oligos attached to a capture feature through an attachment moiety are also provided.

[0014] In any embodiment of CO or any embodiment including CO, the CO may further comprise a conditionally cleavable element, optionally arranged to allow release of the CO (or a substantial portion of the CO) from the element or surface to which the CO is attached (e.g., by an attachment moiety).

[0015] Another embodiment is a capture element (structure, component, etc.), which comprises an element to which CO is attached (through an attachment moiety). By way of example, the element comprises a bead or another addressable capture feature.

[0016] It is contemplated that in several embodiments, the provided capture element is one capture element in an array of different capture elements, and that each CO in each of the multiple different capture elements in the array includes a different tag ID barcode.

[0017] Also provided are capture pairs (e.g., pairs of oligonucleotides) comprising a DNA / RNA hybrid hairpin tag having a structure shown or described herein, or a released hairpin tag derived from the DNA / RNA hybrid hairpin tag, and a capture oligo (CO) having a structure shown or described herein, wherein the sequences of the DNA / RNA hybrid hairpin tag and the CO are at least partially complementary such that the released hairpin tag is captured by the CO when the hairpin tag is released in the vicinity of the CO. Such capture pairs are shown herein, for example, in the figures and corresponding description, and in Appendix A included in U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes this application.

[0018] Another embodiment is a method for detecting and / or quantifying a target in a substantially two-dimensional (2D) sample, the method being as described or illustrated herein.The substantially 2D sample can be a thin tissue section or another slice of a biological sample (e.g., a long-term storage tissue slice, etc.), and a substantially 2D array of a biological sample (e.g., a cell extract or a synthetic mixture, etc., that can be applied to a slide or other surface).A substantially 2D sample can optionally be homogeneous (in terms of content, such as biological macromolecules) across its entire surface area, but in most cases it is heterogeneous, so that some targets are found only in some areas or with varying concentrations in different areas.Thus, the provided method embodiment allows for target localization within the 2D surface of the sample being analyzed.

[0019] Also provided are methods for the detection and / or quantification and / or localization of targets in substantially two-dimensional (2D) samples, which are "one-pot" methods (in which multiple chemical and / or enzymatic reactions occur simultaneously or sequentially) carried out at essentially a single temperature (i.e., isothermally).

[0020] Examples of these methods for detecting and / or quantifying and / or localizing targets in substantially two-dimensional (2D) samples are shown (in whole or in part) in Figures 4A, 4B, 5A, 5B, 6, 7A, 7B, 8, 9A-9E, 12, 13, or in Appendix A to U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes the present application.

[0021] In any of the methods provided, embodiments include the enzymatic activity of one or more of RNase H, DNA polymerase, reverse transcriptase, RNA polymerase, and / or one or more restriction enzymes. Optionally, these enzymatic activities may occur in a single container (a "one-pot" method) and / or at a single temperature (an isothermal method).

[0022] Any of the method embodiments provided may further include sequence analysis of a plurality of nucleic acid molecules that include one or more molecular ID tags.

[0023] Another embodiment is a spatially coded capture array substantially as described or illustrated herein. For example, such a spatially coded capture array can be as shown in FIG. 10 or FIG. 11 or in Appendix A of U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which is hereby incorporated by reference. Some embodiments of the spatially coded capture array are provided, which include capture elements embedded in a biomolecule-permeable matrix. In some examples, the capture elements include beads, the biomolecule-permeable matrix is ​​a gel, or both. Optionally, the spatially coded capture array is provided in the form of a flexible "sticker," which is intended to be used in direct contact with a substantially 2D sample, e.g., for analysis of targets within the sample. Examples of such analysis methods are described.

[0024] Yet another aspect is a spatial encoding workflow essentially as described or illustrated herein, examples of which are shown in Figures 12 or 13, or in Appendix A of U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes this application.

[0025] Spatially encoded feature arrays having a "grid within a grid" arrangement as shown in FIG. 10 and described herein are also described.

[0026] Additional aspects include a computer-readable medium or digital resource, or a digital database that contains spatial location information for features of the spatially encoded array described herein. For example, the computer-readable medium or digital resource, or digital database, in some instances, contains spatial location information for substantially all features of the spatially encoded array.

[0027] Also provided is the use of a computer-readable medium or digital resource, or a digital database, to provide a user with location information for one or more targets that correlates with the spatial location information of a spatially encoded array, e.g., in some cases, the correlation occurs through the use of a spatially encoded array in a workflow or method shown or described herein.

[0028] Another aspect is a method for isothermal spatial encoding of a biological sample substantially as described or shown herein.

[0029] The use of the DNA / RNA hybrid hairpin tags described or illustrated herein for transcriptome analysis of biological samples is also described.

[0030] Also described are spatially encoded surfaces such as capture arrays, e.g., bead-based capture arrays, substantially as described or shown herein.

[0031] Provided herein is a hairpin tag nucleic acid molecule comprising parts A, B, C, D, E, and E operably linked in 5' to 3' order, wherein part A is: (1) a stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site part B comprises a stretch of DNA bases comprising a tag PCR handle; part C comprises a stretch of DNA bases comprising a tag ID barcode, and said stretch of DNA bases forms part of the loop of the hairpin; part D comprises a stretch of DNA bases having the reverse complement of the tag PCR handle, thereby forming part of the stem of the hairpin; and part E comprises (1) a stretch of RNA bases in part A, or (2) the stretch of DNA bases containing the RE recognition site The hairpin comprises a stretch of DNA bases having the reverse complement of at least a portion of either of the following sequences, thereby forming part of the stem of the hairpin.

[0032] Also provided is a set of two or more of the described hairpin tag nucleic acid molecules and / or the described DNA / RNA hybrid hairpin tag nucleic acid molecules, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence. In an example of such a set, the set further includes at least one attenuation tag-like nucleic acid molecule, wherein the attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by the absence of a functional cleavable site. For example, provided is a set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules, wherein the attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by (1) having a DNA base instead of an RNA base in part A, or (2) lacking an RE recognition site in part A.

[0033] Also provided are tagged probes comprising a probe molecule attached to the described hairpin tag nucleic acid molecule or the described DNA / RNA hybrid hairpin tag nucleic acid molecule via the attachment moiety. By way of example, the probe molecule comprises an affinity molecule having binding affinity for a target molecule. Optionally, the affinity molecule comprises an antibody binding domain having affinity for an antigen, and the target molecule comprises the antigen.

[0034] Another embodiment is a released hairpin tag nucleic acid molecule derived from a described hairpin tag or derived from a described DNA / RNA hybrid hairpin tag, wherein the released hairpin tag is separated from the attachment moiety by the enzymatic action of a restriction endonuclease or RNase H enzyme.

[0035] Yet another embodiment is a capture oligo(CO) nucleic acid molecule comprising parts I-II-III-IV operably linked in 5'→3' order, wherein part I is: (1) a single RNA base or a continuous stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site part II comprises a stretch of DNA bases comprising a CO PCR handle; part III comprises a stretch of DNA bases comprising a spatial barcode; and part IV comprises a stretch of DNA bases comprising a tag capture region. Optionally, the CO nucleic acid molecule further comprises an attachment moiety conjugated to the 5' end of part I. For example, the attachment moiety, in some cases, provides amine-reactive crosslinker activity or thiol-reactive crosslinker activity.

[0036] Yet another embodiment is a set of two or more of the provided CO nucleic acid molecules and / or any of the provided DNA / RNA chimeric CO nucleic acid molecules, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

[0037] Yet another provided embodiment is a spatially encoded capture feature that includes a capture feature to which a provided CO nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule is attached via an attachment moiety.

[0038] Also described is a capture pair comprising a provided hairpin tag nucleic acid molecule, or a provided DNA / RNA hybrid hairpin tag nucleic acid molecule, or a provided released hairpin tag nucleic acid molecule; and a provided capture oligo(CO) nucleic acid molecule, or a provided DNA / RNA chimeric CO nucleic acid molecule, or a provided attached CO, wherein the sequences of the hairpin tag nucleic acid molecule and the CO nucleic acid molecule are at least partially complementary such that when the hairpin tag nucleic acid molecule is released from its attached moiety in the vicinity of the CO nucleic acid molecule, the released hairpin tag nucleic acid molecule is captured by sequence complementary bonding to the CO nucleic acid molecule at its 3' end, such that the resulting complex of the released hairpin tag nucleic acid molecule and the CO nucleic acid molecule is eligible for a downstream extension reaction by a polymerase enzyme.

[0039] Yet another aspect is a spatially encoded capture array comprising a defined array of spatially addressed capture features, each capture feature having: predefined, addressable locations on a substantially two-dimensional solid surface; or Beads or other similar discrete solid capture objects spatially distinguishable features, including: Multiple copies of the provided CO nucleic acid molecule or the provided DNA / RNA chimeric CO nucleic acid molecule attached at each feature. wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array.

[0040] Another embodiment is a semi-ordered spatially coded capture array that includes: A grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide comprising: an x-coordinate adaptor oligonucleotide used to label all spatially addressable locations within a row of the grid; and a y-coordinate adaptor oligonucleotide used to label all spatially addressable locations within a column of the grid. the grid being applied to the spatially addressable locations by splint ligation of

[0041] Also provided is a method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample, said method comprising: contacting the substantially 2D sample with at least one tagged probe to generate a substantially 2D stained sample, wherein the tagged probe a hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode, or a DNA / RNA hybrid hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode; and a probe molecule attached via an attachment moiety to the hairpin tag nucleic acid molecule containing a tag ID barcode or the DNA / RNA hybrid hairpin tag nucleic acid molecule containing a tag ID barcode a process comprising: contacting the surface of the substantially 2D stained sample with a transparent, spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: a plurality of spatially distinguishable features; and Multiple copies of capture oligo(CO) nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules attached at each spatially distinguishable feature wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array; placing a flow cell or other solution-containing cover over the sample-array sandwich to form an enclosure containing the sample-array sandwich; adding a solution containing reaction components to the enclosure to form a reaction mixture, the components comprising: a cleaving enzyme selected from RNAse H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ ions; and buffers a process comprising: incubating the sample-array sandwich contacted with the reaction mixture at an assay temperature for 30 to 60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the enclosure; and analyzing the reaction product mixture to detect and / or quantify and / or define the location of the target within the substantially 2D sample. Optionally, in such methods, the enclosure comprises a flow cell. Embodiments of the provided methods provide location information for more than one target within a substantially 2D sample.

[0042] Yet another aspect is a spatial encoding workflow that includes: contacting a hybrid RNA / DNA tag containing a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; placing a capture array containing capture features in contact with the stained sample to create a sample / array sandwich; placing a fluid containment enclosure on top of the sample / array sandwich; introducing an assay solution comprising active RNAse H and an active polymerase into the fluid containment enclosure, thereby contacting the assay solution with the sample / array sandwich; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient for RNAse H activity to at least partially digest the hybrid RNA / DNA tag to generate a cleaved tag, and thereby release the cleaved tag into the assay solution in the vicinity of the capture feature; allowing the cleavable tag to interact with a capture oligo(CO) on the adjacent capture feature to provide a captured cleavable tag; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient to allow polymerase activity to extend the captured cleavable tags using CO as a template to generate extension products; cleaving the extension product with RNAse H in the sample solution once it has been extended sufficiently to generate a complementary RNA / DNA region based on the RNA bases in the CO, thereby releasing a full-length extension product; recovering at least a portion of the released full-length extension products; and amplifying and / or sequencing at least one of the released full-length extension products.

[0043] Also provided are computer-readable media or digital resources and digital databases that contain the spatial location information of the features of the spatially coded arrays described herein. Also disclosed is the use of the provided computer-readable media or digital resources or digital databases to provide a user with location information for one or more targets that correlates with the spatial location information of the spatially coded arrays. As an example of the use of the provided computer-readable media or digital resources or digital databases, the correlation occurs through the use of the spatially coded arrays in the workflows or methods described or shown herein.

[0044] Yet another embodiment is a kit useful for practicing one of the methods provided herein. Exemplary kits include one or more of the following: two or more of the described hairpin tag nucleic acid molecules; two or more of the described DNA / RNA hybrid hairpin tag nucleic acid molecules; a described set of two or more hairpin tags; a set of two or more of the tagged probes described; two or more of the described capture oligo(CO) nucleic acid molecules; two or more of the described DNA / RNA chimeric nucleic acid molecules; a described set of two or more CO nucleic acid molecules; A described set of two or more DNA / RNA chimeric CO nucleic acid molecules; two or more CO nucleic acid molecules as described, each attached to a capture feature; at least one described spatially encoded capture array; At least one of the described semi-aligned spatially encoded capture arrays; or The spatially coded surface described.

[0045] Also, a vessel containing a functional RNAse H enzyme; a container containing a functional polymerase enzyme; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ a vessel containing a solution containing ions; or A container containing a buffer solution Also provided are kit embodiments further comprising one or more of: [Brief explanation of the drawings]

[0046] [Figure 1]Figure 1A: Hybrid hairpin tag structures shown with exemplary sequences. An exemplary nucleic acid tag (Tag1 v6; SEQ ID NO: 7) is shown, incorporating both RNA and DNA bases within a hairpin (stem and loop) structure. The 5' end of the tag includes a flexible linker attached to a molecule (X in the drawing), which can bind to a target immobilized on a surface. From 5' to 3', the linker is followed by a stretch of RNA bases, a downstream constant PCR handle (spanning the set of hybrid hairpin tags), one to three G bases used to space a primer landing site from the unique tag barcode, and the unique tag barcode (unique within the set of hybrid hairpin tags) located within the loop of the hairpin, followed by a 3' region complementary to (i.e., the reverse complement of) the 5' region of the hairpin tag. In the resulting hairpin tag, the RNA and DNA bases of the tag create a DNA / RNA hybrid region (part of the hairpin stem) that is cleavable by RNase H. Figure 1B: The tag is released from the molecule (X) upon cleavage by RNAse H. Digestion of the RNA region of the tag with RNAse H (e.g., to generate SEQ ID NO: 8) exposes a region of ssDNA bases within the hairpin stem designed for capture onto the capture oligo (CO). [Figure 2] Capture of a cleaved hairpin tag by a capture oligo (CO) on a capture bead. The 3' end of the capture oligo (an oligonucleotide designed for capture of the released hairpin tag) includes a capture region complementary to the exposed DNA bases on the released tag after RNase H digestion (see Figures 1A-1B). The sequence of an exemplary capture region attached to the captured hairpin tag (SEQ ID NO: 6) is shown (italicized in the figure). In some embodiments, the capture oligo is attached at its 5' end to a capture feature (shown here with a bead). [Figure 3]Capture beads with capture oligo (CO) structures. An exemplary single-stranded CO structure is shown, comprising a flexible linker at the 5' end for attachment to a capture feature (shown here with a bead), followed by a 5' PCR handle, a spatial barcode, and a 3' capture region. The CO may also comprise a unique molecular identifier (UMI), e.g., as a series of randomized bases or interspersed throughout the spatial barcode region. As described herein, the attachment of the capture oligo to the capture feature (shown here with a bead) can be conditionally cleaved, e.g., by including a photocleavable or chemically cleavable linker, or by including an RNA base or half of a dsDNA restriction enzyme site. The 2D location of each capture feature is predetermined (e.g., within an array), and this information is recorded in the spatial barcode region of the CO. All COs within / located at a capture feature share a unique spatial barcode corresponding to the location of the capture feature to which they are attached (e.g., a bead, an addressable region of a solid surface, etc.). [Figure 4A] Hairpin tag release near capture features. A pool of molecules (e.g., a probe panel or library) is incubated with a sample immobilized on a surface, where each unique molecule in the pool is encoded with a specific molecular-identifying DNA barcode sequence (in the tag loop region). The sample is then washed to remove all unbound and non-specifically bound molecules. Next, one or more enzymes collectively possessing RNase H and polymerase activity are introduced into a common buffer along with appropriate cofactors (dNTPs, Mg2+, etc.). This releases the tag (molecular ID, "released tag," "hairpin tag") from the target-bound molecule (shown as an X in the diagram) on the surface. This release is carried out near CO attached to a capture feature (shown as a bead). [Figure 4B]Hairpin tag capture and extension. In an isothermal "one-pot" reaction, a hairpin tag is released from the sample surface and captured on a nearby CO (based on the sequence of the capture region of the CO and the proximity of the CO to where the tag was released), and the 3' end of the captured tag is extended by an introduced polymerase. In some embodiments, the 3' end of the CO is blocked. Since the melting temperature of the interaction (between the captured hairpin tag and the capture sequence of the capture oligo) is significantly increased by the extension reaction, including DNA polymerase in the one-pot reaction prevents the tag from "hopping" to other nearby capture features. Such hopping, if it occurs, can contribute to a loss of positional fidelity in the analysis (e.g., despite increased diffusion). [Figure 5A] Alternative Constructs for Compatibility. Alternative tags can be constructed for compatibility with the described isothermal one-pot reaction. As shown in this figure, compatible cleavable tags can be created simply by changing the base composition of the tag to include RNA bases between the linker region and the molecular ID region of the tag. In this way, other tag types can be used in the same isothermal one-pot reaction workflow as described in Figure 4B. [Figure 5B] Alternative tag capture and extension. Alternatively designed tags, as outlined in Figure 5A, can be captured on a CO adjacent to a capture feature (e.g., a bead) and extended by a polymerase included in an isothermal one-pot reaction. When capturing alternatively designed tags, such as hairpin oligo capture (shown in Figures 4A-4B), the 3' end of the CO can be blocked or extended during the one-pot reaction. [Figure 6]Alternative tag designs and isothermal amplification for compatibility. Other types of tag designs are also contemplated for use in similar isothermal one-pot reactions. As shown, promoter regions recognized by RNA polymerase (e.g., T7 polymerase) can be designed for incorporation into the tag sequence at one or both ends of the tag. A dsDNA tag with two molecules (X and Y) is shown, each molecule coded (labeled, identified) in a different strand of the dsDNA tag. Here, opposing T7 promoter regions are incorporated to generate amplifiable templates from both strands of the tag. These probes can be bound to a sample immobilized on a surface, and the surface is then washed to remove unbound probes, and the washed surface is exposed to a solution containing T7 polymerase with appropriate cofactors for RNA transcription. This reaction can be carried out in an isothermal one-pot reaction containing reverse transcription (RT) enzyme with appropriate cofactors for DNA elongation, near a capture feature containing CO (e.g., as described herein). [Figure 7A]Conversion of ssDNA Tags for Compatibility and Isothermal Amplification. Tags from existing DNA tagged molecules can be converted for compatibility with the isothermal signaling methods described herein through the incorporation of a promoter region recognized by an RNA polymerase (e.g., T7 polymerase). A commonly used ssDNA tag structure is shown to include two constant regions (often used as PCR handles) flanking a molecule (X) encoding (labeling, identifying) sequence (molecular ID) attached via a linker at the end of the tag (shown here as 5'). This adapter can be incorporated before or after the tagged probe is incubated with the target sample (including the target molecule). Similarly, an extension reaction to create a dsDNA tag by extension of the 3' end of the adapter can be performed before or after the probe is incubated with the sample. After probe binding, unbound probe is removed by washing, and the probe-bound surface is exposed to a solution containing T7 RNA polymerase and RT enzyme along with appropriate cofactors. This transcription reaction can be carried out in an isothermal one-pot reaction that includes a reverse transcriptase (RT) enzyme along with appropriate cofactors for DNA extension, in the vicinity of a capture feature that includes CO. [Figure 7B]Conversion of dsDNA Tags for Compatibility and Isothermal Amplification. Tags on existing DNA-tagged molecules can be converted for compatibility with isothermal signaling through the incorporation of a promoter region recognized by an RNA polymerase (e.g., T7 polymerase). A commonly used dsDNA tag structure is shown to include two constant regions (often used as PCR handles) flanking a molecule (X) encoding (labeling, identifying) sequence (molecule ID) attached through a linker at the end of the tag (shown here as 5'). This adapter can be incorporated before or after the tagged probe is incubated with the sample. After probe binding, unbound probe is removed by washing, and the probe-bound surface is exposed to a solution containing T7 RNA polymerase and RT enzyme with appropriate cofactors. This transcription reaction can be performed in an isothermal one-pot reaction containing the RT enzyme with appropriate cofactors for DNA elongation, near a capture feature containing CO2. [Figure 8] Capture of RNA generated from tag templates. As transcribed RNAs are released into solution, they are captured onto the 3' ends of capture oligos attached to nearby capture features (represented by beads), and the 3' ends are immediately extended by an RT enzyme present in the same reaction mixture. Once the RNA strands are copied onto the 3' ends of the capture oligos via the extension reaction, they are destroyed by RNase H activity (either incorporated within the RT enzyme or by including an enzyme that incorporates RNase H activity, such as E. coli RNase H, in the reaction mixture). The resulting extension products incorporate spatial barcodes, tag information, and spatial information provided by the 3'- and 5'-PCR handles for downstream amplification. Spatially encoded products can be released from the capture features in a variety of ways, as outlined in Figures 9A-9E. [Figure 9A]Release of double-stranded products from capture features by RNase H. Following extension of the 3' end of the tag on the capture oligo to generate spatially encoded dsDNA products, these spatially encoded products can be released into the surrounding solution in the same isothermal one-pot signaling reaction workflow. Here, RNA bases are included at the 5' end of the capture oligo so that after extension by a DNA polymerase (specifically, an RNA- and DNA-dependent DNA polymerase; e.g., Maxima™, available from Thermo Scientific), a DNA / RNA hybrid region is generated, which becomes a substrate for RNase H activity in the reaction. These products are cleaved from the capture feature by RNase H and released into the reaction solution. [Figure 9B] Release of double-stranded products from capture features by endonucleases. An alternative method for releasing spatially encoded dsDNA extension products from capture features is to include half of a dsDNA restriction enzyme (RE) site at the 5' end of the capture oligo. Following extension of the 3' end of the tag on the capture oligo, a fully formed dsDNA RE site can be created. The cognate RE can be introduced during the isothermal signal transduction reaction or after the reaction is complete. Upon introduction, the RE cleaves the spatially encoded dsDNA products from the capture feature, releasing them into the reaction solution. [Figure 9C] Release of double-stranded products from capture features: photo- or chemical. An alternative approach for releasing spatially encoded dsDNA extension products from capture features is to include a photo- or chemically cleavable moiety in the linker that attaches the capture oligo to the capture feature. Here, the cleavage agent is introduced after the one-pot isothermal signal transduction reaction is complete. This method can be combined with other methods for dsDNA capture oligo release (e.g., via RNase H or RE activity) at known percentages for various specific applications. [Figure 9D]Release of single-stranded products from capture features; photo- or chemical. Following capture and extension of the transcript, spatially encoded single-stranded DNA products are generated. These products can be released from the capture features by including a photocleavable or chemically cleavable moiety in the linker that attaches the capture oligo to the capture feature. Here, the cleavage agent is introduced after the one-pot isothermal signal transduction reaction is complete. This method can be combined with other methods for ssDNA capture oligo release (e.g., via RNase H or RE activity after conversion to dsDNA, as outlined below) at known percentages for various specific applications. [Figure 9E] Conversion of single-stranded products for release from capture features. Following capture and extension of the transcripts, spatially encoded single-stranded DNA products are generated. Stationary primers can anneal to the 3' ends of these oligos and be extended by DNA polymerase, converting them into spatially encoded dsDNA products on the capture features. Inclusion of an RNA base or half of a dsDNA RE site at the 5' end of the capture oligo can enable their conversion to dsDNA following enzymatic release of these products from the capture feature. Following extension of the 3' end of the tag on the capture oligo, a fully formed dsDNA RE site can be generated, or a DNA / RNA hybrid region can be generated. Exposure to the appropriate enzyme releases the product into the reaction solution. [Figure 10]Exemplary Spatially Encoded Bead Arrays. One major problem with using capture arrays composed of small (1-10 μm), tightly packed capture beads (or monodisperse beads at high density) is the number of different beads that must be used to create a large surface area capture array. For example, to construct a 1.75 cm x 1.75 cm capture array composed of tightly packed 1 μm beads, approximately 10 B (1 x 10 10 ) individual 1 μm beads are required to completely cover the surface area. Furthermore, there may be very little (ideally none) redundancy in bead spatial barcodes across the capture array, which in this example would require a capture bead library size of at least 10 16 different beads to reduce the probability of spatial barcode redundancy to approximately 1 in 10,000. Furthermore, bead libraries designed for the visual encoding and decoding methods described here (e.g., orthogonal cleavage sequencing (OCS) described in WO 2022 / 187719, as well as other libraries constructed using split-and-pool approaches) are not ideally designed to reach such high levels of diversity. Co-construction of visual and NGS barcodes as described here requires the use of constant bases for splint ligation of each section (e.g., as described in Kershaw & O'Keiffe, Methods Mol Biol. 941:257-269, 2012), which increases the length of the NGS barcode with each round of splitting and pooling (as the diversity in the library increases). Typical applications of this technology may utilize large spatially encoded arrays (e.g., >1 cm × 1 cm, and up to 10 cm × 10 cm), and therefore this issue needs to be addressed. To reduce the number of capture beads required to create a large capture array surface, random bead arrays can be patterned into a grid. The grid structure allows for sub-sectioning of the random bead array into smaller features (35-100 μm features shown).Using 35 μm grid features and 1 μm diameter beads, each grid feature would contain approximately 1,250 individual beads. Similarly, a 100 μm grid feature containing densely packed 3 μm beads would have approximately 1,000 individual beads within each feature. After bead attachment to the surface (grid loading), each feature of the grid receives a unique spatial address. To spatially address each feature of the grid, x and y coordinate adapters are added to the grid, for example, by droplet printing. Briefly, x coordinate adapters (one unique adapter sequence per grid column) are deposited laterally across the bead array by droplet printing onto the grid features, and these adapters are contained within a solution containing a ligase enzyme and a constant nucleic acid splint for splint ligation to the constant regions of the NGS barcodes attached to the beads. Similarly, y-coordinate adapters (one unique adapter sequence per grid row) are deposited longitudinally across the bead array by droplet printing onto the grid features. These adapters are contained within a solution containing a ligase enzyme and a constant nucleic acid splint for splint ligation to the constant region of the x-coordinate adapter already ligated to the NGS barcode attached to the bead. The y-coordinate adapter also contains a 3' capture region for capture of probe tags released from the sample. This allows for unique addressing of each feature of the grid, and this process can be scaled to create ultra-large capture arrays containing densely packed or monodisperse 1 μm beads at high densities. To avoid significant redundancy of spatial barcodes across the array in this configuration, a bead library diversity of around one million can be used even when creating capture array surface areas of 10 cm x 10 cm or greater. [Figure 11]Figure 11A: Bead capture array; capture beads embedded in a polymer-permeable material (e.g., gel). After reading the bead positions within the capture array grid (e.g., using OCS or another method), the position of each bead within the grid is recorded, e.g., in a software package. The NGS capture oligos on the beads contain information operatively linked to a visual barcode sequence, as well as x and y coordinate adapters (see Figure 10) used (in embodiments) for positional addressing via droplet printing. The beads can then be embedded within a gel or other substance that is permeable to the polymer but has sufficient stiffness to confine the beads within the matrix, followed by casting or curing. After embedding the beads, the matrix containing the beads can be removed from the semi-patterned bead array, and the product can be used as a spatially differentiated capture array. By way of example, the thickness of the "sticker" (cast gel / matrix) in various embodiments is 100 μm to 250 μm, or as thin as 50 μm (e.g., if cast). In the described embodiment, the gel is a 0.5% to 2% agarose gel cast using 10 mM Tris pH 7.5 buffer in which high-melting-temperature agarose powder (e.g., Thermo Scientific, e.g., 16500100) has been melted. Other contemplated permeable matrices include polyacrylamide (e.g., 4% polyacrylamide, which has been shown to allow adequate diffusion of macromolecules) or hydrogels of different compositions. Optionally, the permeable matrix (gel) may include functionalization for attachment of immobilization features (such as beads) within the matrix (gel). Figure 11B: Capture oligo information copied and retained within the gel. To generate many capture array gels from a single bead array, extension products made as copies of NGS capture oligos can be retained within the cast gel. Briefly, the NGS capture oligos on the beads are organized in a reverse-complementary orientation, with the sequence encoding the 3' capture region oriented closest to the bead and the 5' PCR handle oriented furthest from the bead.These reverse-complement NGS barcodes can be primed using a common primer that anneals to the 5' constant region on all capture oligos, and this primer can be extended by DNA polymerase to copy the NGS capture oligo information (spatial barcodes). The common primer used for DNA priming and extension contains a moiety for polymerization in a polymer-permeable material such as a gel (e.g., using a 5' acrydite-modified primer for polymerization in a polyacrylamide gel). Following the extension reaction, an appropriate non-polymerized (or non-hardened) gel can be cast over the beads to copolymerize or otherwise incorporate the 5' chemical moiety on the extension product into the gel. The gel now contains precisely oriented copies of the NGS capture oligos assembled in their reverse complements, preserving their relative positions. The gel can then be removed without the beads, and the semi-patterned bead array can be used to cast more gels by repeating the annealing, extension, and gel-casting procedures. [Figure 12]Exemplary spatial coding workflow. Isothermal signal transduction reactions can be performed in close proximity (e.g., in direct contact) with capture beads embedded in a polymeric permeable material such as a gel. The use of a polymeric permeable material such as a gel for the capture array allows for the introduction of spatially coded components (enzymes and cofactors) and the release of spatially coded products by diffusion directly through the material (gel). As shown, an anti-Her2 antibody probe conjugated to tag v6 (SEQ ID NO: 7; shown in Figure 1A) is used to probe Her2+ cells (SKBR3 cells) immobilized on a glass slide. Following probe incubation, the slide was washed, and a thin layer of a solution (approximately 20 μl) containing DNA polymerase (Maxima RT) with dNTPs in 1× RT buffer was placed on the sample, after which a capture bead array gel ("sticker") was "sticked" (placed, overlapped) on the sample (i.e., placed very close to, e.g., directly in contact with, the sample). In this example, the capture oligo contained RNA bases within its 5' region. Next, a solution (approximately 40 μl) containing dNTPs (to avoid dilution of these necessary low-molecular-weight cofactors) and 1× RT buffer was placed on the back of the gel (i.e., the side away from the biological sample). Following a 20-minute incubation at 40°C, this solution was removed and maintained as a negative control. After removing the negative control solution, a solution containing E. coli RNase H and dNTPs (to avoid dilution of these necessary low-molecular-weight cofactors) in 1× buffer was placed on the back of the gel matrix. Following a 20-minute incubation at 40°C, this solution was removed and used along with a negative control sample in a qPCR experiment to quantify the level of extension product present in each solution. The graph shows that approximately 130-fold more extension product was recovered in the RNase H+ sample relative to the negative control (RNase H-) sample.This suggests that the enzyme required to initiate the isothermal signaling reaction, in this case RNase H, can diffuse throughout / through the gel to the sample surface, where it releases the information encapsulated in the probe tag for capture onto the capture beads, allowing the DNA polymerase enzyme to extend the 3' end of the captured tag for extension through the RNA bases of the capture oligo, allowing the release of the extension product through the gel. The released product can then be recovered by removing the solution from the back of the gel and analyzed. [Figure 13]Description of a Simple Spatial Encoding Device and Workflow. The capabilities described herein, including the ability to perform isothermal signaling reactions and release extension products from capture beads into solution, enable a simple and easy spatial encoding workflow, outlined in FIG. 13. Following addition of probes to a sample immobilized on a surface (shown here as a tissue section on a slide), the sample surface is washed to remove unbound probes, and a polymer-permeable, spatially encoded capture array (gel sticker) is placed over the sample. Optionally, a flow cell-like cover can be placed over the encoded surface. This type of device can apply gentle pressure to the encoded surface to ensure and / or increase proximity between the capture features (in the polymer-permeable, spatially encoded capture array) and the sample. Once assembled, the device allows solution to flow over the capture array placed on the tissue / sample, allowing one or more spatially encoded reaction components (enzymes and / or cofactors necessary for one-pot isothermal transduction and release of spatially encoded products into solution, as exemplified herein) to be introduced throughout the polymeric permeable gel, and the spatially encoded products to be released through the gel and into the solution contained within the flow cell. Following incubation at an isothermal reaction temperature (e.g., 37-42°C), the spatially encoded products within the assay solution are recovered (e.g., by aspiration), and the spatially encoded products contained within the recovered solution are analyzed. In some embodiments, this is followed by amplification (e.g., by PCR) for next-generation sequencing library preparation. [Figure 14]"One-pot" isothermal reaction. This figure expands on FIG. 12 and illustrates aspects of this assay embodiment. Consistent with the description in FIG. 13, a tissue sample (e.g., a fresh-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue section on a glass slide or other surface) is stained with a conjugated antibody probe (i.e., an antibody conjugated with a DNA / RNA hybrid hairpin tag), and then a capture array (comprising spatially defined capture features with attached capture oligos—each of which contains a 5′-PCR handle, a spatial barcode, a UMI, and a probe capture region; four capture features are shown) is placed over the stained tissue. A flow cell is placed over the tissue / array sandwich, and an assay solution (comprising RNAse H and polymerase) is introduced. The action of the RNase H enzyme releases the hairpin tag barcodes from the bound probes. The released tags diffuse out of the tissue and (A) interact with spatially encoded array features (i.e., the released hairpin tag barcode is captured on the barcoded capture oligo with the nearest spatially defined feature), where (B) they are extended by polymerase in the assay solution, thereby copying the spatially encoded information (i.e., the polymerase extends the 3' end of each probe, thereby copying the capture oligo's UMI, spatial barcode, and 5'-PCR handle). Upon completion of extension, complementary RNA / DNA regions are created, which are then (C) cleaved by RNAse H in the assay solution (isothermal release of probes with template oligos from spatially defined capture features by RNAse H enables PCR amplicon-based Illumina library preparation). The released, fully extended tags can then diffuse through a permeable gel matrix and be recovered for further analysis, such as amplification and NGS (see graph in Figure 12, showing that no cleavage release occurs without RNAse H). [Figure 15]Figure 15A: Anti-Her2 antibody with an HPv6 tag was applied to MCF7 cells (Her2-negative). This was done to test the issue of background staining of hairpin-tagged antibodies. To assess background labeling, the Her2-HPv6 antibody was visualized with an anti-mouse Alexa 555 secondary antibody. Three separate blocking / washing conditions (DMSO, Denhardt's solution, salmon sperm DNA) were tested for their ability to remove background staining. Images were quantified in Volocity® imaging software, and data were imported into GraphPad Prism for graph generation. (**=p<0.001). Figure 15B: Experimental conditions, quantification, and graphing were performed as described for Figure 15A. In this experiment, anti-ER-HPv6 antibody was tested on SKBR3 cells (ER-negative) to evaluate background staining and blocking methods. [Figure 16A] Figures 16A-16B. Illustration of all 256 loop barcodes. Figure 16A: A graph was generated from 1 million (randomly selected) reads from 26,000,000 paired-end (PE) reads aligned to a reference genome library; the percentage of representation for each barcode was then plotted. This was performed four times, each time using 1 million randomly selected reads, to allow for the generation of error calculations. The percentage of reads obtained for each of the 256 barcodes was then plotted using GraphPad Prism. The corresponding raw data for the reads is provided in Appendix B of Priority Application No. 63 / 487,575, dated February 28, 2023. [Figure 16B] Figures 16A-16B. Illustration of all 256 loop barcodes. Figure 16B: The logo shown was created by uploading 10,000 reads (successfully aligned to the reference genome) to Weblogo (available online at weblogo.berkeley.edu / logo.cgi). [Figure 17A]17A-17C. Expanding the spatially encoded capture array surface area using a dual coordinate oligo system: Further expansion of the spatially encoded array surface area can be achieved through droplet printing of two barcode types in different combinations. In FIG. 17A, a grid pattern of subarrays is shown outlining the physical map of different combinations of oligos (x and y coordinate oligos are shown here), which can optionally be droplet printed directly onto the subarray features, so that each subarray contains multiple capture features (e.g., beads, whereby each bead is coated with a unique capture oligo (CO)). An exemplary subarray feature size is 100 μm × 100 μm, which contains approximately 1,000 3 μm beads or approximately 10,000 1 μm beads, tightly packed together but randomly positioned within the subarray. In all subarrays, each bead contains multiple copies of a unique visual barcode that is functionally linked to a DNA barcode sequence (VBC) contained within the corresponding attached capture oligo nucleic acid, which also appears in multiple copies. The region of the capture oligo corresponding to the unique functionally linked visual barcode is referred to as the VBC in the figure; this region can be further spatially addressed by droplet printing of a unique first oligo barcode (shown here as the x-coordinate barcode oligo (x-BC)) that is ligated to the VBC using a printing oligo. This process is further illustrated in Figure 17B, which shows the addition of two different x-BC oligos (x1 and x2 oligos) by droplet printing onto two adjacent subarrays containing bead capture features that only have the VBC region of the capture oligo. This step is ideally carried out in the presence of an RNA splinting oligo (which may contain a single DNA base at both the 3' and 5' ends for improved stability) and the Splint® ligase enzyme (available from NEB M0375S), which recognizes RNA / DNA hybrids for ligation of adjacent DNA strands templated by the RNA oligonucleotide.After a washing step, a second spatial coordinate oligo is droplet-printed onto each subarray for ligation to the first spatial coordinate oligo. Figure 17C shows a second oligo type, y-coordinate barcode (y-BC) oligo, which is added to each subarray perpendicular to the direction in which the x-coordinate oligo was added, and also involves ligation for attachment. This creates a unique combination of two different types of coordinated oligos that can be added and ligated to VBC oligos to create a scalable surface area of ​​spatially addressed features for creating large capture arrays. Here, the y-BC oligo also contains a capture region for capturing nucleic acids and / or tag oligos from the stained sample. [Figure 17B] See the legend to Figure 17A. [Figure 17C] See the legend to Figure 17A. DETAILED DESCRIPTION OF THE INVENTION

[0047] Array references The nucleic acid and / or amino acid sequences described herein are shown using standard letter abbreviations as defined in 37 CFR §1.822. While only one strand of each nucleic acid sequence is shown, it should be understood that the complementary strand is included where appropriate. All oligonucleotides are shown in the 5'→3' direction. Legend: 5AmMC6 = 5' flexible 6 carbon atom linker with a reactive primary amine (IDT: / 5AmMC6 / ); 5AmMC12 = 5' flexible 12 carbon atom linker with a reactive primary amine (IDT: / 5AmMC12 / ); 5Phos = 5' phosphorylation added, e.g., to allow the oligo to be a substrate for T4 DNA ligase; 3InvdT = 3' inverted dT modification, resulting in a 3'-3' linkage that inhibits both 3' exonuclease degradation and DNA polymerase extension; 5BioTEG = 5' modification containing biotin and a triethylene glycol (TEG) spacer that increases the oligo-biotin distance to 15 atoms; 5Biosg = 5' biotin added so that the oligo can be captured by binding to avidin; "r" indicates that the subsequent base is RNA rather than DNA; + indicates that the subsequent base is a locked nucleic acid (LNA) base.

[0048] In the sequence listing: TIFF2026508331000002.tif173164TIFF2026508331000003.tif191164

[0049] Detailed Description The hybrid tag structure disclosed herein encompasses both DNA and RNA bases and can be attached to solid supports or various polymeric probes, enabling their molecular encoding (identification, labeling). The tag is designed as a hairpin, so that stretches of DNA bases complementary to the RNA bases in the tag are held in close proximity to create a stable RNA / DNA hybrid region; the RNA bases in the tag are flanked on both sides by DNA bases. These aspects protect the RNA bases in the tag from degradation (primarily by ribonucleases lacking RNAse H activity). This structure allows the tag to be released from its attachment site upon introduction of RNAse H. Furthermore, the hairpin creates a self-contained coding element that can be used without the need for hybridization of a recognition strand, which is currently required for DNAPaint workflows and other cyclic immunofluorescence (IF) and digital encoding workflows that use nucleic acid-tagged probes.

[0050] The exemplary tag designs and experimental examples presented herein relate to the use of hybrid hairpin tags to encode antibody probes that can be used, for example, in workflows for the detection and quantification of protein targets in tissue sections or other substantially two-dimensional (2D) sample configurations. This workflow also uses previously described visual molecular barcode types that allow for the identification of many different features (beads) immobilized on a surface (e.g., as published in WO 2022 / 187719) and other methods for providing visual molecular barcodes. Following visual identification, the location of each feature (e.g., each bead) can be determined and recorded (mapped and reliably addressed), and the surface containing the addressed (or mapped) features can be used for spatially resolved assays. In embodiments, the visual barcode attached to each feature corresponds to (operably linked to) sequence information within capture oligos that are retained throughout the next generation sequencing (NGS) workflow: each bead (or other addressable immobilized feature) contains at least one visual barcode and at least one corresponding capture oligo that is unique to that bead.

[0051] In some embodiments, more than one unique visual barcode corresponds to more than one unique capture oligo.

[0052] In the assays described herein, a tissue sample (e.g., for a standard IHC experiment) is prepared and exposed to a cocktail or panel of antibody-DNA / RNA hybrid tag-conjugated probes, washed to remove unbound probes, and incubated in a solution compatible with downstream processing (transfer buffer). A surface containing spatially resolved beads (or other features) is created, mapped, and coated at low temperature (below room temperature, e.g., on ice) with a solution containing (in a typical embodiment) transfer buffer, RNAse H, and DNA polymerase. The spatially resolved feature (e.g., bead) array is then placed "bead-side down" so that the features (beads) are placed in direct contact with the (tissue) sample. By way of example, the (tissue) sample is mounted on / supported by a solid surface, e.g., a standard microscope slide. The sandwich (bead array / tissue slide) is then incubated at elevated temperature (e.g., room temperature to 42°C) for isothermal transfer of cleaved tags (in an exemplary embodiment, by RNAse H) from the probes bound to the tissue sample onto spatially coded capture oligos immobilized on features (e.g., beads) of the spatially coded (bead) array. In this manner, hybrid DNA / RNA hairpin tags can be released from the tissue-bound probes and captured directly onto the bead / feature nearest to each released tag.

[0053] In some embodiments, DNA polymerase is also present in the isothermal cleavage / capture solution, so that once captured on the nearest bead in the array, each tag has its 3' end extended, effectively copying spatial information from the bead to the tag via DNA extension. The 3' end of the capture oligo is blocked and therefore unextendable. This exemplary "touchdown" extension reaction is expected to stabilize tag capture and increase assay resolution by reducing lateral diffusion of released tags. This extension step may solve or significantly reduce the problem of tag "hopping" to or from nearby beads (capture features) after release from the probe. This is because, during extension, the length of the newly synthesized DNA / DNA duplex interaction increases, and therefore the melting temperature (Tm) of the interaction (between the tag oligo and the capture oligo) increases with each base added to the 3' end of each captured tag during the extension reaction.

[0054] Contained within the released tag (from 5' to 3') are: (1) a sequence that acts as a 3' constant PCR handle; (2) a short sequence corresponding to the probe type (in this example, a barcode that identifies the antibody); and (3) a constant capturable region that becomes exposed as single-stranded DNA after RNAse H digestion. The capture oligo on each bead of the spatially encoded feature (bead) array is attached (immobilized) by its 5' end to the immobilization feature (bead) through standard linker attachment chemistry (here, biotin-streptavidin) and contains (from 5' to 3' in multiple embodiments): (1) a short stretch of three DNA bases; (2) eight RNA bases (although, as described herein, this can be varied); (3) a constant 5' PCR handle; (4) a spatial barcode corresponding to the visual barcode on the bead; (5) optionally, a stretch of randomized bases that creates a unique molecular identifier (UMI); and (6) a 3' constant capture region for capture of the released tag.

[0055] In the example, a unique molecular identifier (UMI), a bead-specific position-identifying barcode region, and a 5' constant PCR handle are copied onto the probe by an extension reaction to create a continuous sequence encompassing both the 3' and 5' constant PCR handles. "Constant" in all cases herein means that the region / element is common to all probes and capture oligos in the assay.

[0056] Because the capture oligo also contains an RNA base at its 5' end, after the polymerase extends through the DNA-templated base of the capture oligo, it extends through the RNA base of the capture oligo and thereby creates a DNA / RNA hybrid species. Because RNAse H is also present in the isothermal transfer reaction solution, this newly synthesized DNA / RNA hybrid is cleaved by RNAse H, thereby releasing the capture oligo from the immobilization feature (bead). After release, the released capture oligo remains hybridized to the extended full-length tag.

[0057] This overall process involves 1) isothermal cleavage of tags from probes bound to the tissue / sample being analyzed; 2) capture of the released tags onto a spatially coded array, such as a spatially coded bead array; 3) extension of spatial coordinates onto the tags; and 4) release of the spatially coded full-length tags, including both 5' and 3' PCR handles (constant regions), for PCR-based amplification and recovery of only the full-length tags using a set of common primers. The amplification primers can include, for example, Illumina flow cell adapter sequences, making the spatially coded biomarker assay resolvable and quantitative by next-generation sequencing. More generally, step 3) can be considered a step of combining tag information and spatial barcode information via an extension reaction, which includes additional aspects beyond those identified above. An option can be included to block the 3' end of the tag, which is not extended during the extension step; only the CO is extended. In such embodiments, the extension product release step is a separate step. This is because the method relies on the presence of a second RNA / DNA hybrid created during the extension step for product release via a one-pot reaction.

[0058] Methods for controlling or influencing the direction of the extension step include: 1) blocking the 3' end of the capture oligo to prevent CO extension (see, e.g., the description in Figure 4B), thereby allowing tag-only extension (as shown herein); or 2) blocking the 3' end of the tag so that it is not extendable when released as a hairpin, which may allow CO-only extension. Regarding the product release step shown, the method relies on the generation of extension products that create DNA / RNA hybrid regions by incorporating RNA bases into CO, and therefore, optionally, the release step is not part of an isothermal reaction. One option described herein is to photorelease these, as shown herein.

[0059] In the RNA capture embodiment (with amplification by the T7 promoter region), extension occurs by reverse transcription, which can extend the CO rather than the 3' end of the captured RNA. This is not a hairpin format, but rather the actual captured RNA, but the direction of extension occurs from the 3' end of the CO.

[0060] The figures provided with this specification show a representative overall workflow scheme including the following components: Encoding and decoding beads with spatially defined barcodes to create spatially coded arrays (e.g., using visual molecular barcodes such as those described in WO 2022 / 187719); probe-tag compositions (RNA / DNA hybrid hairpins) that enable an isothermal signaling process; generation and cleavage of prototype tags; and one-pot reactions showing cleavage of tags from a surface (bead) and capture onto another surface (bead) bearing capture oligos, which leads to extension and release of full-length extension products from the capture beads into the supernatant.

[0061] Two exceptional elements of the workflow described herein are the "sticker" (generally a spatially coded, thin (<250 μm), flexible, 3D polymeric permeable matrix containing a 2D array of capture features at predetermined or "addressed" locations) and the DNA / RNA hybrid tag design. When combined with a spatial coding system (such as the spatially coded beads described in WO 2022 / 187719), the workflow provides a one-step isothermal cleavage, extension, and release within the sandwich. This workflow is very user-friendly. Because pre-reading of the spatial bead array can be performed offline, this platform does not require customized equipment. Due to the nature of the visual barcode, users cannot re-determine the positions after they are read (reading destroys the visual barcode). In several embodiments, consumers receive the spatially coded bead array to use in combination with reagents in a kit format, for example, for spatially resolved biomarker expression in tissue samples. The array is a single-use consumable product.

[0062] Spatially encoded bead arrays such as those described herein and uses thereof are also provided.

[0063] There are several other uses for spatially coded bead capture arrays; this may be the currently cheapest way to make spatially coded bead arrays (reading bead locations by sequencing is much more expensive, or similarly making microarrays to code locations), whereby each bead has an NGS capture oligo that includes a spatial barcode (operably linked) that corresponds to the visual bead barcode on the bead.

[0064] Provided herein is a hairpin tag nucleic acid molecule comprising parts A, B, C, D, E, and E operably linked in 5' to 3' order, wherein part A is: (1) a stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site part B comprises a stretch of DNA bases comprising a tag PCR handle; part C comprises a stretch of DNA bases comprising a tag ID barcode, and said stretch of DNA bases forms part of the loop of the hairpin; part D comprises a stretch of DNA bases having the reverse complement of the tag PCR handle, thereby forming part of the stem of the hairpin; and part E comprises (1) a stretch of RNA bases in part A, or (2) the stretch of DNA bases containing the RE recognition site and a stretch of DNA bases having the reverse complement of at least a portion of either of the following sequences, thereby forming part of the stem of the hairpin.

[0065] Optionally, such hairpin tag nucleic acid molecules may further comprise one or more of the following: an attachment moiety conjugated to the 5' end of part A (in embodiments, the attachment moiety provides amine-reactive crosslinker activity or thiol-reactive crosslinker activity); a linker between the attachment moiety and part A (in embodiments, the linker comprises PEG(n), where n=1-20); a tag ID barcode that is at least 4 bases in length, or in embodiments, the tag ID barcode is 4, 5, 6, 7, 8, or more than 8 bases in length; and / or a tag PCR handle in part B and its reverse complement in part D, each at least 10 bases in length (in embodiments, the tag PCR handle in part B and its reverse complement in part D, each 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length).

[0066] The hairpin tag nucleic acid molecules described herein can optionally be constructed at least in part using a templated ligation reaction.

[0067] In a representative hairpin tag nucleic acid molecule embodiment, part A comprises a stretch of DNA bases comprising an RE recognition site, and the RE recognition site is at least 4 bases in length. For example, the RE recognition site may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length. Optionally, the RE recognition site in any hairpin tag nucleic acid molecule can be a type IIS restriction enzyme recognition site, a site for an RE that generates a 3' overhang, or both.

[0068] Additional exemplary hairpin tag nucleic acid molecules are DNA / RNA hybrid hairpin tag nucleic acid molecules, where part A comprises a single RNA or a stretch of RNA bases, and the stretch of RNA bases in part A is at least 5 bases in length. Thus, contemplated DNA / RNA hybrid hairpin tag nucleic acid molecules exist where the stretch of RNA bases in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length.

[0069] Also provided is a set of two or more of the described hairpin tag nucleic acid molecules and / or the described DNA / RNA hybrid hairpin tag nucleic acid molecules, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence. In an example of such a set, the set further includes at least one attenuation tag-like nucleic acid molecule, wherein the attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by the absence of a functional cleavable site. For example, provided is a set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules, wherein the attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by (1) having a DNA base instead of an RNA base in part A, or (2) lacking an RE recognition site in part A.

[0070] Also provided are tagged probes comprising a probe molecule attached to the described hairpin tag nucleic acid molecule or the described DNA / RNA hybrid hairpin tag nucleic acid molecule via the attachment moiety. By way of example, the probe molecule comprises an affinity molecule having binding affinity for a target molecule. Optionally, the affinity molecule comprises an antibody binding domain having affinity for an antigen, and the target molecule comprises the antigen.

[0071] Also provided are tagged probes, wherein the probe molecule comprises one or more of: an antibody or its binding fragment, a nucleic acid, a small molecule, an organic or inorganic chemical, a putative drug target, a identified pharmaceutical drug, or a biological macromolecule complex.For example, the probe molecule can be one of a set of probe molecules, each of which comprises one of a plurality of members of a small molecule library, a drug target library, a biological affinity molecule library, a natural product library, a bioactive compound library, a genome library, a transcriptome library, a metabolomics library, or a drug screening library.

[0072] In yet another embodiment of the tagged probe, the target molecule comprises a biological molecule, an inorganic object, or an addressable feature of an array.For example, the target molecule can comprise a biological molecule, and the biological molecule comprises one or more proteins, lipids, carbohydrates, nucleic acid molecules, or a combination of proteins, lipids, carbohydrates, and / or nucleic acid molecules.Optionally, in some cases, the target molecule is one of multiple molecules that constitute a complex, and the complex is located outside or inside one or more cells in a tissue sample.

[0073] In any tagged probe embodiment, the tagged probe may further comprise an amplification sequence comprising a polymerase promoter sequence. By way of example, the amplification sequence comprises a T7 promoter sequence, such as a T7 promoter adapter.

[0074] Another embodiment is a released hairpin tag nucleic acid molecule derived from a described hairpin tag or derived from a described DNA / RNA hybrid hairpin tag, wherein the released hairpin tag is separated from the attachment moiety by the enzymatic action of a restriction endonuclease or RNase H enzyme.

[0075] Yet another embodiment is a capture oligo(CO) nucleic acid molecule comprising parts I-II-III-IV operably linked in 5'→3' order, wherein part I is: (1) a single RNA base or a continuous stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site part II comprises a stretch of DNA bases comprising a CO PCR handle; part III comprises a stretch of DNA bases comprising a spatial barcode; and part IV comprises a stretch of DNA bases comprising a tag capture region. Optionally, the CO nucleic acid molecule further comprises an attachment moiety conjugated to the 5' end of part I. For example, the attachment moiety in some cases provides amine-reactive crosslinker activity or thiol-reactive crosslinker activity.

[0076] In a further option, the CO nucleic acid molecule comprises a linker between the attachment moiety and part I. For example, an exemplary linker comprises PEG(n), where n=1-20.

[0077] In a further embodiment of the CO nucleic acid molecule, the spatial barcode is at least 4 bases long. For example, the spatial barcode can be 4, 5, 6, 7, 8, or more than 8 bases long.

[0078] Optionally, in embodiments of the CO nucleic acid molecule, the CO PCR handle in part II is at least 5 bases in length. For example, the CO PCR handle in part II can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length.

[0079] CO nucleic acid molecules that further comprise a unique molecular identifier (UMI) are also contemplated.

[0080] In any of the embodiments of a CO nucleic acid molecule, examples are contemplated in which the sequence has no more than two consecutive bases with internal sequence self-complementarity. For example, the sequence of the CO nucleic acid molecule has no more than three, no more than four, no more than five, no more than six, no more than seven, no more than eight, no more than nine, no more than ten, no more than eleven, or no more than twelve consecutive bases with internal sequence self-complementarity.

[0081] In any of the embodiments of the CO nucleic acid molecule, examples are constructed at least in part using a templated ligation reaction.

[0082] In any of the CO nucleic acid molecule embodiments, optionally, part I comprises a stretch of DNA bases that includes a RE recognition site, and the RE recognition site is at least 4 bases in length. For example, the RE recognition site can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length. Optionally, the RE recognition site is a type IIS restriction enzyme recognition site, a site for an RE that leaves a 3' overhang, or both.

[0083] CO nucleic acid molecules that are DNA / RNA chimeric CO nucleic acid molecules are also contemplated, where part I comprises a single RNA base. DNA / RNA chimeric CO nucleic acid molecules in which part I comprises a contiguous stretch of RNA bases are also provided. By way of example, such DNA / RNA chimeric CO nucleic acid molecules can include a contiguous stretch of RNA bases in part I that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases in length, or greater than 20 bases in length.

[0084] Yet another embodiment is a set of two or more of the provided CO nucleic acid molecules and / or any of the provided DNA / RNA chimeric CO nucleic acid molecules, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

[0085] Also provided are CO nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules, each of which is attached to a capture feature through an attachment portion of the nucleic acid molecule. For example, in various embodiments, the capture feature is a bead, a chemically functionalized spot on a glass surface, a defined region of a chemically functionalized permeable gel, a bead or other inorganic object embedded within or on the surface of a permeable gel, or one of a series of spatially defined objects attached to a gel. Also contemplated are attached CO nucleic acid molecules, where the capture feature is a spatially addressable feature within an array, e.g., a microarray, e.g., a microarray having at least 100 addressable capture features.

[0086] Yet another provided embodiment is a spatially encoded capture feature comprising a capture feature to which a provided CO nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule is attached via the attachment moiety. For example, the capture feature comprises a bead or an addressable location on a substantially two-dimensional surface. For example, a representative spatially encoded capture feature embodiment comprises a spatially encoded capture feature within an array of at least 100 different spatially encoded capture features, wherein each CO nucleic acid molecule on each of the at least 100 different spatially encoded capture features of the array comprises a different spatial barcode.

[0087] Also described is a capture pair comprising a provided hairpin tag nucleic acid molecule, or a provided DNA / RNA hybrid hairpin tag nucleic acid molecule, or a provided released hairpin tag nucleic acid molecule; and a provided capture oligo(CO) nucleic acid molecule, or a provided DNA / RNA chimeric CO nucleic acid molecule, or a provided attached CO, wherein the sequences of the hairpin tag nucleic acid molecule and the CO nucleic acid molecule are at least partially complementary such that when the hairpin tag nucleic acid molecule is released from its attached moiety in the vicinity of the CO nucleic acid molecule, the released hairpin tag nucleic acid molecule is captured by sequence complementary bonding to the CO nucleic acid molecule at its 3' end, such that the resulting complex of the released hairpin tag nucleic acid molecule and the CO nucleic acid molecule is eligible for a downstream extension reaction by a polymerase enzyme.

[0088] Yet another aspect is a spatially encoded capture array comprising a defined array of spatially addressed capture features, each capture feature having: Predefined, addressable locations on a substantially two-dimensional solid surface; or beads or other similar discrete solid capture objects spatially distinguishable features, including: Multiple copies of the provided CO nucleic acid molecule or the provided DNA / RNA chimeric CO nucleic acid molecule attached at each feature. wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array. In some examples, one or more CO nucleic acid molecules comprise: Droplet printing of CO2 at predefined, addressable locations on a substantially two-dimensional solid surface; or CO deposition onto beads through the deposition site to spatially addressed capture features within the array.

[0089] Optionally, the spatially coded capture array comprises beads, each comprising a visual barcode operatively linked to a CO. For example, an example spatially coded capture array comprises a visual barcode that allows beads to be assigned to a location within the capture array.

[0090] Examples of spatially encoded capture arrays in which beads or other similar discrete capture entities are embedded in a biomolecule-permeable matrix are also contemplated. For example, the capture entities may include beads, the biomolecule-permeable matrix may include a gel, or both. In certain examples, the gel-containing biomolecule-permeable matrix is ​​formatted as a flexible sticker.

[0091] Further exemplary spatially encoded capture arrays include a biomolecule-permeable matrix that is structurally stable at a selected temperature between 4 and 45°C; permeable to proteins such as functional RNAse H and polymerase; permeable to ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); and Mg 2+ It is permeable to ions; substantially inert to biological molecules; and sufficiently flexible to allow a relatively thin layer of the matrix to be directly applied to a substantially two-dimensional sample or surface.For example, a spatially coded capture array can be constructed as a three-dimensional thin gel, whose width and length are substantially greater than its thickness, and wherein spatially distinguishable capture features are arranged in a substantially single plane across the entire gel surface defined by the length and width.In some instances, at least a first spatially distinguishable capture feature is directly attached to and / or contacts a second spatially distinguishable capture feature.

[0092] In any of the spatially coded capture arrays provided, the matrix can comprise a hydrogel or polyacrylamide gel. For example, the thickness of the matrix or gel is about 2 mm or less. In particular examples, the thickness of the matrix or gel is about 1 mm or less, 500 μm or less, 250 μm or less, 200 μm or less, 150 μm or less, 125 μm or less, 100 μm or less, or less than 100 μm. Advantageously, the thickness of the matrix or gel in some examples is 100-200 μm, 100-150 μm, or about 125 μm.

[0093] In any of the examples of spatially encoded capture arrays provided, it is contemplated that the array may be reinforced by an inert mesh or other support structure.

[0094] In the examples of spatially coded capture arrays provided, the predefined, addressable locations on a substantially two-dimensional solid surface have a surface area of ​​about 1 μm x 1 μm or less; or the beads or other similar discrete solid capture objects have a diameter of about 20 μm or less.

[0095] In the examples of spatially coded capture arrays provided, the beads or other similar discrete solid capture objects have diameters of 18 μm or less, 15 μm or less, 12 μm or less, 10 μm or less, 8 μm or less, 5 μm or less, 3 μm or less, 1 μm or less, or about 100 nm. For example, the beads or other similar discrete solid capture objects can have diameters of 1-3 μm.

[0096] Another embodiment is a semi-aligned spatially encoded capture array comprising: A grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide comprising: an x-coordinate adaptor oligonucleotide used to label all spatially addressable locations within a row of the grid; and y-coordinate adapter oligonucleotides used to label all spatially addressable locations within a column of the grid The grid is applied to the spatially addressable locations by splint ligation of a smeared region. Examples of such semi-aligned spatially encoded capture arrays are constructed at least in part using the methods provided in Figures 17A-17C. In some embodiments, the semi-aligned spatially encoded capture array has a grid-within-a-grid format, as shown in Figure 10.

[0097] Also provided is a semi-aligned spatially coded capture array that includes: An array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray comprising a uniquely identifiable capture feature, the location of which is determined at least in part by the identity of the subarray within the semi-aligned spatially coded capture array.

[0098] Another example is a semi-aligned spatially coded capture array that includes: A set of two or more subarrays, each containing a plurality of capture features, wherein each capture feature in each subarray is attached to a capture oligo that contains a unique position tag, the capture features in each subarray are randomly arranged, and the capture features in each subarray further contain a subarray identifying oligo tag attached to the capture oligo on each feature in the subarray by splint ligation.

[0099] There is also provided a method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample, said method comprising: contacting the substantially 2D sample with at least one tagged probe to generate a substantially 2D stained sample, the tagged probe comprising: a hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode, or a DNA / RNA hybrid hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode; and a probe molecule attached via an attachment moiety to the hairpin tag nucleic acid molecule containing a tag ID barcode or the DNA / RNA hybrid hairpin tag nucleic acid molecule containing a tag ID barcode a process comprising: contacting the surface of the substantially 2D stained sample with a transparent, spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: a plurality of spatially distinguishable features; and Multiple copies of capture oligo(CO) nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules attached at each spatially distinguishable feature wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array; placing a flow cell or other solution-containing cover over the sample-array sandwich to form an enclosure containing the sample-array sandwich; adding a solution containing reaction components to the enclosure to form a reaction mixture, the components comprising: a cleaving enzyme selected from RNAse H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ ions; and buffers a process comprising: incubating the sample-array sandwich contacted with the reaction mixture at an assay temperature for 30 to 60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the enclosure; and Analyzing the reaction product mixture to detect and / or quantify and / or define the location of the target within the substantially 2D sample. Optionally, in such methods, the enclosure comprises a flow cell. Embodiments of the provided methods provide location information for more than one target within a substantially 2D sample.

[0100] Embodiments of the method are carried out at a single temperature (isothermally) or within a range of about 5°C within a single temperature. Illustratively, assay temperatures range from 20 to 55°C, or from 37 to 42°C.

[0101] By way of example, in any one of the method embodiments, the at least one polymerase provides the enzymatic activity of a DNA polymerase, a reverse transcriptase, or an RNA polymerase.

[0102] For example, in any of the method embodiments, analyzing the reaction product mixture comprises sequence analysis of a plurality of nucleic acid molecules comprising the spatial barcode and tag ID barcode.For example, in some cases, analyzing the reaction product mixture comprises next-generation sequencing (NGS) of a plurality of nucleic acid molecules comprising the spatial barcode and tag ID barcode.For example, in some methods, the plurality of nucleic acid molecules comprising the spatial barcode and tag ID barcode are full extension products released from spatially distinguishable features by cleavage at the cleavage site of CO (e.g., cleavage based on RNAse H enzyme activity or restriction endonuclease activity).In the exemplary method, analyzing comprises assigning the spatial location of at least one nucleic acid molecule comprising the spatial barcode and tag ID barcode within a substantially 2D sample.

[0103] Also provided are methods for isothermal spatial encoding of biological samples, including methods for detection and / or quantification and / or localization of targets in a substantially two-dimensional (2D) sample, wherein the substantially 2D sample is a biological sample.

[0104] Another aspect is the use of a set of hairpin tag nucleic acid molecules provided herein, or a set of capture oligo(CO) nucleic acid molecules provided herein, or both, for transcriptome analysis of a biological sample.

[0105] Also described are spatially encoded surfaces such as capture arrays, e.g., bead-based capture arrays, substantially as described or shown herein.

[0106] Yet another aspect is a spatial encoding workflow that includes: contacting a hybrid RNA / DNA tag containing a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; placing a capture array containing capture features in contact with the stained sample to create a sample / array sandwich; placing a fluid containment enclosure on top of the sample / array sandwich; introducing an assay solution comprising active RNAse H and an active polymerase into the fluid containment enclosure, thereby contacting the assay solution with the sample / array sandwich; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient for RNAse H activity to at least partially digest the hybrid RNA / DNA tag to generate a cleavable tag, and thereby release the cleavable tag into the assay solution in the vicinity of the capture feature; allowing the cleavable tag to interact with a capture oligo(CO) on the adjacent capture feature to provide a captured cleavable tag; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient to allow polymerase activity to extend the captured cleavable tags using CO as a template to generate extension products; cleaving the extension product with RNAse H in the sample solution once it has been extended sufficiently to generate a complementary RNA / DNA region based on the RNA bases in the CO, thereby releasing a full-length extension product; recovering at least a portion of the released full-length extension products; and amplifying and / or sequencing at least one of the released full-length extension products.

[0107] Also provided are computer-readable media or digital resources, and digital databases, that contain spatial location information for features of the spatially encoded arrays described herein. For example, the computer-readable media or digital resources, or digital databases, in some instances, contain spatial location information for substantially all features of the spatially encoded arrays.

[0108] Also disclosed is the use of the provided computer-readable medium or digital resource, or digital database, to provide a user with location information for one or more targets that correlates with the spatial location information of the spatially encoded array. As an example of the use of the provided computer-readable medium or digital resource, or digital database, the correlation occurs through the use of the spatially encoded array in a workflow or method described or illustrated herein.

[0109] Yet another embodiment is a kit useful for practicing one of the methods provided herein. Exemplary kits include one or more of the following: two or more of the described hairpin tag nucleic acid molecules; two or more of the described DNA / RNA hybrid hairpin tag nucleic acid molecules; a described set of two or more hairpin tags; a set of two or more of the tagged probes described; two or more of the described capture oligo(CO) nucleic acid molecules; two or more of the described DNA / RNA chimeric nucleic acid molecules; a described set of two or more CO nucleic acid molecules; A described set of two or more DNA / RNA chimeric CO nucleic acid molecules; two or more CO nucleic acid molecules as described, each attached to a capture feature; at least one described spatially encoded capture array; At least one of the described semi-aligned spatially encoded capture arrays; or The spatially coded surface described.

[0110] In an example of such a kit, the spatially coded capture array, semi-aligned spatially coded capture array, or spatially coded surface is in the form of a flexible sticker.

[0111] Also, a vessel containing a functional RNAse H enzyme; a container containing a functional polymerase enzyme; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ a vessel containing a solution containing ions; or A container containing a buffer solution Also provided are kit embodiments, further comprising one or more of: RNAse H enzyme, polymerase enzyme, restriction endonuclease enzyme, rNTPs and / or dNTPs, Mg. Optionally, in such kits, at least one of the containers contains at least one of: 2+ Optionally, the complete assay solution is provided in a single container.

[0112] Kit embodiments may also optionally include one or more of the following: components useful for preparing samples for analysis using the methods provided herein; a solution-containing cover suitable for placement over a sample-array sandwich on a slide to form a fluid-containing enclosure for the sample-array sandwich; a flow cell cover; or a glass slide or other surface suitable for receiving a substantially two-dimensional sample.

[0113] Additional details and options are used to describe aspects of the present disclosure.

[0114] Methods and systems for isothermal spatial encoding and assay of biological samples - Patent Application 20070122999 Isothermal method: isothermal signal transduction, spatial encoding, and release of the spatially encoded product into solution. As described herein, a new type of nucleic acid tag is used to encode molecules or probes, and this tag contains a hairpin structure, whereby chimeric RNA / DNA molecules create regions of RNA / DNA hybridization through base complementarity. This unit is highly stable and resistant to RNA exonuclease activity, and a specific barcode (a stretch of four consecutive unpaired bases is provided as an example, but longer lengths are contemplated) can be included within the loop region of the hairpin to distinguish different probes from each other in multiplex assays. The sequence of the hairpin tag also contains the first of two PCR handles used for downstream amplification of the spatially encoded product. Methods for constructing various versions of this hairpin tag are provided, and its usefulness in workflows when conjugated to (for example) antibody probes is demonstrated.

[0115] After binding a probe conjugated with an RNA / DNA hybrid-containing hairpin tag to a surface and washing away unbound conjugates, the hairpin tag can be released from the probe as a smaller, DNA-only hairpin (released tag) while still bound to the sample surface by using an enzyme containing RNAse H activity. In some embodiments, prior to its release by RNAse H, a spatially encoded capture array is placed near (e.g., in direct contact with) the sample surface containing the bound probe conjugates. As described herein, in some embodiments, the capture array surface is an array made of 1 μm beads coated with capture oligos. As demonstrated herein, the capture surface may also include an enzyme-permeable matrix (e.g., a gel) in which the capture beads are embedded. This may be in the form of a sticker containing the capture beads in the matrix, which is flexible enough to allow it to be applied in direct contact with the sample surface to be assayed.

[0116] Also contemplated are embodiments in which multiple different tag barcodes are included on the same antibody, so that the normal distribution of tags after amplification can be analyzed (e.g., if they are all released from the same antibody type and should be in equimolar ratios.) This embodiment is largely outside the scope of normal workflow, but can be used, for example, as an internal control or calibration.

[0117] In embodiments, the capture oligo binds to a capture feature (such as a bead) through its 5' end and includes a 3' capture region designed for specific base complementarity with the 3' end of the released DNA hairpin, as well as a positional barcode and the second of two PCR handles used for downstream amplification of the spatially encoded product. The capture oligo may also include a stretch of 5' RNA bases located between the second PCR handle and the 5' bead attachment portion.

[0118] The position of the beads / features in the capture array is predetermined, and this bead position information is contained in the sequence of the capture oligos (spatial barcode region or positional barcode region) attached to the capture beads or features.Each capture bead or feature contains a unique spatial barcode that is common to all capture oligos on that bead or within that feature address.The position of the beads or features in the capture array can be uploaded and stored in a software package or database, for example, in the form of a DNA sequence (positional barcode or spatial barcode) corresponding to the specific position of each bead or feature.

[0119] Once placed in proximity and RNAse H is introduced, information from the sample (the hairpin released from the probe) is transferred to the (nearest) spatially encoded capture bead or feature through specific hybridization with a capture region contained within the 3' sequence of the capture oligo bound to the capture bead. The polymerase enzyme and a cofactor (Mg) required for extension of the hairpin 3' end by DNA polymerase are then introduced. 2+ , dNTPs) are present in the same reaction mixture as that templated by the capture oligo, thereby copying the positional information and a second PCR handle onto the released hairpin oligo. Optionally, a 5' RNA base can be placed in the capture oligo to create a second substrate for recognition and cleavage by RNAse H activity present in the isothermal one-pot reaction mixture. Inclusion of an RNA base at the 5' end of the capture oligo allows the spatially encoded probe to be released from the capture bead into the surrounding solution.

[0120] When using a capture array in which capture oligos (or beads) are embedded within a polymeric permeable surface or gel, the capture surface can be placed near the probe-bound sample prior to the introduction of enzymes and other reaction components, allowing the enzymes / components to diffuse through the permeable surface / gel. A flexible encoding surface can be useful for conforming to the sample surface, thereby placing spatially encoded capture features (beads, as shown in several embodiments herein) in close proximity to the sample. The provided "one-pot" isothermal encoding reaction can then be initiated by absorbing the one-pot encoding reaction solution through the gel while incubating at the reaction temperature (e.g., 37-42°C). In the reaction, information from the sample is transferred via RNAse H digestion of the hairpin tags and subsequent capture of the released hairpin tags near where they were bound to the sample, which are then spatially encoded via polymerase-mediated extension and, optionally, released into the surrounding solution after spatial encoding. Applying a coded polymeric permeable capture surface or gel directly onto a sample allows one or more of the enzymes (such as at least the trigger enzyme, E. coli RNAse H, as demonstrated herein) and / or cofactors required for an isothermal reaction to diffuse through the coded (spatially discriminating) surface and contact the appropriate substrate without moving the capture surface relative to the sample.

[0121] After the reaction, the spatially encoded reaction products diffuse out of the gel (or other permeable matrix) into the surrounding solution, where they can be collected by aspiration of the solution. A volume of solution sufficient to cover the spatially encoded gel (capture array) is required, but optionally, a flow cell may be present that covers the entire area of ​​the spatially encoded gel, thus defining the volume of solution. Ideally, this is a low volume of solution so that the concentration of the diffused products remains high. However, in embodiments employing the described optional capture step, volume is less critical.

[0122] In the illustrated embodiment, the solution volume is approximately 40 μl. If the volume is significantly larger, an additional (bead) capture step can be performed to capture the diffused, spatially encoded product, e.g., on beads. This can be achieved by the fact that the spatially encoded product can be released from the capture beads after the extension step, which creates new DNA / RNA hybrid regions. The DNA region that becomes exposed after removal of the RNA region (after extension in the reaction solution and exposure to RNAse H activity) can be captured using beads (or another capture surface) containing immobilized oligos designed to contain complementary regions for capturing the extended and released product. After capture in 2x capture buffer containing 20 mM Tris pH 7.5 and 1 M NaCl (added 1:1 by volume to the aspirated solution containing the spatially encoded, released extension products), an optional wash in 1x buffer can be performed, followed by reducing the beads to a low volume (e.g., by magnet or centrifugation to pellet the beads) for resuspension in a uniform, low-volume (<25 μl) elution solution or buffer containing little to no salt (e.g., water or 10 mM Tris pH 7.5). More salt in the capture step increases the likelihood of capturing spatially encoded extension products by hybridization to bead-immobilized oligos. Less salt in the elution buffer increases the likelihood of dehybridization of captured products from bead-immobilized oligos upon exposure to heat (>75°C). The smaller elution volume provides consistency between experiments and allows for the recovery of all products that diffuse out of the gel during the isothermal reaction.

[0123] These elution products can be further processed, for example, by capture and / or PCR amplification. These amplification products can then be prepared for next-generation sequencing using known amplicon-based or ligation-based sequencing library preparation techniques. For example, see the information available online at illumina.com / techniques / sequencing / ngs-library-prep.html, which describes multiple types of library preparation for Illumina sequencing, including both amplicon-based and ligation-based preparations. The resulting sequence data library contains continuous reads (counts) that contain individual probe hairpin sequences fused with the positional information contained in the capture oligos attached to the beads on which the hairpins are captured. Next, a predetermined map of bead positions (barcodes) is used (e.g., through access to a license or subscription service) to reconstruct a digital image by overlaying the counts for each probe obtained by sequencing at their captured positions onto a spatially coded array. Thus, this workflow allows for the reconstruction of digital images through the use of next-generation sequencing.

[0124] The hairpin nucleic acid tags described herein encompass both RNA and DNA to create substrates for cleavage by RNAse H (see Figures 1A-1B). Advantages arising from this design include: the ability to reveal rationally designed stretches of DNA sequences through RNAse H digestion for optimal capture of released hairpins at isothermal temperatures; the ability to capture and extend such sequences onto capture oligos bound within capture features contained within an array of spatially addressed capture features; optimization of the hairpin capture region Tm under isothermal reaction conditions; and a method that functions without compromising antibody recognition of targets in the sample being analyzed.

[0125] As described herein, the provided hairpins have been shown to be stable for at least 6 weeks when conjugated to an antibody and stored in phosphate buffered saline (PBS) plus bovine serum albumin (BSA) at 4°C.

[0126] Described herein is the use of hairpin tags containing both RNA and DNA for the isothermal release of information from surface-bound DNA-encoded molecules following a selective binding event. Advantages arising from this include the ability to "trigger" hairpin release upon contact of the tag with an enzyme containing RNAse H activity; and the ability to trigger hairpin release at a temperature compatible with capturing the released tag on a capture oligo.

[0127] Capture of information released from surface-bound DNA-encoded molecules (Figure 2) after a selective binding event is also provided by using capture oligos (structures shown in Figure 3) coupled with an extension reaction performed by a polymerase enzyme contained within the same reaction mixture (Figure 4). Advantages arising from this include: the extension reaction immediately "locks on" each hairpin to the first (or one of the first) capture oligos it comes into contact with via polymerase extension, thereby reducing lateral diffusion of the probe or "hopping" of the probe to other capture oligos; and the ability to copy positional information from a nearby capture oligo to the released DNA hairpin oligo (and vice versa) within the same reaction mixture in which the hairpin oligo was released from the sample surface by templated extension of the 3' end of the capture oligo (and one version of the capture oligo; if the 3' end of the capture oligo is not blocked).

[0128] When using a polymerase enzyme that includes reverse transcriptase activity (e.g., RNA-dependent DNA polymerase activity), it becomes possible to read through the RNA bases contained within the capture oligo template during the extension reaction. Here, a polymerase that includes both RNA- and DNA-dependent DNA polymerase activity was used, which allows the incorporation of deoxyribonucleotide triphosphate bases (dNTPs) in response to either DNA or RNA bases within the capture oligo template. The data described herein demonstrates the following: PCR amplification for the recovery of full-length products after hairpin capture and extension on capture beads; qPCR results from + / - experiments; gels of amplified full-length products; and Sanger sequencing traces of full-length extension products prepared for NGS (see Appendix A in U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which is hereby incorporated by reference).

[0129] Release of spatially encoded products from spatial capture arrays (Figures 9A–9E show various ways to achieve this) can occur within the same isothermal reaction mixture. This can be achieved through the inclusion of RNA bases within the 5′ region of the capture oligo, so that polymerase extension through this region creates new DNA / RNA hybrid species, which then become substrates for RNase H, which is already included in the one-pot isothermal signaling reaction (Figures 9A and 9E). This can also be achieved through the inclusion of an encoded restriction enzyme site (at the 5′ end of the single-stranded capture oligo), so that polymerase extension generates a dsDNA restriction site for recognition and cleavage by the corresponding restriction enzyme during the one-pot isothermal signaling reaction or after the extension step has been performed (Figures 9B and 9E). The inclusion of a photocleavable or chemically cleavable linker for attachment of the 5′ end of the capture oligo to the capture feature enables a third mechanism for release of spatially encoded products (Figures 9C and 9D).

[0130] An additional advantage is that no user interaction is required for product recovery. In previously known workflows, after capture, the two surfaces are separated, the capture array is washed, and the captured probes are eluted from the capture array at elevated temperatures. The methods disclosed herein eliminate the need for these steps.

[0131] Not all capture oligos can be modified to include a 5' RNA base for spontaneous release of the product into solution after spatial encoding; rather, perhaps a certain percentage of the capture oligos in each capture feature (here, on each bead) include a 5' RNA base for spontaneous release of the product, while a certain percentage of these contain only DNA bases but are attached to the capture feature by a photocleavable linker. This embodiment illustrates a very useful possibility. This allows the user to later return to interesting samples that were screened at a relatively "shallow" read depth (i.e., not all of the captured information was released during the isothermal reaction, but the resulting library was sequenced and a digital image was reconstructed from this partial read) and select a region of interest (ROI) through software displaying the digital image. Thus, it is possible to employ an instrument for defined, site-specific photorelease of all remaining spatially encoded products within the selected region of interest. This has been achieved, for example, by using digital micromirror arrays to focus wavelengths of UV light at defined locations across the surface; arrays of this type can be manufactured or purchased commercially with a resolution of 10 μm or better. Additional data may then be obtained from the remaining sample thus released.

[0132] It is also contemplated to provide compatibility between other DNA-encoded molecules (those that do not include the tag features described herein) and the isothermal spatial coding system described herein as a way to expand the application of the technology described herein. To do this, molecular ID tags can be designed to include RNA bases therein (FIG. 5A), which function seamlessly with the one-pot isothermal signaling reaction described herein (FIG. 5B). In one example, this is achieved by including a stretch of 5' RNA bases within a linear (non-hairpin) tag attached to an encoded molecular library. This tag type includes, from the 5' to 3' end of the tag, a 5' linker used to attach the tag to the molecular library, followed by a stretch of 5' RNA bases (constant region on all tags) that encodes the reverse complement of the sequence to be captured, followed by a variable region that encodes the individual molecule attached to the tag, followed by a second constant region composed of DNA bases. Before or after the binding assay (screening or selection), a dsDNA region can be generated by hybridization of an oligo complementary to the 3' constant region of the tag, or can be incorporated as a constant hairpin structure. This constant "primer" or hairpin structure at the 3' end is best incorporated before the binding assay and can be extended by an extension reaction with a polymerase (including both RNA- and DNA-dependent DNA polymerases to extend through both base types contained in the tag), creating a DNA / RNA hybrid region at the 5' end of the tag in the vicinity of the attached molecular library. The extension reaction can be performed before or after the binding assay. After binding, the tagged probes or molecular library can be spatially resolved using the approach described above. Upon introduction of an enzyme containing RNase H activity, these tags are released from the sample surface in the same manner as above, thereby revealing the current 3' ends of the extension products (constant capturable regions on all tags) and therefore eligible for inclusion in the same isothermal signaling workflow described herein.Other sequences can also be designed for inclusion within the tag sequence, as shown in Figure 6, which has a number of additional advantages outlined below: In Figure 6, two opposing RNA polymerase promoters are used, which may be most optimal.

[0133] An additional method for achieving this involves adding adapter molecules to other DNA tags to make them compatible with one-pot isothermal signal transduction reactions. These various adapter molecules can be provided in a variety of forms, examples of which are described below.

[0134] The first form of adapter is simply an RNA / DNA hybrid hairpin tag, as described herein, appended to the free end of the DNA encoding the probe (Figure 1). This adapter type can be fused to other DNA tags as a continuous polymer or attached to other DNA tags or molecules through compatible covalent reaction chemistries. In this case, the tag barcode is now encoded within the loop of the hairpin attached to the DNA-encoded probe. This approach may work well for converting and encoding antibodies or other smaller panels of probes (up to tens of thousands of probes in a mixture), such as reagents from other companies (e.g., SomaLogic), or specific transcriptome probes. This form of adapter has already been shown to work well in one-pot isothermal signaling reactions.

[0135] amplification: A second form of adapter confers an amplification feature to this method. Here, an adapter containing a polymerase promoter sequence (e.g., T7 or S6) is ligated onto the DNA strand of a DNA-encoded molecule through a templated ligation reaction (Figures 7A-7B). To achieve adapter ligation to a ssDNA tag (Figure 7A), the ligation reaction must be templated by a splinting sequence, so the sequence information for ligation must be known. Fortunately, most encoded molecule libraries contain two constant regions within their DNA tag structure (for downstream recovery after screening or selection by PCR amplification), and these constant regions flank the information corresponding to the encoded library member. One of these constant regions (region 1) can be used for adapter ligation, and the other constant sequence (region 2) can be used for capture. For ligation of this adapter type to a dsDNA tag (Figure 7B), sequence information for the constant regions is not required. Once ligated, this promoter sequence is competent for initiating transcription of the probe-encoding sequence upon introduction of an appropriate polymerase. In the example of a T7 adapter, the adapter consists of a double-stranded DNA sequence encoding the T7 polymerase promoter and has an overhanging, compatible "sticky end" for ligation to the encoding DNA strand of the molecular library. Alternatively, the adapter may consist of a single, continuous DNA strand in a hairpin structure, whereby the stem of the hairpin (the dsDNA region) encodes the T7 polymerase promoter sequence and thereby provides an overhanging, compatible "sticky end" available for attachment to the encoding DNA strand of the molecular library. The same structure can be used to encode any polymerase promoter sequence. This type of adapter can be ligated onto the encoding DNA strand of the molecular library before or after binding of the probe to the sample surface.After washing unbound probe from the sample, a capture surface of the same type as used in the previous example can be placed in proximity to the probed sample.

[0136] To initiate this isothermal signal transduction reaction, an appropriate polymerase (herein T7 RNA polymerase) is required to bind appropriate cofactors (in this case NTPs for transcription, Mg 2+ (e.g., nucleotides) are introduced onto the surface, and up to 1,000 copies of RNA are estimated to be generated from each adapter-modified DNA tag (through its associated probe) bound to the surface. In this case, the 3' capture region of the capture oligo in the capture array is designed to specifically capture the RNA generated from the coding tag via a second constant region not used in adapter ligation (Figure 8). The RNA generated by this reaction is captured in close proximity onto spatially encoded capture features in the presence of a polymerase containing reverse transcriptase activity. Upon contact (capture of the RNA onto the capture oligo), the reverse transcriptase extends the information contained within the released RNA onto the capture oligo of the capture array by extending the 3' end of the capture oligo (CO). This process creates a contiguous DNA molecule containing the information released (amplified) from the adapter-modified probe (probe or molecular ID barcode) and the information from the spatially encoded CO molecule closest to its location on the sample surface. The constant region of the coding tag used in ligation then becomes one of two PCR handles for library amplification, while the other PCR handle is provided on the capture oligo. These consecutive molecules constitute a spatially coded library and can be released into solution from the capture array by chemical cleavage, photolysis, RNase H, or restriction enzyme cleavage (Figures 9D and 9E). The spatially coded library products are then collected by aspiration of the solution as described above for downstream NGS library preparation and sequencing.

[0137] Application of methods to include transcriptomes This general initial approach (the DNA / RNA hybrid hairpin tag approach) can be expanded to include transcriptomics probing within the spatial coding workflow described. This approach involves using probe pairs that recognize RNA sequences adjacent to a target RNA (as has been done by others). Each probe pair is designed to generate the described type of hairpin structure upon recognition of its target RNA sequence only when bound to its target in close proximity; the RNA bound to the probe generates a template for proximity ligation of a matching probe pair to create the disclosed hairpin structure. When multiplexed, each hairpin formed by ligation of proximity-ligated probe pairs contains a barcode in the loop region that corresponds to the RNA being probed. To accommodate larger libraries consisting of tens of thousands of probes, the barcode in the hairpin loop can be extended to 8-10 bp. The isothermal signaling reaction is initiated in the same manner as with antibody probes tagged with DNA / RNA hybrid hairpin oligo tags. This may ultimately enable multi-omics assay formats that can be performed simultaneously on the same sample surface using the described isothermal signal transduction reactions. If necessary or desirable, depending on the sample type, this approach can be performed in tandem (probing first RNA and then protein from the same sample, or vice versa), or on serial sections for tissue assays, if not simultaneously.

[0138] Other versions of creating RNA / DNA hybrid species by proximity have also been designed and demonstrated. When used within a similar workflow, these tag types can also be useful for probing nucleic acid sequences in a sample and / or for determining interactions between tagged molecules with spatial resolution across the entire sample. They can consist of two complementary regions forced together by proximity, designed to avoid complementarity at the temperature of the hybridization step (these complementary regions are less than 10 bp in length, such that one of the two complementary regions consists of RNA bases and the other consists of DNA bases within the complementary region). Thus, when used in this workflow, their proximity is driven by the probe specificity of each probe for its respective target, and when the two tags are brought together closely (close enough to create an RNA / DNA species by proximity), contact with an enzyme containing RNase H activity releases information about their proximity, which can then be spatially encoded through capture of the released information using downstream steps in the isothermal spatial encoding workflow described above.

[0139] Spatially Encoded Surfaces As described herein, in some examples, a spatially coded capture surface is an array of features, each containing bound capture oligonucleotides. The features of the array may comprise densely packed randomly arranged beads or spotted microarrays, or both technologies may be utilized for full surface deployment (described below). The capture oligos (COs) in each feature of the capture array contain a capture region (CR) for capturing information emitted from the sample by base complementarity. This CR may be common to all COs contained within the spatially coded capture surface. Every CO also contains a spatial barcode (SB) unique to each individual feature (common to all COs within a lawn of COs at each feature location), which is used to assign capture feature locations by one or more forms of sequencing. The COs also contain one of two constant regions (CRs) common to all COs in the array, which can be used as PCR handles (PHs) necessary for recovery and / or amplification of spatially coded products following the spatial coding reaction. The CO may also include a continuous or non-contiguous stretch of randomized bases containing a unique molecular identifier (UMI), which can be used to eliminate amplification bias caused by PCR (known in the art; see, e.g., Kivioja et al., Nat. Methods 9:72-74, 2012, doi.org / 10.1038 / nmeth.1778). One preferred structural orientation of the nucleic acid element of the CO in the capture array is, from 5' to 3', a 5' linker for attachment of the CO to the surface (feature), followed by any stretch of 4 to 8 5' RNA bases, followed by one of two PH regions, followed by an SB region, followed by a CR used to capture information released from the sample. In this manner, information released from the sample during an isothermal spatial encoding reaction by RNase H or RNA amplification can be captured on the CO at each spatially resolved capture feature.

[0140] While embodiments using eight RNA bases are provided herein, it is contemplated that this number may be reduced, up to a length cleavable by RNAse H (in some instances, this is as short as a single RNA base from the hybrid strand). Also contemplated are embodiments in which the number of RNA bases is increased beyond eight to expose more (captureable) bases after extension and release of the spatially encoded extension product (as described herein).

[0141] When RNase H is used, the 3' ends of the oligos captured from the sample can be extended by a polymerase to create products consisting of contiguous sequences that encompass both the information of the released probe and the positional information provided by the capture feature. Furthermore, both RNases were required for downstream recovery and / or amplification of the spatially encoded products.

[0142] If amplification of tag information by the first polymerase is enabled through the attachment of an adapter region to the probe nucleic acid tag to include a suitable promoter region (as described in Section 5, "Amplification," above, this is T7 RNA polymerase), information released from the sample is captured through a newly synthesized sequence common to all probe tags. The COs in the capture array contain a common 3' CR for capture of information released from the sample through complementary base pairing. In this case, an enzyme containing RNA-dependent DNA polymerase activity (reverse transcriptase activity) is included in the same reaction mixture and is used to extend the 3' end of the COs to include information templated by the RNA product released from the bound probe. These fully extended COs that encounter the released RNA sequence cannot be extended again. Furthermore, the RNase H activity in the reverse transcriptase destroys the released RNA after its copying via the extension reaction, thus reducing its chances of serving as a template for subsequent extension reactions. This effectively results in one sequencing count per released RNA.

[0143] When the COs of the capture array contain a stretch of 4-8 RNA bases within their 5' ends, extension of the captured oligos through the CO regions consisting of both base types in the CO template requires a polymerase enzyme that encompasses both RNA- and DNA-dependent DNA polymerase activities (or two polymerase enzymes that encompass these activities together). Preferably, a single enzyme that encompasses both activities (such as Maxima™ RT polymerase) is used under conditions that are compatible with the other enzymes used in the isothermal reaction mixture.

[0144] Optionally, a percentage or all of the COs include this stretch of 5'-RNA bases for spontaneous release of the extended spatially encoded product into solution by an enzyme that includes RNAse H activity.

[0145] Optionally, a percentage or all of the CO can be attached to features of the capture array via photocleavable linkers, allowing these extended spatially encoded products to be released after the isothermal spatial encoding reaction is complete.

[0146] Optionally, a percentage or all of the COs contain a 5' restriction enzyme (RE) site formed after extension by a polymerase enzyme (which need not necessarily have RNA-dependent DNA polymerase activity, but only DNA-dependent DNA polymerase activity), so that the spatially encoded product can be released by an RE that recognizes this newly formed site after polymerase extension. This RE can be included in the same reaction mixture as the isothermal signaling reaction or can be introduced after the isothermal spatial encoding reaction is complete.

[0147] Different versions of direct visual sequencing of spatial barcodes contained within features of a capture array that can be used to determine the location of randomly arranged capture features include those known in the art (e.g., sequencing-by-synthesis (SBS), sequencing-by-ligation (SBB), or orthogonal cleavage sequencing (OCS) as described in WO2022 / 187719). This allows for the identification of each individual feature location within the capture array, and these location coordinates are recorded as a unique, contiguous DNA sequence within a software package that creates a physical map of feature locations (different from microarrays). While this product can be used as a capture surface, subsequent modification of the capture array is preferred. Determining the location of randomly arranged capture features (such as beads coated with capture oligos randomly arranged on the surface) is not necessary in the case of microarrays, as the locations of the capture features are predetermined.

[0148] In the case of direct sequencing using SBS or SBB method, the positional barcode in the capture oligo can comprise 30 or less consecutive bases.This is because the positional barcode in the capture oligo can be directly sequenced by visual decoding (next-generation sequencing).However, the visual barcode constructed for use in decoding by OCS experiment is constructed cooperatively with the capture oligo spatial barcode throughout the three rounds of splitting and pooling of the bead-based library.This approach was first described in WO2022 / 187719; its use for encoding capture oligo for next-generation sequencing applications is further described herein (see Appendix A included in U.S. Provisional Patent Application No. 63 / 487,575, dated February 28, 2023, which is a priority application of this application). Briefly, a barcode segment consisting of dsDNA encompassing a designed cleavage site (for RE recognition) is attached to one of five detectable labels (identimers) and placed in a plate or tube, whereby the five differentially labeled identifier segments are mixed 1:1 in a non-redundant combination. For example, AF405-labeled Id1 is mixed with AF488-labeled Id1, or AF550-labeled Id1, or AF647-labeled Id1, or AF750-labeled Id1, but not with additional AF405-labeled Id1. This can improve visual detection during OCS experiments. This is done for each differentially labeled identifier segment within a strand, providing a total of 10 equally mixed dual-colored options for each segment. Herein, six identifier segments of this type are used to create a combinatorial library consisting of 1 million beads. This bead library can be generated by three rounds of splitting and pooling instead of six (for six identifier segments).Because segments Id1 and Id2 are pre-ligated during identifier ligation round 1 to form 100 choices (Id1 + 2), segments Id3 and Id4 are pre-ligated during identifier ligation round 2 to form 100 choices (Id3 + 4), and segments Id5 and Id6 are pre-ligated during identifier ligation round 3 to form 100 choices (Id5 + 6), this results in a total library diversity of 1 million differentially labeled beads after just three rounds of splitting and pooling. Here, two different labels are removed from each bead in each cycle, thereby enabling robust results from OCS experiments. Before, during, or after the first round of identifier attachment to the beads, 100 different single-stranded oligonucleotides are attached to the beads (using identical or unique / orthogonal / non-competitive attachment chemistries), with each of the 100 different options (Id1+2) distinguished by a unique nucleic acid sequence that represents the first segment of the NGS capture oligos that will be constructed in conjunction with the visual identifier barcode. The first segment of the NGS capture oligo (NGS1) contains the following: a 5' attachment chemistry compatible with attachment to the bead, an optional photocleavable linker, followed by the optional inclusion of an RNA base within the 5' end of the oligo (a constant region composed of either RNA or DNA), followed by one of two PCR handles (constant region) required for downstream PCR, followed by a 5-base sequence used to encode each of the 100 identifier combinations (Id1+2), followed by a 4-base site common to all 100 different versions of NGS1, designed for orthogonal ligation to subsequent NGS barcode segments. After attachment of the first coordinated (co-encoded) identifier segment (Id1+2) and its corresponding NGS segment (NGS1), the beads are washed, pooled, mixed, and then divided into the next 100 different sets of options.Due to the fact that enzymatic ligation of subsequent identifier segments to NGS barcode segments can be completely orthogonal, these reactions can occur simultaneously in the same reaction mixture. For example, the identifier segment contains a sticky end for ligation that has a different length and base composition than the sticky end created for NGS segment ligation. This is achieved not only by carefully designing the sticky ends for each ligation (identifier vs. NGS segment) to be orthogonal by base composition and length, but also by using RNA splints to template the ligation of NGS segments. This allows T4 DNA ligase (whose preferred substrate is the identifier segment dsDNA) to be used together with Splint® enzyme (whose preferred substrate is the RNA-sprinted DNA-DNA ligation junction). This will further improve the specificity of the reactions occurring for both segments when performed in tandem or simultaneously in the same mixture. Thus, orthogonal enzymatic ligation of Id3+4 and NGS2 can be performed simultaneously in the same well, resulting in Id3+4 consisting of 100 different options encoded within the spatial barcode region of NGS2. NGS2 contains a 5' region complementary to the constant RNA splint that can be used for templated ligation of NGS1 and NGS2 on the bead, a region encoding each of the 100 different options corresponding to Id3+4, followed by a 3' end for templated ligation to the next NGS segment in the strand. The beads are then washed, pooled, mixed, and divided into the final 100 different wells for orthogonal ligation of Id5+6 and NGS3 segments. NGS3 contains a 5' region complementary to a constant RNA splint that can be used for templated ligation of NGS2 and NGS3 on the bead, a 5-base region encoding each of 100 different options corresponding to Id5+6, followed by a 3' end for capture or templated ligation to the next NGS segment in the strand (outlined in the capture array below).The beads are then washed and pooled to form a library of one million diverse beads, whereby each bead type in the library contains a unique sequence consisting of a visual label with coordinated NGS capture oligos (identifier strands) that contain DNA sequence information corresponding to a color code.

[0149] Benefits include: significantly reduced time and cost for bead reading via the OCS workflow compared to SBB or SBS (next-generation sequencing). This is because the OCS workflow required to read the identifier strand sequence on the beads involves the introduction of a different unique / orthogonal / non-competitive restriction endonuclease (RE) in each cycle and subsequent imaging. These REs are significantly less expensive than the components required for next-generation sequencing. Furthermore, all cleavage agents required for bead reading via the OCS workflow required herein can operate in the same reaction buffer (1x CutSmart® buffer from NEB). Furthermore, the entire OCS bead decoding workflow is performed at a low, isothermal reaction temperature (37°C), thus eliminating the need for elevated temperatures or any temperature changes during decoding. Finally, the cycle time for decoding this type of identifier strand is around 5 minutes per cycle; decoding an identifier strand composed of six segments as described herein via the required OCS workflow takes approximately 30 minutes. After decoding of the identifier strands on the beads to determine the bead position on the surface, the NGS capture oligos, containing information (color code) about which identifier strand was on that bead, remain intact for subsequent nucleic acid capture (e.g., during the isothermal spatial encoding workflow described herein).

[0150] Alternatively, as described above, bead position can be determined using conventional NGS reagents. Conventional NGS sequencing experiments require expensive reagents for accurate DNA sequence determination, including reversibly terminated and / or labeled nucleotides, high temperatures (typically 65°C) during decoding, multiple different reagents are introduced during each decoding cycle, and these reagents cannot be mixed (e.g., cleavage solution cannot be mixed with incorporation solution), thereby requiring more sophisticated fluidics and NGS cycle times significantly longer than 5 minutes each. To read the large surface area of ​​1 μm beads via NGS—for example, a 1.75 cm × 1.75 cm surface composed of densely packed 1 μm beads containing approximately 10 billion individual 1 μm beads—the length of the NGS position barcode must be greater than 24 bp to reach the diversity (possible combinations of position barcodes) of at least 200 trillion different beads in the library to reduce the possibility of barcode redundancy within the large surface area, ultimately requiring more than six decoding cycles.

[0151] In an exemplary existing technology, all beads are labeled with a different combination of fluorophores before decoding by OCS (e.g., as described in WO2022 / 187719), so high dynamic range (HDR) imaging should be used when determining identifier strand sequences via an OCS workflow for accurate decoding. Because the diversity of the bead library for the described identifier strands and OCS workflow is only 1 million, how can a capture array cover a 1.75 cm × 1.75 cm surface area with such low diversity of beads? This is solved using semi-patterned bead arrays, in which x- and y-coordinate oligonucleotide barcodes are printed directly onto randomly arranged beads within each feature of the semi-patterned bead array. These x- and y-coordinate barcodes are directly ligated onto bead capture oligos, imparting additional positional information to the beads within each feature of the semi-patterned bead array. This is more clearly outlined below in the capture array section. The problem with this approach is that each randomly ordered bead capture array must be read individually, which can be made much more suitable for the manufacture of many arrays by copying the information contained within the bead capture oligo array onto an acceptor array, as described in more detail below.

[0152] Capture Array: The coordinated identifier / NGS bead library can be immobilized on a surface at a desired density using one of many different attachment chemistries (covalent or non-covalent) for downstream visual encoding and decoding experiments, such as those using OCS. Preferably, the beads are randomly arranged in a semi-patterned fashion, so that the patterned features of the array consist of defined areas for bead immobilization. These bead immobilization features can consist of squares or other shapes of defined size (e.g., a 100 μm × 100 μm square in which approximately 1,000 closely packed 3 μm beads can be randomly arranged, or a 35 μm × 35 μm square in which approximately 1,200 1 μm beads can be randomly arranged). Once randomly arranged within the square, a droplet printer can add additional oligos to the array at x and y coordinates. These x and y coordinate oligos can be directly ligated to the NGS barcodes on the beads to provide additional positional information. For example, an array containing 35 μm × 35 μm squares spaced 1–3 μm apart in a 500 × 500 grid covers a total surface area of ​​over 1.75 cm × 1.75 cm (Figure 10). Each square of the grid contains a unique combination of oligos, whereby the x-coordinate oligos are ligated via splinting to the NGS barcodes of the beads, which contain the identifier strand information. The x-coordinate oligos are added first to a row of the grid and contain the 5′ region used for templated splint ligation to the NGS3 barcodes (3′ acceptor regions) of the beads, the 5-base region corresponding to that row of the grid, and the 3′ region for templated splint ligation to the y-coordinate oligos. The y-coordinate oligos are added to the grid after the x-coordinate oligos are ligated to the beads and include a 5' region for splint ligation to the x-coordinate oligos on the beads, a 5-base region corresponding to the columns of the grid, and a 3' capture region for capture of the oligos (e.g., during isothermal signal transduction reactions described herein).Thus, through the use of additional spatial information provided by the x and y coordinate oligos deposited within the square, very large surface areas can be covered without risking bead redundancy and by reading a relatively low diversity library of differentially barcoded beads (1 million as described herein). The square grid can be expanded to increase the total surface area of ​​the capture array. This overall approach (a square grid encompassing randomly arranged beads) reduces the overall diversity of beads required to generate a large surface area capture array and significantly reduces the time required to read each capture array.

[0153] Reading location of identifier / NGS cooperatively encoded beads: Identifier strands on beads can be identified by imaging before and after exposure to unique / orthogonal / non-competitive REs, one at a time, in a series of cycles (as described in WO2022 / 187719 and briefly discussed herein). Importantly, the cleavage agents used (e.g., REs) do not cleave the NGS barcodes. Thus, after the OCS experiment used to read the bead's location, the corresponding NGS capture oligos encoding the bead's location remain intact and eligible for capturing the information released during the one-pot isothermal signaling / spatial encoding reaction.

[0154] Use of Capture Arrays: There are various ways to functionalize spatially encoded capture arrays for use. After reading the bead positions within a semi-patterned random bead array, the beads can be embedded in a gel, detached from the surface where their positions were determined (as in the example shown herein), and used directly as a capture array (Figure 11A). Rigid surfaces such as patterned silicon wafers, glass, or plastic are less suitable for the transfer reaction because they are not flexible (like gels or membranes), and therefore do not conform well to the sample. In addition, even if the beads are covalently bound to the capture array surface, maintaining bead immobilization on the rigid surface during the transfer reaction is difficult. This is because, with any transfer approach, achieving subcellular resolution requires bringing the capture array very close to the sample (within just a few microns or in contact). This physical contact can literally wipe the immobilized beads off the rigid surface. However, if beads are embedded in a flexible matrix such as a gel and then peeled off from the surface on which they were originally immobilized, the beads tend to remain in their original relative positions during the transfer reaction. Furthermore, due to isothermal and potential integrity (in some versions, spatially encoded products are released from the capture beads during the isothermal reaction), a semi-permeable gel can be used throughout the reaction. In this manner, the reagents (enzymes and / or their necessary cofactors) required to initiate and / or carry out the complete isothermal transfer reaction can be introduced across the semi-permeable gel matrix by diffusion. Simply by incubating the array and sample (sandwich) in the isothermal transfer reaction solution at an isothermal temperature (37-42°C), the reaction proceeds and the product is released from the capture features into the surrounding solution, thereby allowing its diffusion through the gel and its subsequent recovery by aspiration of the solution (Figure 12). See also Appendix A in U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which is hereby incorporated by reference.

[0155] After aspiration, further analysis can be as simple as adding isothermal reaction components directly to beads containing attached RNA / DNA hybrid hairpin tags or to samples containing antibody-tag conjugates, incubating for a period of time at isothermal reaction temperature, and then separating the solution from the sample surface (or beads via a magnet), and capturing the tags released into the aspirated solution on capture beads containing capture oligos added to the aspirated solution. These tags are captured in the presence of all isothermal reaction components at the reaction temperature, so that the tags are extended on the capture beads to include the spatial barcode region and PCR handle encoded in the capture oligo. These full-length extension products are then cleaved from the beads by RNAse H activity (the capture oligos contain a 5'-RNA base to enable this after the extension reaction), and these products are eligible for amplification using designed primers.

[0156] There are other ways to improve the stability of the beads (how well the beads remain bound) within the gel matrix than those presented here. Exemplary approaches are described below.

[0157] In the examples presented here, the spatially encoded surface consists of 1 μm beads containing a single capture oligotype (not spatially encoded) bound to a glass surface or embedded in a gel (0.5-2% agarose / 10 mM Tris pH 7.5). These examples demonstrate the biochemistry of a "one-pot" isothermal reaction. To create a robust spatially encoded capture array, its production can be industrialized and requires software components developed to read the bead positions and reconstruct a digital image using next-generation sequencing counts.

[0158] Constructing capture oligos by printing oligos at x and y coordinates As described elsewhere (both herein and in the OCS PCT application), visual barcodes can be operatively linked to capture oligos on the same bead, whereby the visual code is encoded within the DNA sequence of the corresponding capture oligo. To expand the surface area of ​​capture arrays encompassing monodisperse, randomly arranged, low-diversity capture beads (e.g., less than 10 million different beads), it is possible to create subarrays of the beads for further spatial addressing (shown herein in Figures 17A-17C). Fabrication of subarrays of defined size can be achieved using photolithography techniques known in the art, using substrates ranging from silicon wafers to glass, and alternative methods for fabricating subarrays from PDMS can be used. Further spatial addressing can be achieved by droplet printing of x- and y-coordinate oligos at known locations, thereby corresponding individual combinations of x- and y-coordinate oligos to specific subarrays. In this example, the visual barcode (VBC) region of the capture oligo contains (5' to 3') an attachment moiety and linker, a stretch of RNA bases, a 5' PCR handle, a variable region (a stretch of DNA bases) specific to the visual barcode on the bead, followed by a region (a stretch of DNA bases) with complementarity to the x-sprinting oligo used to ligate the x-coordinate barcode (x-BC) oligo. After a solution containing a ligase enzyme (preferably Splint® enzyme) and a unique x-BC oligo is droplet-printed unidirectionally across the subarray, this oligo is splint-ligated to the VBC oligo via base complementarity between the splinting oligo (preferably an RNA splinting oligo) and the 3' end of the VBC oligo. The x-BC oligo may include (5' to 3') a stretch of DNA bases complementary to the x-sprinting oligo, a unique x-coordinate oligo barcode region (which differs between each x-BC oligo), and a stretch of DNA bases complementary to the y-sprinting oligo.Each row (in the case of horizontal printing) or column (in the case of vertical printing) of the subarray receives a unique x-BC oligo for attachment to the VBC oligo on the bead via splint ligation. Following ligation, the array (including all subarrays) is then washed to prepare for printing a unique y-coordinate barcode (y-BC) oligo onto each subarray. The y-BC oligo is then printed in the same manner as the x-BC oligo (e.g., using a ligase enzyme in a buffer), but perpendicular to the direction in which the x-BC oligo was printed. This results in a unique combination of x-BC and y-BC oligos in every subarray. The y-BC oligo may contain a stretch of DNA bases complementary to the y-splinting oligo (5' to 3'), a unique y-coordinate oligo barcode region (which differs between each y-BC oligo), and a stretch of DNA bases complementary to the released hairpin tag (capture region). After ligation, the array (including all subarrays) is washed and prepared for optional splint removal. If the splinting oligos are composed of RNA and the ligation step is performed using Splint® enzyme, the remaining splint oligos can be removed with an RNAse enzyme, or a denaturation step can be performed to remove the capture oligo splints. The visual barcode can be decoded before or after the x- and y-coordinate oligos are ligated, imparting additional spatial information to the visually barcoded beads.

[0159] Attenuation of "high" abundance signals Also contemplated herein are attenuation tag-like nucleic acid molecules and their use for attenuating the signal generated by high-abundance targets in analysis.Generally, such attenuation tag-like nucleic acid molecules differ from hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules by the absence of functional cleavable site, i.e., by the substitution of RNA bases that allow cleavage / release by RNAse H, or by the omission / modification of the restriction endonuclease recognition site that serves as the cleavable site of the tag.

[0160] When multiplexing probes or conjugates for spatially encoded readout, this type of attenuation is used to "see" (detect, readout) all tag sequences, regardless of whether they are high or low abundance. This type of probe attenuation has been demonstrated before, but not for the first time in a system such as the one described herein.

[0161] For example, the assays herein can be used in an attenuated format by including "DNA base-only" tags mixed in (e.g., a predetermined portion or percentage) with hairpin DNA / RNA hybrid tags, so that these conjugates behave identically in the assay (having similar sizes and structures, including any hairpin structure), but the signal from highly abundant targets can be attenuated. Similarly, the signal from a hairpin tag that relies on a stretch of DNA bases containing a restriction enzyme (RE) recognition site for cleavage can be attenuated by including a similar tag that does not contain the RE recognition site.

[0162] There are risks in expanding probe content to a large number of differentially coded probes. Because the described system allows for unprecedented expansion, it is important to account for the fact that highly expressed biomarkers do not "drown out" reads from biomarkers with low expression levels. For example, attenuation of very high-count probes can be achieved by mixing two conjugate types (one type conjugated with a hairpin tag containing only DNA bases and the other type conjugated with a hairpin tag containing an RNA / DNA hybrid region) in a known ratio (which can then be back-calculated during analysis).

[0163] Improving bead stability (immobilization) within the gel matrix: Beads can be made more stable within the gel matrix by adding components to the gel that specifically bind to moieties on the beads. For example, beads can be multifunctionalized through chemical modification to include a variety of different solvent-accessible unique / orthogonal / non-competitive reactive chemistries. Trifunctional beads can include a first reactive chemical handle for attachment to the identifier strand, a second (unique / orthogonal / non-competitive) reactive handle for attachment to the NGS barcode, and a third (unique and distinct from the other two, orthogonal) reactive handle for covalent or non-covalent immobilization within the gel matrix.

[0164] Generation of Capture Arrays: Advantageously, it is not necessary to generate a new bead array and read it by OCS every time a capture array is made. A preferred method involves creating a bead library as described above and immobilizing the beads to read their position within the semi-patterned array as described above, but instead of embedding the beads in a gel and physically transferring the beads within the gel (as in the previously described capture arrays above), the information contained within the NGS capture oligos is copied into the gel (Figure 11B). This can be achieved by creating an NGS barcode in a reverse-complementary orientation, where the capture region and PCR handle of the capture oligo are swapped relative to each other. In this configuration, the capture region (in the reverse complement) is oriented toward the 5' end closest to the bead, and the constant PCR handle is oriented toward the 3' end furthest from the bead. A constant priming oligo with complementarity to the 3' end of the reverse-complementary NGS barcode oligo can hybridize to all beads on the surface. This priming oligo may include a chemical modification for polymerization into a gel, such as polyacrylamide. Next, all reverse-complementary NGS barcode oligos can be converted to dsDNA by an extension reaction. The full length (correct complement) of the NGS barcode is then attached to the reverse-complementary NGS barcode oligo on the bead by hybridization. The acrydite-modified primers used in the extension reaction (this modification is available from commercial oligonucleotide manufacturers such as IDT) can be directly polymerized into polyacrylamide gels cast at various percentages. A 4% polyacrylamide gel will allow permeability of macromolecules during the isothermal signaling reaction. These gels can be cast very thinly (<200 μm thick), allowing the NGS barcode (location and information) to be directly polymerized into the gel.Cast gels can be peeled from the beads (in the presence of dsDNA denaturants such as >50% formamide, >3 M urea, or 0.1 M sodium hydroxide) without dislodging the beads from the array surface, allowing for gentle removal of the gel from the bead bed, which may allow multiple gels to be cast from the same bead array by repeating the stretching and gel-casting steps. Thus, the most ideal capture array would not contain beads but would contain information from the beads that was enzymatically copied and subsequently transferred to the gel by polymerization (Figure 11B). To impart rigidity to 4% polyacrylamide gels or other gels that may ultimately be used, a semipermeable membrane can be placed over the gel before polymerization or gel hardening, creating a layered "sticker" that can be used as a capture array. This rigidifying matrix can be composed of a biologically inert material with a defined pore size, such as cellulose, various meshes composed of suitable plastics, or PTFE.

[0165] Instrumentation. General instrumentation requirements for reading the features (e.g., beads) described herein are typical and conventional, including those previously described in WO 2022 / 187719, as well as those for other DNA sequencing technologies. In some embodiments, it may be beneficial for the instrument to be capable of scanning large surface areas (up to 10 cm x 10 cm with the described grid system) densely packed with 1 μm beads using high dynamic range imaging (HDR) to resolve immobilized barcodes, with the ability to see at least, for example, up to five or more colors (e.g., in some embodiments, standard fluorophores), or even more in embodiments employing quantum dots with appropriate filters, during each decoding cycle. Solution circulation is performed under automated microfluidics, regulated by instrument control software that also controls the timing of image acquisition.

[0166] Software. It will be appreciated that the methods, systems, and workflows described herein are supported by, for example, instrument control software (including, for example, as described in the literature, with modifications to accommodate the feature encoding and decoding cycles described herein), image analysis software used to decode the beads, software for creating a 2D map of the bead array in the form of DNA sequences (spatial barcode sequences) at each feature / bead location, software for aligning NGS reads to create a list based on the spatial barcode sequences (e.g., to allow tag counts to be associated with physical locations within the 2D feature array map), and software for displaying the digital image by overlaying tag read counts with physical locations within the 2D feature (bead) array map to generate a digital image.

[0167] The term "orthogonal" refers to a multi-component system in which one component has chemical reactivity with a particular reagent under a specific set of reaction conditions, while at least one other component of the multi-component system has limited or no reactivity with the reagent, even though all components of the multi-component system exist in the same environment. Additional terms used include "unique" (e.g., reactivity acting on different targets, such as REs cleaving at distinct nucleic acid sites) and "non-competitive" (e.g., reactivity that may occur under the same conditions but does not act in overlapping or competing reactions (e.g., with respect to components such as reaction sites)). Isoschizomers are unique enzymes (usually pairs) (in terms of enzyme structure) but have the same target sequence recognition and cleavage activity. Thus, two isoschizomers have the same biological activity but are (structurally) unique enzymes. Isoschizomers are not considered "non-competitive" because they cleave the same target sequence, and therefore may not be suitable for use in the single workflow described herein.

[0168] Similarly, the phrase "orthogonal reactivity" refers to a multi-component system in which, although all components of the system are in the same environment, some components in the system have chemical reactivity with a particular reagent under a particular set of reaction conditions, while at least one or more components in the system do not.

[0169] The following illustrative embodiments and examples are included to demonstrate particular aspects of the present disclosure. In light of the present disclosure, those of ordinary skill in the art should recognize that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0170] Exemplary Embodiment Set 1: 1. A DNA / RNA hybrid hairpin tag having a structure as shown or described herein. 2. 2. The DNA / RNA hybrid hairpin tag of embodiment 1, comprising (in 5'→3' order) an attachment moiety, a stretch of RNA bases, a tag PCR handle, a tag ID barcode, a tag PCR handle complementary sequence, and a stretch of DNA bases complementary to at least a portion of the stretch of RNA bases. 3. The DNA / RNA hybrid hairpin tag of embodiment 1 or embodiment 2, having a structure as shown in FIG. 1A or having the sequence of tag v1, v2, v3, v5, or v6. 4. A released hairpin tag derived from the DNA / RNA hybrid hairpin tag of any one of embodiments 1-3, for example as shown in FIG. 1B. 5. A tagged element comprising an element having the DNA / RNA hybrid hairpin tag of embodiment 2 or 3 attached thereto via said attachment moiety. 6. The tagging component of embodiment 5, wherein said element comprises one or more of a biological molecule (such as a protein or nucleic acid), a cell or tissue, an affinity molecule (such as an antibody), a bead, or another addressable feature. 7. A capture oligo (CO) having a structure as shown or described herein. 8. 8. The CO of embodiment 7, comprising (in 5'→3' order) an attachment moiety, a first stretch of DNA bases, a tag ID barcode, and a second stretch of DNA bases complementary to at least a portion of said first stretch of DNA bases. 9. The CO of embodiment 7 or embodiment 8, having the structure shown in FIG. 2 or FIG. 3. 10. The CO of embodiment 8 or embodiment 9, wherein the CO is attached to a capture feature through the attachment moiety. 11. The CO of any one of embodiments 7 to 10, further comprising a conditionally cleavable element. 12. A capture element comprising the element of any one of embodiments 8-11 having CO attached thereto through said attachment moiety. 13. The capture element of embodiment 12, wherein the element comprises a bead or another addressable capture feature. 14. a capture element in an array of different capture elements; each CO in each of a plurality of different capture elements of the array comprises a different tag ID barcode; The capture element of embodiment 12 or embodiment 13. 15. a DNA / RNA hybrid hairpin tag having a structure as shown or described herein, or a released hairpin tag derived from said DNA / RNA hybrid hairpin tag, and a capture oligo (CO) having a structure as shown or described herein; the sequence of the DNA / RNA hybrid hairpin tag and the sequence of the CO are at least partially complementary so that when the hairpin tag is released in the vicinity of the CO, the released hairpin tag is captured by the CO; Capture pair. 16. A method for the detection and / or quantification of a target in a substantially two-dimensional (2D) sample as described or shown herein. 17. The method of embodiment 16, which is a "one-pot" method carried out essentially at a single temperature (ie, isothermally). 18. The method of embodiment 16 or 17, as shown (in whole or in part) in any of Figures 4A, 4B, 5A, 5B, 6, 7A, 7B, 8, 9A-9E, 12, 13, or in Appendix A to U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes the present application. 19. 19. The method of any one of embodiments 16-18, wherein the method also provides location information for one or more targets within said substantially 2D sample. 20. 20. The method of any one of embodiments 16-19, comprising the enzymatic activity of one or more RNase H, DNA polymerase, reverse transcriptase, RNA polymerase, and / or one or more restriction enzymes. twenty one. 21. The method of any one of embodiments 16-20, further comprising sequence analysis of the plurality of nucleic acid molecules comprising one or more molecular ID tags. twenty two. 10. A spatially encoded capture array substantially as herein described or shown. twenty three. The spatially encoded capture array of embodiment 22 shown in Figure 10 or 11, or in Appendix A of U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes this application. twenty four. 24. The spatially encoded capture array of embodiment 22 or 23, comprising capture elements embedded in a biomolecule-permeable matrix. twenty five. 25. The spatially encoded capture array of embodiment 24, wherein the capture elements comprise beads, the biomolecule-permeable matrix is ​​a gel, or both. 26. 26. The spatially encoded capture array of embodiment 24 or embodiment 25, in the form of a flexible "sticker" intended to be used in direct contact with a substantially 2D sample for analysis of targets within said sample. 27. 10. A spatial coding workflow essentially as described or illustrated herein. 28. The spatial encoding workflow of embodiment 27 shown in Figure 12 or Figure 13, or in Appendix A of U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which precedes this application. 29. A spatially encoded feature array having a grid-within-a-grid arrangement as shown in FIG. 10 and described herein. 30. A computer-readable medium or digital resource, or digital database, containing spatial location information for features of a spatially encoded array described herein. 31. 31. The computer-readable medium or digital resource, or digital database of embodiment 30, comprising spatial location information for substantially all features of said spatially encoded array. 32. Use of the computer-readable medium or digital resource, or digital database of embodiment 30 or embodiment 31 to provide a user with location information about one or more targets that correlates with the spatial location information of the spatially coded array. 33. The method of embodiment 32, wherein the correlation occurs through the use of a spatially encoded array in a workflow or method described or illustrated herein. 34. 10. A method for isothermal spatial encoding of a biological sample substantially as herein described or shown. 35. Use of the DNA / RNA hybrid hairpin tags described or shown herein for transcriptome analysis of biological samples. 36. 10. A spatially encoded surface, such as a capture array, e.g., a bead-based capture array, substantially as described or shown herein.

[0171] Exemplary Embodiment Set 2: 1. A hairpin tag nucleic acid molecule comprising parts A, B, C, D, and E operably linked in 5' to 3' order, Part A is (1) a stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site a cleavable site comprising any of: Part B contains a stretch of DNA bases that includes a tag PCR handle; part C comprises a stretch of DNA bases comprising a tag ID barcode, and said stretch of DNA bases forms part of the loop of said hairpin; Part D comprises a stretch of DNA bases having the reverse complement of the tag PCR handle, thereby forming part of the stem of the hairpin; and Part E is (1) the stretch of RNA bases in part A, or (2) the stretch of DNA bases containing the RE recognition site and a stretch of DNA bases having the reverse complement of at least a portion of any of the following sequences, thereby forming part of the stem of the hairpin: The hairpin tag nucleic acid molecule. 2. The hairpin tag nucleic acid molecule of embodiment 1, further comprising an attachment moiety conjugated to the 5' end of part A. 3. The hairpin tag nucleic acid molecule of embodiment 2, wherein the attachment moiety provides amine-reactive crosslinker activity or thiol-reactive crosslinker activity. 4. 3. The hairpin tag nucleic acid molecule of embodiment 2, further comprising a linker between said attachment moiety and part A. 5. The hairpin tag nucleic acid molecule according to aspect 4, wherein the linker comprises PEG(n), where n=1 to 20. 6. 2. The hairpin tag nucleic acid molecule of embodiment 1, wherein the tag ID barcode is at least 4 bases in length. 7. 7. The hairpin tag nucleic acid molecule of embodiment 6, wherein the tag ID barcode is 4, 5, 6, 7, 8, or more than 8 bases in length. 8. 2. The hairpin tag nucleic acid molecule of embodiment 1, wherein said tag PCR handle in part B and its reverse complement in part D are each at least 10 bases in length. 9. 9. The hairpin tag nucleic acid molecule of embodiment 8, wherein said tag PCR handle in part B and its reverse complement in part D are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length, respectively. 10. The hairpin tag nucleic acid molecule of any one of embodiments 1 to 9, constructed at least in part using a templated ligation reaction. 11. 11. The hairpin tag nucleic acid molecule according to any one of aspects 1 to 10, wherein part A comprises the stretch of DNA bases including the RE recognition site, and the RE recognition site is at least 4 bases in length. 12. 12. The hairpin tag nucleic acid molecule of embodiment 11, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length. 13. 12. The hairpin tag nucleic acid molecule of embodiment 11, wherein the RE recognition site is a type IIS restriction enzyme recognition site, a site for an RE that generates a 3' overhang, or both. 14. a DNA / RNA hybrid hairpin tag nucleic acid molecule, Part A comprises the stretch of RNA bases, and the stretch of RNA bases in part A is at least 5 bases long. A hairpin tag nucleic acid molecule according to any one of embodiments 1 to 10. 15. 15. The DNA / RNA hybrid hairpin tag nucleic acid molecule of embodiment 14, wherein the stretch of RNA bases in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length. 16. A set of two or more hairpin tag nucleic acid molecules according to any one of aspects 1 to 13 and / or DNA / RNA hybrid hairpin tag nucleic acid molecules according to aspect 14 or aspect 15, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence. 17. 17. The set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules of embodiment 16, further comprising at least one attenuation tag-like nucleic acid molecule, which attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by the lack of a functional cleavable site. 18. 18. The set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules of embodiment 17, wherein the attenuation tag-like nucleic acid molecules differ from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by (1) having DNA bases instead of RNA bases of part A, or (2) lacking an RE recognition site of part A. 19. A tagged probe comprising a probe molecule attached to a hairpin tag nucleic acid molecule according to any one of embodiments 2 to 13, or to a DNA / RNA hybrid hairpin tag nucleic acid molecule according to embodiment 14 or embodiment 15, via the attachment moiety. 20. 20. The tagged probe of embodiment 19, wherein the probe molecule comprises an affinity molecule having binding affinity for a target molecule. twenty one. 21. The tagged probe of embodiment 20, wherein the affinity molecule comprises an antibody binding domain having affinity for an antigen, and the target molecule comprises the antigen. twenty two. 20. The tagged probe of embodiment 19, wherein the probe molecule comprises one or more of an antibody or binding fragment thereof, a nucleic acid, a small molecule, an organic or inorganic chemical, a putative drug target, an identified pharmaceutical drug, or a biological macromolecular complex. twenty three. 20. The tagged probe of embodiment 19, wherein the probe molecule is one of a set of probe molecules, each of which comprises one of a plurality of members of a small molecule library, a drug target library, a biological affinity molecule library, a natural product library, a bioactive compound library, a genome library, a transcriptome library, a metabolomics library, or a drug screening library. twenty four. 21. The tagged probe of embodiment 20, wherein the target molecule comprises a biological molecule, an inorganic object, or an addressable feature of an array. twenty five. 25. The tagged probe of embodiment 24, wherein the target molecule comprises a biological molecule, the biological molecule comprising one or more proteins, lipids, carbohydrates, nucleic acid molecules, or a combination of proteins, lipids, carbohydrates, and / or nucleic acid molecules. 26. 26. The tagged probe of embodiment 25, wherein the target molecule is one of a plurality of molecules that constitute a complex, and the complex is located outside or inside one or more cells in a tissue sample. 27. 27. The tagged probe according to any one of embodiments 19 to 26, further comprising an amplification sequence comprising a polymerase promoter sequence. 28. 28. The tagged probe of embodiment 27, wherein the amplified sequence comprises a T7 promoter sequence, such as a T7 promoter adapter. 29. 16. A released hairpin tag nucleic acid molecule derived from a hairpin tag according to any one of aspects 2 to 13 or derived from a DNA / RNA hybrid hairpin tag according to aspect 14 or aspect 15, wherein the released hairpin tag has been separated from the attachment moiety by the enzymatic action of a restriction endonuclease or an RNase H enzyme. 30. A capture oligo(CO) nucleic acid molecule comprising parts I-II-III-IV operably linked in 5' to 3' order, Part I is (1) a single RNA base or a continuous stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site a cleavable site comprising any of: Part II comprises a stretch of DNA bases including a CO PCR handle; Part III comprises a stretch of DNA bases comprising a spatial barcode; and Part IV comprises a stretch of DNA bases comprising a tag capture region; The capture oligo(CO) nucleic acid molecule. 31. The CO nucleic acid molecule of embodiment 30, further comprising an attachment moiety conjugated to the 5' end of part I. 32. The CO nucleic acid molecule of embodiment 31, wherein the attachment moiety provides an amine-reactive crosslinker activity or a thiol-reactive crosslinker activity. 33. 32. The CO nucleic acid molecule of embodiment 31, further comprising a linker between the attachment moiety and part I. 34. 34. The CO nucleic acid molecule of embodiment 33, wherein the linker comprises PEG(n), where n=1 to 20. 35. 31. The CO nucleic acid molecule of embodiment 30, wherein the spatial barcode is at least 4 bases in length. 36. 36. The CO nucleic acid molecule of embodiment 35, wherein the spatial barcode is 4, 5, 6, 7, 8, or more than 8 bases in length. 37. 31. The CO nucleic acid molecule of embodiment 30, wherein the CO PCR handle in part II is at least 5 bases in length. 38. 38. The CO nucleic acid molecule of embodiment 37, wherein the CO PCR handle in part II is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length. 39. 39. The CO nucleic acid molecule of any one of embodiments 30 to 38, further comprising a unique molecular identifier (UMI). 40. 39. The CO nucleic acid molecule of any one of embodiments 30 to 38, wherein the sequence of the CO nucleic acid molecule has no more than two consecutive bases with internal sequence self-complementarity. 41. 41. The CO nucleic acid molecule of embodiment 40, wherein the sequence of the CO nucleic acid molecule has no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, or no more than 12 consecutive bases with internal sequence self-complementarity. 42. 42. The CO nucleic acid molecule of any one of embodiments 30 to 41, constructed at least in part using a templated ligation reaction. 43. 43. The CO nucleic acid molecule of any one of aspects 30 to 42, wherein part I comprises the stretch of DNA bases that includes the RE recognition site, and the RE recognition site is at least 4 bases in length. 44. 44. The CO nucleic acid molecule of embodiment 43, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length. 45. 44. The CO nucleic acid molecule of embodiment 43, wherein the RE recognition site is a type IIS restriction enzyme recognition site, a site for an RE that leaves a 3' overhang, or both. 46. a DNA / RNA chimeric CO nucleic acid molecule, Part I comprises a single RNA base; A CO nucleic acid molecule according to any one of embodiments 30 to 42. 47. a DNA / RNA chimeric CO nucleic acid molecule, Part I comprises the contiguous stretch of RNA bases; A CO nucleic acid molecule according to any one of embodiments 30 to 42. 48. 48. The DNA / RNA chimeric CO nucleic acid molecule of embodiment 47, wherein the contiguous stretch of RNA bases in part I is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length. 49. A set of two or more CO nucleic acid molecules according to any one of aspects 30 to 45 and / or DNA / RNA chimeric CO nucleic acid molecules according to any one of aspects 46 to 48, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence. 50. A CO nucleic acid molecule according to any one of embodiments 31 to 45 or a DNA / RNA chimeric CO nucleic acid molecule according to any one of embodiments 46 to 48, attached to a capture feature via the attachment moiety. 51. 51. The CO nucleic acid molecule of embodiment 50, wherein the capture feature is one of a bead, a chemically functionalized spot on a glass surface, a defined region of a chemically functionalized permeable gel, a bead or other inorganic object embedded within or on the surface of a permeable gel, or a series of spatially defined objects attached to a gel. 52. 51. The CO nucleic acid molecule of embodiment 50, wherein the capture features are spatially addressable features in an array. 53. 53. The CO nucleic acid molecule of embodiment 52, wherein the array is a microarray having at least 100 addressable capture features. 54. A spatially encoded capture feature comprising a capture feature to which a CO nucleic acid molecule described in any one of embodiments 31 to 42 or a DNA / RNA chimeric CO nucleic acid molecule described in any one of embodiments 43 to 48 is attached via the attachment moiety. 55. 55. The spatially encoded capture feature of embodiment 54, wherein the capture feature comprises a bead or an addressable location on a substantially two-dimensional surface. 56. a spatially encoded capture feature in an array of at least 100 different spatially encoded capture features; each of the CO nucleic acid molecules on each of the at least 100 different spatially encoded capture features of the array comprises a different spatial barcode; A spatially encoded capture feature according to embodiment 54 or embodiment 55. 57. a hairpin tag nucleic acid molecule according to any one of embodiments 2 to 13, or a DNA / RNA hybrid hairpin tag nucleic acid molecule according to embodiment 14 or embodiment 15, or a released hairpin tag nucleic acid molecule according to embodiment 29; and A capture oligo(CO) nucleic acid molecule according to any one of embodiments 31 to 42, or a DNA / RNA chimeric CO nucleic acid molecule according to any one of embodiments 43 to 48, or an attached CO according to any one of embodiments 50 to 73. a capture pair comprising: the sequence of the hairpin tag nucleic acid molecule and the sequence of the CO nucleic acid molecule are at least partially complementary such that when the hairpin tag nucleic acid molecule is released from its attachment moiety in the vicinity of the CO nucleic acid molecule, the released hairpin tag nucleic acid molecule is captured by sequence-complementary bonding at its 3' end to the CO nucleic acid molecule, such that the resulting complex of the released hairpin tag nucleic acid molecule and the CO nucleic acid molecule is competent for a downstream extension reaction by a polymerase enzyme; said capture pair. 58. A spatially coded capture array comprising a defined array of spatially addressed capture features, each capture feature comprising: Predefined, addressable locations on a substantially two-dimensional solid surface, or Beads or other similar discrete solid capture objects spatially distinguishable features, including: Multiple copies of the CO nucleic acid molecule of any one of embodiments 31 to 42 or the DNA / RNA chimeric CO nucleic acid molecule of any one of embodiments 43 to 48 attached at each feature. Including, CO nucleic acid molecules in each feature have a unique spatial barcode sequence compared to CO nucleic acid molecules in other features within the array. The spatially encoded capture array. 59. one or more CO nucleic acid molecules, droplet printing of the CO onto the predefined addressable locations on the substantially two-dimensional solid surface; or Attachment of the CO onto beads through the attachment moiety. 59. The spatially coded capture array of embodiment 58, wherein the signal is applied to spatially addressed capture features within the array by 60. 60. The spatially coded capture array of embodiment 58 or embodiment 59, wherein said array comprises beads each comprising a visual barcode operatively linked to said CO. 61. 61. The spatially encoded capture array of embodiment 60, wherein the visual barcode allows beads to be assigned to locations within the capture array. 62. 59. The spatially encoded capture array of embodiment 58, wherein the beads or other similar discrete capture entities are embedded in a biomolecule-permeable matrix. 63. the capture object comprises beads; the biomolecule-permeable matrix comprises a gel; or It's both. 63. The spatially encoded capture array of embodiment 62. 64. 64. The spatially encoded capture array of embodiment 63, wherein the biomolecule-permeable matrix comprising the gel is formatted as a flexible sticker. 65. The biomolecule-permeable matrix is Structurally stable at selected temperatures between 4 and 45°C; Permeable to proteins such as functional RNAse H and polymerase; permeable to ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ Permeable to ions; is substantially inert to biological molecules; and be sufficiently flexible to allow for the direct application of a relatively thin layer of said matrix to a substantially two-dimensional sample or surface; 63. The spatially encoded capture array of embodiment 62. 66. 66. The spatially encoded capture array of embodiment 65, constructed as a three-dimensional thin gel, the width and length of which are substantially greater than the thickness of the gel, and the spatially distinguishable capture features are arranged in substantially a single plane across the entire surface of the gel defined by the length and width of the gel. 67. 67. The spatially encoded capture array of embodiment 66, wherein at least a first of the spatially distinguishable capture features is directly attached to and / or in contact with a second of the spatially distinguishable capture features. 68. 68. The spatially encoded capture array of any one of embodiments 62-67, wherein the matrix comprises a hydrogel or a polyacrylamide gel. 69. 69. The spatially encoded capture array of embodiment 68, wherein the thickness of the matrix or gel is about 2 mm or less. 70. 70. The spatially coded capture array of embodiment 69, wherein the thickness of the matrix or gel is about 1 mm or less, 500 μm or less, 250 μm or less, 200 μm or less, 150 μm or less, 125 μm or less, 100 μm or less, or less than 100 μm. 71. 71. The spatially encoded capture array of embodiment 70, wherein the thickness of the matrix or gel is 100-200 μm, 100-150 μm, or about 125 μm. 72. 72. The spatially encoded capture array of any one of aspects 58-71, wherein the array is reinforced by an inert mesh or other support structure. 73. the predefined addressable locations on a substantially two-dimensional solid surface have a surface area of ​​about 1 μm×1 μm or less; or the beads or other similar discrete solid capture objects have a diameter of about 20 μm or less; 73. The spatially encoded capture array of any one of embodiments 58 to 72. 74. 74. The spatially coded capture array of embodiment 73, wherein the beads or other similar discrete solid capture objects have a diameter of 18 μm or less, 15 μm or less, 12 μm or less, 10 μm or less, 8 μm or less, 5 μm or less, 3 μm or less, 1 μm or less, or about 100 nm. 75. 75. The spatially encoded capture array of embodiment 74, wherein said beads or other similar discrete solid capture objects have a diameter of 1-3 μm. 76. A semi-aligned spatially coded capture array, including: A grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide comprising: an x-coordinate adaptor oligonucleotide used to label all spatially addressable locations within a row of the grid; and y-coordinate adapter oligonucleotides used to label all spatially addressable locations within a column of the grid the grid being applied to the spatially addressable locations by splint ligation of 77. 77. The semi-aligned spatially encoded capture array of embodiment 76, constructed at least in part using the method provided in Figures 17A-17C. 78. 77. The semi-aligned spatially encoded capture array of embodiment 76, having a grid-within-a-grid format as shown in FIG. 10. 79. A semi-aligned spatially coded capture array, including: An array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray comprising a uniquely identifiable capture feature, the location of which is determined at least in part by the identity of the subarray within the semi-aligned spatially coded capture array. 80. A semi-aligned spatially coded capture array, including: A set of two or more subarrays, each containing a plurality of capture features, wherein each capture feature in each subarray is attached to a capture oligo containing a unique position tag, the capture features in each subarray are randomly arranged, and the capture features in each subarray further contain a subarray identifying oligo tag attached by splint ligation to the capture oligo on each feature in the subarray. 81. 1. A method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample, comprising: contacting the substantially 2D sample with at least one tagged probe to generate a substantially 2D stained sample, the tagged probe comprising: a hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode, or a DNA / RNA hybrid hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode; and a probe molecule attached via the attachment moiety to the hairpin tag nucleic acid molecule containing a tag ID barcode or the DNA / RNA hybrid hairpin tag nucleic acid molecule containing a tag ID barcode. a process comprising: contacting the surface of the substantially 2D stained sample with a transparent, spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: a plurality of spatially distinguishable features; and Multiple copies of capture oligo(CO) nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules attached at each spatially distinguishable feature wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array; placing a flow cell or other solution-containing cover over the sample-array sandwich to form an enclosure containing the sample-array sandwich; adding a solution containing reaction components to the enclosure to form a reaction mixture, the components comprising: a cleaving enzyme selected from RNAse H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ ions; and buffer a process comprising: incubating the sample-array sandwich contacted with the reaction mixture at an assay temperature for 30 to 60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the enclosure; and Analyzing the reaction product mixture to detect and / or quantify and / or define the location of the target in the substantially 2D sample. 82. 82. A method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample according to embodiment 81, wherein said enclosure comprises a flow cell. 83. 83. The method of embodiment 81 or embodiment 82, wherein the method is carried out at a single temperature (isothermally) or within a range of about 5°C within a single temperature. 84. 84. The method of any one of aspects 81 to 83, wherein the assay temperature has a range of 20 to 55°C or 37 to 42°C. 85. 85. The method of any one of embodiments 81-84, wherein the method provides location information for more than one target within a substantially 2D sample. 86. 86. The method of any one of embodiments 81 to 85, wherein the at least one polymerase provides a DNA polymerase, reverse transcriptase, or RNA polymerase enzymatic activity. 87. 87. The method of any one of embodiments 81-86, wherein analyzing the reaction product mixture comprises sequence analysis of a plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes. 88. 88. The method of embodiment 87, wherein said analyzing the reaction product mixture comprises next generation sequencing (NGS) of a plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes. 89. 89. The method of embodiment 87 or embodiment 88, wherein said plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes are full extension products released from spatially distinguishable features by cleavage at the cleavage site of a CO. 90. 90. The method of embodiment 89, wherein said cleavage comprises RNAse H enzymatic activity or restriction endonuclease activity. 91. 91. The method of any one of aspects 81-90, wherein said analyzing step comprises assigning a spatial location of at least one nucleic acid molecule comprising a spatial barcode and a tag ID barcode within the substantially 2D sample. 92. A method for isothermal spatial encoding of a biological sample, comprising the method of any one of embodiments 81 to 91, wherein the substantially 2D sample is a biological sample. 93. 49. Use of a set of hairpin tag nucleic acid molecules according to any one of embodiments 16 to 18, or a set of capture oligo(CO) nucleic acid molecules according to embodiment 49, or both, for transcriptome analysis of a biological sample. 94. 10. A spatially encoded surface, such as a capture array, e.g., a bead-based capture array, substantially as described or shown herein. 95. Spatial coding workflows, including: contacting a hybrid RNA / DNA tag containing a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; placing a capture array containing capture features in contact with the stained sample to create a sample / array sandwich; placing a fluid containment enclosure on top of the sample / array sandwich; introducing an assay solution comprising active RNAse H and an active polymerase into the fluid containment enclosure, thereby contacting the assay solution with the sample / array sandwich; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient for RNAse H activity to at least partially digest the hybrid RNA / DNA tag to generate a cleavable tag, and thereby release the cleavable tag into the assay solution in the vicinity of the capture feature; allowing the cleavable tag to interact with a capture oligo(CO) on the adjacent capture feature to provide a captured cleavable tag; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient to allow polymerase activity to extend the captured cleavable tags using CO as a template to generate extension products; cleaving the extension product with RNAse H in the sample solution once it has been extended sufficiently to generate a complementary RNA / DNA region based on the RNA bases in the CO, thereby releasing a full-length extension product; recovering at least a portion of the released full-length extension products; and amplifying and / or sequencing at least one of the released full-length extension products. 96. A computer-readable medium or digital resource, or a digital database comprising spatial location information for features of a spatially encoded array according to any one of embodiments 58 to 80. 97. 97. A computer-readable medium or digital resource, or digital database according to embodiment 96, comprising spatial location information for substantially all features of the spatially encoded array. 98. Use of a computer-readable medium or digital resource, or a digital database, as described in embodiment 96 or embodiment 97, to provide a user with location information about one or more targets that correlates with the spatial location information of a spatially coded array. 99. The method of embodiment 98, wherein the correlation occurs through the use of a spatially encoded array in a workflow or method described or illustrated herein. 100. A kit comprising one or more of the following: A hairpin tag nucleic acid molecule according to any one of two or more aspects 1 to 13; two or more DNA / RNA hybrid hairpin tag nucleic acid molecules of embodiment 14 or embodiment 15; a set of two or more hairpin tags according to any one of aspects 16 to 18; a set of two or more tagged probes according to any one of aspects 19 to 28; A capture oligo(CO) nucleic acid molecule according to any one of two or more aspects 30-46; two or more DNA / RNA chimeric CO nucleic acid molecules of embodiment 47 or embodiment 48; a set of two or more CO nucleic acid molecules according to any one of embodiments 30 to 45; a set of two or more DNA / RNA chimeric CO nucleic acid molecules according to any one of embodiments 46 to 48; two or more attached CO nucleic acid molecules of any one of embodiments 50-53, each attached to a capture feature; at least one spatially encoded capture array of any one of embodiments 58-75; At least one semi-aligned spatially encoded capture array according to any one of embodiments 76-80; or 95. The spatially coded surface of embodiment 94. 101. The kit of embodiment 100, wherein the spatially coded capture array, semi-aligned spatially coded capture array, or spatially coded surface is in the form of a flexible sticker. 102. The kit according to embodiment 100, further comprising one or more of the following: a vessel containing a functional RNAse H enzyme; a container containing a functional polymerase enzyme; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ a vessel containing a solution containing ions; or A container containing a buffer solution. 103. At least one of the containers contains an RNAse H enzyme, a polymerase enzyme, a restriction endonuclease enzyme, rNTPs and / or dNTPs, Mg 2+ 102. The kit of embodiment 101, comprising at least two of: a cation; a buffer; or a solution. 104. The kit of any one of embodiments 100-103, further comprising one or more of the following: Components useful for preparing samples for analysis using the methods provided herein; a solution-containing cover suitable for placement over the sample-array sandwich on a slide to form a fluid-containing enclosure for the sample-array sandwich; a flow cell cover; or a glass slide or other surface suitable for receiving a substantially two-dimensional sample. [Example]

[0172] Example 1: Construction of a DNA / RNA hybrid hairpin tag Exemplary DNA / RNA hybrid hairpin tags were constructed by ligation of two separate nucleic acids: a first nucleic acid (tag stem oligo) containing RNA bases within its 5' region, and a second nucleic acid (tag loop oligo) consisting entirely of DNA, encompassing bases at its 3' end designed for complementarity with the RNA bases of the first nucleic acid, followed by a hairpin loop region and a stem region at its 5' end. In all cases, the first nucleic acid (tag stem oligo) contained a 5' flexible linker linking it to a reactive primary amine (Integrated DNA Technologies (IDT): / 5AmMC6 / or IDT: / 5AmMC12 / ), and in some cases (as outlined below), the second nucleic acid (tag loop oligo) contained an internal amino-modified base (IDT: / iAmMC6T / ) for covalent labeling with amine-reactive chemistries. Prior to labeling nucleic acids, any remaining free amino groups carried over from the synthesis reaction (if standard desalted oligos were ordered from the vendor) were removed by sodium acetate / EtOH precipitation using the published protocol. The precipitated amino-modified oligos (and any internally amino-modified oligos or oligos ordered as purified and delivered lyophilized by the manufacturer) were resuspended in HO to 400 μM and then diluted 1:1 with 200 mM sodium phosphate buffer, pH 8.5. The resulting samples contained 200 μM of each amino-modified oligo in a final N-hydroxysuccinimide (NHS) conjugation buffer composition of 100 mM sodium phosphate, pH 8.5, for downstream labeling with NHS-modifying reagents.

[0173] To each 100 μl oligo sample, 10 μl of the appropriate NHS-containing labeling reagent (20 mM in anhydrous DMF) was added at approximately a 10-fold molar excess for overnight conjugation at room temperature. The oligo-labeling reaction was then quenched by adding 25 μl of 1 M Tris pH 7.5 to each sample. All oligo-labeling reactions were analyzed on a 15% TBU gel (ThermoFisher EC6885BOX), where unmodified oligos of the same type were run in parallel with modified oligos in adjacent lanes to observe a clear upward shift of the bands in the lanes corresponding to their successful and complete modification with the appropriate NHS reagent (no bands corresponding to unmodified oligos were observed in any of the modified oligo lanes); 2 pmol per band was loaded and stained with SYBR™ Gold (ThermoFisher S11494). To remove excess labeling reagent from each reaction, oligos were precipitated with sodium acetate / EtOH, resuspended in 100 μl of HO, and then desalted using a 0.5 ml 7.5K MWCO Zeba™ column (ThermoFisher 89883) equilibrated with HO to further remove excess labeling reagent. Oligos labeled with surface or probe-attached chemistries (here, biotin or trans-cyclooctene (TCO) were used) were subjected to a second desalting step by repeating the above desalting procedure for more complete removal of any remaining free label. Oligos labeled with fluorophores were typically subjected to a single desalting step. These two oligos (tag stem oligo and tag group oligo) were enzymatically covalently linked together using T4 DNA ligase (M0202S) or Splint® ligase (NEB M0375S) with relatively equal efficiency as determined by denaturing nucleic acid gel analysis using 10% TBU gels (ThermoFisher EC68752BOX).

[0174] Various versions of the DNA / RNA hybrid hairpin tag (referred to here as v1–v6) were tested to compare their relative performance when conjugated to surfaces and antibody probes for use in analyte binding experiments and in tissue staining workflows. From these experiments, it was concluded that smaller, more compact tag versions, containing fewer unpaired bases within the stem and loop regions, exhibited fewer nonspecific interactions with tissue samples under optimized staining procedures. Tag version v6 was selected for downstream experiments due to its compact size (and the relatively few solvent-exposed single-stranded bases that could result in nonspecific interactions with the sample) and its overall performance in the tissue staining procedure. Tag version v6 (whose full sequence is provided herein) can be ordered as a full-length tag from a vendor (purchased here from IDT) to include a 5' flexible linker bearing a primary amino group for conjugation with an NHS-bearing labeling agent (IDT: / 5AmMC12 / ) without the need to ligate two separate species. However, for all dual-labeled v6 tags used here, the described two-step labeling and ligation approach, followed by gel purification of the ligation product, was employed.

[0175] Example 2: Optimization of tag capture region To determine optimal capture of truncated hairpin tags by capture oligos, a series of oligos was designed and ordered from IDT (shown below: Ab_DRD-tag1 truncated v1–v6). These capture experiments were performed at an isothermal assay temperature (42°C) and in an isothermal assay buffer (1x RNAse H buffer from NEB). Oligos within this series of hairpins differed only by the length of their capturable region, allowing for a titration series around the capture Tm. The underlined sequences correspond to the region of each oligo in the series that was captured by the capture oligo in the experiment. The salt-adjusted Tm of these sequences was calculated using the Northwestern Oligo Calc website, analyzed at an oligo concentration of 50 nM and a salt (monovalent cation: Na+) concentration of 75 mM. Ab_DRD-tag1 truncated v1; salt adjusted Tm=60.4 (SEQ ID NO: 8) Ab_DRD-tag1 truncated v2; salt adjusted Tm=54.3 (SEQ ID NO: 9) Ab_DRD-tag1 truncated v3; salt adjusted Tm=52.9 (SEQ ID NO: 10) Ab_DRD-tag1 truncated v4; salt adjusted Tm=46.4 (SEQ ID NO: 11) Ab_DRD-tag1 truncated v5; salt adjusted Tm=43.8 (SEQ ID NO: 12) Ab_DRD-tag1 truncated v6; salt adjusted Tm=40.9 (SEQ ID NO: 13)

[0176] To perform capture experiments, MyOne T1 streptavidin Dynabeads were coated with a single biotin-modified capture oligotype (DRD1_CO_6sUMI - DNA bases; SEQ ID NO: 14) consisting of only DNA bases at a bead concentration of 1 mg / mL and an oligo concentration of 200 nM in SA binding buffer (10 mM Tris pH 7.5, 500 mM NaCl) while mixing at room temperature. The capture beads were washed as outlined above, and 1 mg / mL of the beads were mixed with various hairpin tags (truncated v1–v6; SEQ ID NO: 8–13) at a hairpin tag concentration of 50 nM for incubation in 1× RNAse H buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl, 10 mM DTT, pH 8.3) at 42°C for 30 minutes. After incubation, the samples were quickly transferred from the temperature block to a magnetic tube stand and allowed 3-4 minutes for the magnet to fully capture the beads. The supernatant was collected, and the beads were resuspended in 1x RNAse H buffer equal to the volume of the collected supernatant. Equal volumes of these two fractions were run on a 10% TBU denaturing gel (ThermoFisher EC68752BOX). The amount of hairpin oligo contained in the bead fraction was compared with the amount of hairpin oligo contained in the supernatant fraction to generate a ratio for each hairpin oligo tested. Gel analysis revealed that hairpin oligo v4 was the most efficiently captured of the six hairpins tested under these conditions, and therefore the length and composition of this capture region served as the basis for designing the final capture length of the optimized v6 hairpin tag.

[0177] Example 3: Capture Oligos The capture oligos (COs) used here were either composed entirely of DNA (DRD1_CO_6sUMI - DNA bases; SEQ ID NO: 14) or were designed to include RNA bases within their 5' ends (DRD1_CO_6sUMI - RNA bases; SEQ ID NO: 38). The capture oligos contained a flexible linker at the 5' end, which had a primary amino group for downstream conjugation with an NHS-modified label. In the experiments shown here, a 3' inverted T base modification was included to cap the 3' end of the COs (preventing extension). COs in NHS conjugation buffer (200 μM oligo in 100 mM sodium phosphate buffer, pH 8.5) were modified with NHS-LC-biotin (ThermoFisher 21336) at a 10-fold molar excess of labeling agent, as described in the labeling procedure outlined above, and allowed to react overnight at room temperature. The conjugation reaction was then quenched with 1 M Tris pH 7.5 as described above and precipitated with sodium acetate / EtOH using standard techniques. The precipitated pellet was resuspended in 100 μl of HO. The resuspended oligos were then subjected to two sequential desalting steps per sample using 0.5 mL 7.5 MWCO Zeba desalting columns (ThermoFisher 89883) to remove as much free biotin as possible. To construct a CO containing a 5'-RNA base, the two oligos were splint-ligated together using T4 DNA ligase and a splinting oligo designed to be complementary to both the 3' end of the biotinylated oligo containing the RNA base and the 5' end of the downstream oligo fragment encoding the remainder of the capture oligo sequence. Following ligation, the full-length biotinylated CO product encompassing the 5'-RNA base was gel-purified for use in downstream immobilization experiments.

[0178] Example 4: Capture beads Capture beads were generated by coating MyOne T1 streptavidin Dynabeads (ThermoFisher 65601) with CO. Briefly, the beads were washed according to the manufacturer's instructions, sonicated to reduce bead aggregation, and resuspended to 1 mg / mL in SA binding buffer (500 mM NaCl, 10 mM Tris pH 7.5) containing 200 nM biotinylated CO. The tube was rotated for 30 min at room temperature for continuous mixing. Once coated with CO, the beads were washed six times in SA binding buffer, with sonication steps between washes to remove any free oligos. The capture beads were then resuspended to a concentration of 1–2 mg / mL in the appropriate buffer (different buffers were used for different intended applications), sonicated to reduce aggregation, and used for various downstream tag capture experiments.

[0179] Example 5: Conjugation of DNA / RNA hybrid hairpin tags with antibody probes Gel analysis of tag-conjugated antibodies typically consists of three lanes of a 3-8% Tris-acetate gel (ThermoFisher WG1602BOX) for each conjugation reaction: lane 1 contains protein size markers, lane 2 contains 1 μg of the original, unconjugated antibody, and lane 3 contains approximately 2-3 μg of the tag-conjugated reaction mixture. In typical results, lane 2 contains two bands: one migrates at approximately 65 kD relative to the protein size marker, corresponding to the BSA carrier protein in the antibody solution, and the second migrates at approximately 150 kD, corresponding to the unconjugated antibody. Lane 3 consistently contains two major bands shifted higher in the gel (relative to the migration of the original, unconjugated antibody) corresponding to antibodies modified with either one tag molecule or two tag molecules, as well as a very faint band (usually <5%) corresponding to the original, unconjugated antibody and the carrier BSA band at approximately 65 kD. This typical result demonstrated successful conjugation of the DNA / RNA hybrid hairpin tag to the antibody probe, such that the reaction mixture primarily contained one or two tags per antibody molecule (as expected). For analysis of antibodies conjugated with fluorescently labeled DNA / RNA hybrid hairpin tags, the gel was first imaged using a fluorescent gel scanner (ThermoFisher iBright Imaging System) using the appropriate excitation wavelength and emission filter for each fluorophore, followed by staining with Coomassie Blue. Two fluorescent bands were typically observed in the lane loaded with the conjugation reaction, and after Coomassie Blue staining, a banding pattern similar to that seen in the nonfluorescent analysis gel of the conjugate (described above) was observed.

[0180] Example 6: In vitro binding assay To determine whether conjugation to the DNA / RNA hybrid hairpin tag affects antibody binding to the target analyte, an in vitro binding assay was performed using an octet instrument (OctetRED 384, forteBIO, Division of Pall Life Sciences), in which anti-human IgG (Jackson ImmunoResearch 109-005-003) was conjugated to DNA / RNA hybrid tag v1 (SEQ ID NO: 3) and used in the experiment along with samples of unconjugated antibody and conjugated antibody preincubated with RNAse H (NEB M0297S). The unmodified antibody and both conjugate samples were tested in a 3-fold dilution series ranging from 18 μM to 74 nM for binding to biotinylated human IgG immobilized on a high precision streptavidin coated Octet chip (SATORIUS - Octet® High Precision Streptavidin (SAX) Biosensors - 18-5117). The procedure was performed at 37°C and 500 RPM and included a 15-minute chip pre-equilibration step in equilibration buffer (10 mM Tris pH 7.4 supplemented with 80 mM NaCl), followed by a 1-minute analyte loading step in loading buffer (1x PBS supplemented with 100 nM biotinylated human IgG), followed by a 1-minute wash step in blocking buffer (50% Superblock / 50% PBS supplemented with 1 mg / ml BSA), followed by a 10-minute association step (exposure to antibody samples at different concentrations in blocking buffer), followed by a 10-minute dissociation step. These results showed similar association kinetics among all three samples at all concentrations tested, although a slight decrease in the dissociation rate of the conjugate was observed. It was hypothesized that the use of 10% DMSO in the dissociation step might increase the dissociation rate of the conjugate, and this was observed experimentally.It was hypothesized that including a small amount of detergent (0.01–0.2% Tween-20) in the dissociation step or increasing the salt concentration (to 300 mM NaCl) might further increase the dissociation rate of the conjugate. These components improved the dissociation rate of the conjugate to a lesser extent than that observed with the addition of 10% DMSO. This lower dissociation rate was observed only with DNA / RNA hybrid hairpin tag v1 (SEQ ID NO: 3) and may not apply to tag v6 (SEQ ID NO: 7).

[0181] Example 7: Stability of DNA / RNA hybrid tag-conjugated antibodies To test the stability of conjugates stored at 4°C for several weeks, Tagv6-conjugated antibodies (α-ER [EPR4007] (Abcam: ab108398) and α-HER2 / ErbB2 (R&D Systems: MAB1129)) were stored in PBS containing 0.25% BSA at 4°C for 7 weeks. Unconjugated antibodies (1 μg per lane) and HPv6-conjugated antibodies (2–3 μg per lane) were run in separate lanes on a 3–8% tris-acetate gel and visualized by Coomassie blue staining. Lanes containing stored conjugated antibodies showed that the majority of the antibodies (>95%) were still stably conjugated to either one or two Tagv6 molecules after 7 weeks.

[0182] Example 8: Exemplary antibody staining procedure The following protocol was used for antibody staining on cells in 2D culture: 1) Fix the cells with 4% PFA for 10 minutes. 2) Permeabilize by incubating with 0.25% Triton-X in 1x PBS for 15 minutes at room temperature. 3) Block with Image-iT™ FX Signal Enhancer (Life Technologies, I36933) for 30 minutes at room temperature. 4) Block with BSA (50 mg / ml) in SuperBlock™ (Life Technologies, 37580) and fragmented salmon DNA (Thermo, AM9680) in a ratio of 93%:2%:5% for 30 minutes at room temperature. 5) Allow the antibody-tag conjugate to bind overnight at 4°C or for 2 hours at room temperature. 6) Wash away unbound antibody with three 5 minute washes in PBST (0.02% Tween-20 in 1x PBS).

[0183] For FFPE tissue sections, the following steps were performed: 1) FFPE slides were baked at 65°C for 30 minutes. 2) Washing: 3 times in xylene (5 min), 2 times in 100% EtOH (5 min), 2 times in 95% EtOH (3 min), 2 times in 70% EtOH (3 min), and 2 times in HO (3 min). 3) Antigen retrieval was performed in sodium citrate buffer (pH 6.0) under high pressure at 110°C for 15 minutes. 4) After antigen retrieval, the procedure continues from step 3 of the antibody staining procedure above.

[0184] The labeled antibodies were used at the following concentrations: α-ER [EPR4007] (Abcam:ab108398) - used at 1:150 α-HER2 / ErbB2 (R&D Systems:MAB1129) - used at 1:100

[0185] Example 9: Imaging All imaging was performed on a Leica THUNDER widefield deconvolution microscope using one of four objectives: HC PL APO 20x 0.8 NA, HCX PL FLUOTAR L 40x 0.6 NA CORR PH2, HC APO 40x 1.25 NA GLYC CORR CS2, or HC PL APO 63x 1.4 NA OIL. The excitation light source was provided by a Lumencor SPECTRA X light engine housing the following LED sources: 395, 440, 470, 510, 550, 640, and 750. The emission filters consisted of a quad cube (Ex: 375-407, 462-496, 542-566, 622-654; Dc: 415, 500, 572, 660; Em: 420-450, 506-532, 578-610, 666-724) and a Y7 cube (Ex: 672-748, Dc: 760, Em: 765-855). The left side of the emission signal from the quad cube was cleaned up using a final high-speed filter wheel equipped with the following LP filters: 440, 510, 590, 700, and 100%. Images were then collected with a Leica DFC9000 sCMOS camera.

[0186] Example 10: Tag recovery and qPCR analysis of released tags In some experiments, DNA / RNA hybrid hairpin tags were cleaved from MyOne T1 streptavidin Dynabeads (ThermoFisher); in others, these tag types were cleaved from conjugated probes (antibodies) bound to analytes in biological samples (e.g., fixed cells or tissue sections) immobilized on glass slides. Following labeling of the analytes in the samples (here, Her2 or ER were probed) with the appropriate antibodies conjugated to two differentially barcoded HPv6 tags (Her2-v6 tag1 and ER-v6 tag2, respectively), the samples were briefly washed with 1x RNAse H buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, pH 8.3). Following this wash, RNAse H (NEB M0297S) in 1x RNAse H buffer (75 units of enzyme in 300 μl of buffer) was placed directly on the sample for tag cleavage (release). The reaction was performed on a heat block set at 37°C for 30 minutes, and the solution was removed from the sample (cells or tissue sections) by aspiration. The 300 μl of aspirated solution was then split in half, and one half received capture beads (0.1 mg / mL final) coated with a capture oligo (DRD1_CO_6sUMI - DNA base; SEQ ID NO: 14) containing only DNA bases. The other half (150 μl) received capture beads (0.1 mg / mL final) coated with a capture oligo (DRD1_CO_6sUMI - RNA base; SEQ ID NO: 38) containing RNA at its 5' end. DRD1_CO_6sUMI - DNA base (SEQ ID NO: 14) DRD1_CO_6sUMI - RNA base (SEQ ID NO: 38)

[0187] The DRD1_CO_6sUMI-RNA base oligo was generated by split ligation of the following two small oligos: CO_5_RNA (SEQ ID NO: 15) CO_3_invT (SEQ ID NO: 16) CO_Splint (used to ligate CO_5_RNA and CO_3_invT) (SEQ ID NO: 17)

[0188] To these two samples, dNTPs (200 μM each, final concentration) and 1 μl of Maxima Reverse Transcriptase (NEB) [200 U / μl] containing RNAse H activity were added for templated extension of the 3' end of the capture tag by the capture oligo. The extension reaction was allowed to proceed at 37°C for 45 minutes. Following this incubation, the beads were pulled to a magnet, and the supernatant (approximately 125 μl) was collected by aspiration from each of the two reactions. The remaining beads were resuspended in 125 μl of 1× RNAse H buffer and incubated at 85°C for 3 minutes to melt the extension product from the capture oligo. The samples were quickly transferred from the heat source to the magnet, and the supernatant was collected as soon as the beads were completely pulled by the magnet. For each sample, these two fractions were designated as the supernatant and beads, respectively. The fractions were then subjected to qPCR for quantification. Briefly, 10 μl fractions were carried over to a qPCR assay using Thermo Fisher Scientific PowerUp SYBR Master Mix and the following primers: 5' DRD LNA v4 (SEQ ID NO: 18; where + indicates locked nucleic acid (LNA)) 3' DRD consensus primer v4 (SEQ ID NO: 19)

[0189] Initial tests of tag recovery, capture, extension, and amplification were performed using Hpv6 antibodies conjugated to α-ER [EPR4007] (ab108398) abcam or α-HER2 / ErbB2 (MAB1129) used to stain a mixture of SKBR3 (Her2+, ER-) and MCF-7 (Her2-, ER+) cultured cells immobilized on glass slides.

[0190] Example 11: α-HER2-v6 tag1 antibody conjugate staining of SKBR3 vs. MCF7 cultured cells For experiments with SKBR3 and MCF7 cells, approximately 50,000 cells were grown on coverslips to approximately 70% cell density, fixed and permeabilized as described above, and used for downstream staining experiments. To capture tags released from probe-tag conjugates bound to biological samples, capture beads were coated with one of two different capture oligo types as described above: one containing only DNA bases (DRD1_CO_6sUMI - DNA base; SEQ ID NO: 14) and the other containing RNA bases at the 5' end (DRD1_CO_6sUMI - RNA base; SEQ ID NO: 38). The inclusion of only DNA bases in the capture oligo allows for capture and extension of the 3' end of the captured tag, while the inclusion of RNA bases at the 5' end of the capture oligo allows for capture, extension, and release of the extension product via a complete "one-pot isothermal" reaction, with the creation of a new DNA / RNA hybrid region formed after tag extension by polymerase. The RNAse H activity of E. coli RNAse H (NEB M0297S) and Maxima polymerase enzyme (ThermoFisher Maxima EP0741) not only liberates the tag from the surface-bound probe, but also liberates the extended tag attached to the capture oligo via the newly formed DNA / RNA hybrid region. Following the α-HER2-v6 tag1 staining experiment, the sample was washed with 1x RNAse H buffer and exposed to a solution containing E. coli RNAse H to cleave the tag from the bound conjugate as described above. This solution was removed from the sample after incubation at isothermal reaction temperature, divided into two equal volumes, and two different types of capture beads were added to capture the released α-HER2-v6 tag1 hairpin in the presence of Maxima RT and dNTPs.In experiments using a capture oligo composed solely of DNA bases, qPCR results demonstrated that the level of released α-HER2-v6 tag1 hairpin in the bead fraction after probing of SKBR3 cells was increased by approximately 13.7-fold relative to the bead fraction after probing of MCF7 cells with this conjugate, consistent with known expression levels of the respective HER2 proteins. These results suggested that the tag cleavage (hairpin release), capture, and extension steps of the method were functioning as expected.

[0191] In probed SKBR3 cells, the released α-HER2-v6 tag1 hairpin signal was observed to be 15-fold higher in the bead fraction than in the supernatant fraction, and a 40-fold increase in signal over background was observed by qPCR. Following experiments using a capture oligo containing an RNA base at the 5' end, qPCR results demonstrated that the released α-HER2-v6 tag1 hairpin signal in the supernatant fraction after probing SKBR3 cells with an anti-Her2-v6 tag conjugate was approximately 11.7-fold higher than in the supernatant fraction after probing MCF7 cells. This recovered signal was similar to the amount of signal obtained from the bead fraction when a DNA-only capture oligo was used. Furthermore, the released α-HER2-v6 tag1 hairpin signal in the supernatant fraction was observed to be approximately 1.5-fold higher than in the bead fraction, indicating that after extension of the hairpin captured on the capture oligo, approximately 60% of the extension product was released into the supernatant. These results demonstrate that, as expected, during the isothermal one-pot signaling reaction, the truncated α-HER2-v6 tag1 hairpin was released from the bound probe and captured by the capture oligo encompassing the 5'-RNA base. The captured tag was then extended, and the extended product was then released from the capture beads. The extension product release step was then optimized to obtain a >90% conversion of extension product from the bead fraction to the supernatant fraction in future experiments. In these final experiments, we observed that the released extension product signal obtained after probing SKBR3 cells in the supernatant fraction was approximately 120-fold increased over background signal.

[0192] Example 12: α-ER-v6 tag2 antibody conjugate staining of SKBR3 vs. MCF7 cultured cells To test different antibody-tag conjugates, the same cell culture samples and capture beads prepared and used in the α-HER2-v6 tag1 antibody conjugate staining of SKBR3 versus MCF7 cultured cells were stained with the α-ER-v6 tag2 antibody conjugate. Following experiments using a capture oligo composed exclusively of DNA bases, qPCR results demonstrated that the detectable released α-ER-v6 tag2 hairpin signal in the bead fraction obtained after staining of MCF7 cells was approximately 0.82-fold higher than that in the bead fraction obtained after staining of SKBR3 cells. Within MCF7 cells, there was an approximately 3.5-fold increase in the released α-ER-v6 tag2 hairpin signal on the capture beads compared to the supernatant fraction, representing an approximately 10-fold increase over background signal. Following experiments using a capture oligo containing RNA bases at the 5' end, qPCR results demonstrated that the released α-ER-v6 tag2 hairpin signal in the supernatant fraction after staining of MCF7 cells was approximately 0.97-fold higher than that observed in the supernatant fraction after staining of SKBR3 cells. In MCF7 cells, the released α-ER-v6 tag2 hairpin signal in the supernatant fraction was approximately 5.9-fold higher than that in the bead fraction. These results suggested that estrogen receptor expression levels were similar between MCF7 and SKBR3 cells, but this was not consistent with previously published studies and was also inconsistent with in-house experiments in which these same cells were stained with an unconjugated ER antibody and visualized using a fluorescently labeled secondary antibody (ThermoFisher A32732).These initial studies indicated that accurate staining and counting of tags released from antibody conjugates used to probe low-expression level proteins may require additional blocking steps, as experiments using the α-HER2-v6tag1 conjugate to probe HER2 protein levels (which are known to be expressed at much higher levels in SKBR3 cells than the ER protein expressed in the nuclei of MCF7 cells) did not require additional blocking steps beyond the use of a blocking solution containing SuperBlock™ supplemented with 1 mg / mL BSA and salmon sperm DNA.

[0193] Example 13: Improvement of blocking and washing conditions for antibody conjugate staining The addition of nucleic acid tags to antibodies can lead to nonspecific interactions between the conjugated antibody and other nucleic acids or positively charged molecules in the sample. These nonspecific interactions can result in increased "background" signals in staining experiments. Therefore, a series of experiments was designed to determine more optimal blocking conditions for staining with the antibody-tag conjugates used here. Briefly, different blocking conditions were tested for nonspecific staining by exposing MCF7 cells (which have low HER2 protein expression levels) to the α-HER2-v6 tag 1 conjugate or SKBR3 cells (which have low or no ER protein expression) to the α-ER-v6 tag 2 conjugate. Initial characterization of the background signals was performed by quantitative immunofluorescence (IF) staining, and interesting results were further confirmed by qPCR analysis.

[0194] After staining MCF7 cells with α-HER2-v6 tag 1, the following blocking or washing steps were tested: control staining was blocked with SuperBlock™ (PBS) blocking buffer (ThermoFisher 37515) and then washed with PBST. From these experiments, a significant amount of background staining was observed in both IF staining and qPCR assays (Figures 15A and 15B).

[0195] The following conditions were then tested and the resulting changes in background signal were quantified via IF staining: Wash with 10% DMSO (1.2-fold reduction in background staining) Further blocking with Denhardt's solution (1.3-fold reduction in background staining) Further blocking with salmon sperm DNA (67-fold reduction in background staining)

[0196] The use of salmon sperm DNA as a blocking agent was further tested in experiments in which SKBR3 cells (highly expressing HER2 protein) were stained with α-HER2-v6 tag1, and this blocking agent had little effect on the overall staining signal. To confirm the observed benefits of using salmon sperm DNA as a blocking agent, MCF7 and 1954 cells were blocked and stained with α-HER2-v6 tag1, washed, RNAse H was added, the released hairpins were captured and extended on capture oligos bound to capture beads, and the samples were quantified by qPCR as described above. These experiments yielded the following findings:

[0197] Upon staining of MCF7 cells (HER2 negative) with α-HER2-v6tag1, the addition of salmon sperm DNA as a blocking agent reduced background staining by approximately 62-fold via qPCR quantification, which is very similar to the levels observed in IF staining experiments.

[0198] Upon staining of 1954 cells (HER2 positive) with α-HER2-v6 tag1, the addition of salmon sperm DNA as a blocking agent reduced the positive staining signal by approximately 1.2-fold via qPCR quantification, which further confirmed the IF results that showed that this blocking agent did not interfere with specific staining by the antibody.

[0199] Although the addition of salmon sperm DNA was effective in reducing background staining when using the α-HER2-v6 tag1 conjugate, this blocking agent was not sufficiently effective to recover accurate signal from experiments using the α-ER-v6 tag2 conjugate. To further improve the blocking conditions, IF experiments were performed after staining of SKBR3 cells (ER protein negative) to determine whether background staining by the α-ER-v6 tag2 conjugate could be further removed. Here, the control condition included salmon sperm DNA in the blocking solution, and the following washing and blocking conditions were tested: 10% DMSO wash - (1.13-fold reduction in background staining) 20% DMSO wash - (1.03-fold reduction in background staining) Image-iT™ FX Signal Enhancer - (4.9-fold reduction in background staining) RNAse A treatment - (1.9-fold reduction in background staining) DNAse A treatment - (1.27-fold reduction in background staining)

[0200] In these experiments, the background signal observed in the nucleus and cytoplasm of these cells was calculated independently, and it was clear that the blocking step was equally effective in both of these compartments of the cell. The above experiments (α-ER-v6 Tag2 antibody conjugate staining of SKBR3 vs. MCF7 cultured cells) were repeated using Image-iT™ FX Signal Enhancer as a blocking agent to test whether the signal from these experiments could be restored. Addition of Image-iT™ FX Signal Enhancer as a blocking agent (in an end-to-end workflow with qPCR readout) resulted in an approximately 1.8-fold increase in ER expression in MCF7 (ER+) cells compared to SKBR3 (ER-) cells. While significant, this restoration of ER signal from these experiments may not fully reflect the ER levels in the cells. Therefore, further optimization of blocking and washing conditions would be beneficial.

[0201] Example 14: Library construction: To test for sequence bias during hairpin recovery and PCR amplification, a hairpin library was designed to mimic the structure of the released hairpin species. This library consisted of four random nucleotide positions (Ns) within the hairpin loop, representing the positions of various tag barcodes. This library was prepared by IDT via mechanical mixing of four different bases at each position. The ordered oligo library was then PAGE-purified in-house using a 10% TBU gel. The library amplification bias test oligo sequence is shown in SEQ ID NO: 37.

[0202] The PAGE-purified oligos were then diluted to 0.5 pM in 1x RNAse H buffer. 22.5 μl of this sample was added to 25 μl of Q5® Hot Start High-Fidelity 2x Master Mix and 2.5 μl each of the following primers: DRD forward overhang P5 tag (SEQ ID NO: 20) DRD reverse overhang P7 tag (SEQ ID NO: 21)

[0203] Primer stock concentrations were 1 μM, with a final concentration in the reaction of 50 nM. The following PCR cycling conditions were used: 98°C (30 s) (1×), 98°C (10 s) → 72°C (15 s) (25×), 72°C (4 min) (1×). The reaction produced a single band of approximately 156 bp, visualized on a 6% TBE gel stained with SYBR Gold. The PCR reaction (50 μl) was purified with AMPure XP beads at a 1.5× ratio to remove excess (unextended) primers. The washed product from this "first stage" of library preparation was eluted from the beads in IBI Scientific PCR-grade water (30 μl), yielding approximately 5 ng / μl of product.

[0204] For the "second stage" of library preparation, 1 μl (5 ng) of the AMPure XP purified product was added to a 50 μl PCR reaction with Q5® Hot Start High-Fidelity 2× Master Mix, and 18 cycles of PCR were performed under the same cycling conditions as in the "first stage" of library construction, except that the following primers were used: P7-G7 index primer (SEQ ID NO: 22) P5-G7 index primer (SEQ ID NO: 23)

[0205] When visualized on a 6% TBE gel stained with SYBR Gold, this reaction yielded a major band reaching approximately 225 bp. This 225 bp band was further purified through a two-step size selection with AMPure XP beads (0.6x ratio → 1.5x). The purified 225 bp band was subjected to a bidirectional Sanger sequencing reaction using the following primers: KAPA Primer 1 (SEQ ID NO: 24) KAPA primer 2 (SEQ ID NO: 25)

[0206] Example 15: Sanger sequencing of extension products Sanger sequencing was performed using Azenta / GeneWiz to analyze the PCR-amplified loop library test oligos. The trace file showed a random, uniform mixture of A, T, C, and G bases at the barcode position "NNNN." While it is difficult to determine whether any specific barcodes were omitted by Sanger sequencing, it is clear that there is likely a bias toward G or C at the first position after the priming site within the tag. To address this issue, for example, an optimized version of Tag v6 can include one or more G bases immediately downstream of the priming site. Next-generation sequencing (NGS) analysis of the hairpin loop library test oligos allowed us to elucidate this phenomenon at a higher resolution.

[0207] Example 16: NGS analysis of extension products The same bands subjected to Sanger sequencing were also subjected to next-generation sequencing (NGS) on an Illumina NovaSeq 6000 S4 flow cell (150 paired-end). This analysis yielded a total of 52,000,000 viable reads in Geneious Prime. To construct a reference genome library for analysis, sequences corresponding to all 256 possible barcodes (4 bases) were generated in the R programming language, with 10 nucleotides on either side of the barcode to match the expected adjacent v6 tag sequence of the template. The paired-end (PE) reads and the "256 barcode" reference genome were imported into Geneious Prime® 2023.0.4 for trimming and alignment. Paired-end reads were trimmed to 10 nucleotides on both sides of the barcode position, and these reads were aligned using the "Geneious" mapper with five iterations of refinement. This alignment demonstrated that 98.6% of the reads mapped to the reference genome library. The percentage occurrence of all 256 barcode tags was determined by dividing the number of reads obtained for each barcode sequence by the total number of reads mapped to the reference genome library. Appendix B submitted herewith is a table containing the read numbers.

[0208] The graph shown in Figure 16A was generated from 1 million randomly selected reads from 26,000,000 PE reads aligned against a reference genome library; the percentage of occurrence of each barcode was then plotted. This was done five times, with 1 million randomly selected reads each time, to allow for error calculations. The percentage of reads obtained for each of the 256 barcodes was then plotted using GraphPad Prism.

[0209] Figure 16B is a graphic "sequence logo" (a graphical representation of patterns within a multiple sequence alignment; see Crooks et al., Genome Res. 14(6):1188-1190, 2004) created by uploading 10,000 reads (successfully aligned to a reference genome) to Weblogo (online at weblogo.berkeley.edu / logo.cgi).

[0210] Example 17: Capture Surface To create biotinylated surfaces, amino-modified glass slides (AutoMate Scientific Po-104 000 406) or glass coverslips (AutoMate Scientific 104 000 406) were first modified with 1 mM NHS-LC-biotin in NHS conjugation buffer. In these experiments, MyOne T1 streptavidin beads, resuspended to 2 mg / ml in SA binding buffer, were attached to the biotinylated surface by allowing the beads to settle onto the surface. The bead-coated surface was then washed to remove all unbound beads. Generally, this procedure resulted in a dense lawn of beads immobilized on the biotinylated surface, as determined by bright-field imaging at various magnifications. Next, the beads were coated with a single biotinylated capture oligotype (500 nM concentration in SA binding buffer) to create a dense monolayer of capture beads attached to the surface for various proof-of-concept experiments. These initial experiments were aimed at obtaining visual confirmation of successful information (tag) transfer from the sample surface (slide) to the capture array via an isothermal signaling reaction. To test this, fluorescently labeled DNA / RNA hybrid hairpin tags modified to include a 5' TCO attachment moiety and an internal fluorophore (AF-647 or AF-550) within their loop region were immobilized on methyltetrazine-modified glass slides. While the slides were kept on ice, a solution containing all the components necessary for the isothermal signal transduction reaction (containing 500 U / mL RNAse H (Slide 1; Experiment 1, see pages 39-41 of Appendix A to U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which is hereby superseded) or not (Slide 2; Experiment 2, see pages 39-41 of Appendix A to U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which is hereby superseded), 200 μM dNTPs, and 5000 U / mL Maxima [RNAse] H minus RT (ThermoFisher EP0751)) was added dropwise to the sample surface.Next, a glass capture array (thin cover slip) consisting of immobilized capture beads coated with a single capture oligotype (containing only DNA bases to allow visualization of the capture process) was placed face down, creating a "sandwich" that brought the capture beads into close proximity with the sample surface in the presence of the two solutions tested. These sandwich-type slide capture arrays were then transferred to a heat block (Thermal mixer with Blocks, ThermoFisher 13687711) preset to 42°C and 0 RPM and incubated at the isothermal transfer reaction temperature. After 30 minutes of incubation, the capture array was detached from the slide by adding 1x PBS buffer (ThermoFisher J61196.AP) dropwise, allowing the buffer to widen the gap between the capture array and the sample slide, allowing for its gentle separation. The slide and capture array were then washed with additional 1x PBS buffer and imaged using the imaging parameters described. Transfer of tags from the sample surface to the capture array was observed only in the presence of a solution containing RNAse H (Slide 1; Experiment 1, see pages 39-41 of Appendix A to U.S. Provisional Patent Application No. 63 / 487,575, filed February 28, 2023, which supersedes this application). In earlier attempts to transfer information from the sample surface to this type of capture array (as described above, fluorescently labeled tag v6 (SEQ ID NO: 7) DNA / RNA hybrid hairpin tags attached to glass slides were used in the described isothermal transfer reaction; data not shown), it was found that capture beads attached to hard surfaces such as glass were not optimal for high-resolution information transfer. Indeed, large features (several millimeters in diameter) containing fluorescently labeled tags could be transferred with high efficiency from glass slides to capture beads attached to hard surfaces, preserving the 2D information contained within the sample surface.However, early attempts to use such glass capture arrays to transfer cellular-level information at subcellular resolution (from a sample surface consisting of fixed Her2+ cells stained with an anti-Her2 antibody conjugated to fluorescently labeled tag v6 (SEQ ID NO: 7)) were successful only when the two surfaces were placed in very close proximity; this required pressing the two surfaces together during the transfer process. This pressure forced the capture beads into the sample's cells, causing them to fluoresce strongly due to the captured tag signal.

[0211] It was hypothesized that capture beads immobilized within a flexible material, such as a gel, would increase the proximity of the capture beads to the sample surface without the need for significant pressure. Furthermore, it was hypothesized that if the flexible material used in the capture array could be made permeable to polymers, the capture array could be placed in close proximity to the sample slide, and then the enzyme and coenzyme components necessary for (at least initiating) the isothermal signaling reaction could be added across the permeable gel to initiate the signaling reaction. Furthermore, if the capture oligos contained RNA bases at their 5' ends, the isothermal signaling reaction of the extension products (the complete reaction in which the tag is released from the probe bound to the sample surface, captured on the capture oligo, extended to include the capture oligo information, and released as a product into the surrounding solution) could be carried out entirely through the gel matrix. These subsequent experiments testing the flexible capture array are outlined below.

[0212] In other experiments, a flexible polymeric permeable gel was used instead of glass as the capture array matrix material. MyOne T1 streptavidin beads were first coated with a single DNA-only capture oligotype as described above, then washed two additional times in 10 mM Tris pH 7.5 and resuspended to 2 mg / ml in 10 mM Tris pH 7.5. This capture bead suspension was then sonicated to reduce bead aggregation and applied directly to an amino-modified glass surface (AutoMate Scientific Po-104 000 406). The capture beads were allowed to settle onto the surface for several hours, where they were likely attached primarily by electrostatic interactions between the DNA on the capture beads and the amino groups on the surface. The bead-coated surface was then washed with 10 mM Tris pH 7.5 to remove any unbound capture beads. Overall, this procedure resulted in the creation of a dense monolayer of capture beads immobilized on the amino-modified surface, as determined by bright-field imaging at various magnifications. Bead lawns were created within a specific shape, as defined by a 125 μm thick sticker (GRACE BIO-LABS 6544008) encompassing eight individual 9 mm diameter holes. The capture bead surface was then placed (bead-coated surface facing up) on a heat block set at 45 °C. While evaporating all remaining buffer (10 mM Tris pH 7.5) from the bead-coated surface, molten agarose (high melting point - catalog number and manufacturer) prepared in 10 mM Tris pH 7.5 with agarose percentages ranging from 0.5 to 2% was cooled to approximately 40-45 °C and slowly dripped directly onto the bead surface.

[0213] A standard glass slide or cover slip was then used to gently press down on the molten gel, flattening the agarose to approximately the thickness of a sticker. After cooling to room temperature, the slide or cover slip used to flatten the gel was gently removed in one direction, revealing a shaped, cast agarose gel "pad" containing a dense lawn of embedded beads. Throughout this exemplary gel-casting procedure, electrostatic interactions maintain adhesion of the captured beads to the amino-modified glass surface, provided the molten agarose is prepared in a buffer containing little to no salt (10 mM Tris pH 7.5 was used successfully here) and is cooled enough to prevent thermal dissociation of the beads from the surface upon exposure to the molten agarose (below 50 °C provided successful results here). Adding molten agarose to a bead monolayer at too low a temperature can also result in mechanical disruption of bead adhesion, so that the slide or cover glass used to flatten the semi-hardened agarose into a thin pad physically disrupts the bead monolayer. Therefore, although these experiments were suboptimal (in many cases, the beads were not stably embedded within the gel), this type of array was still used as a proof-of-concept for obtaining subcellular capture of information from cells (using capture oligos containing only DNA bases). When properly prepared, flexible bead capture arrays can be peeled off the glass surface (like a thin "skin" of the capture beads) and used in capture experiments.

[0214] As outlined above, initial transfer experiments using flexible capture arrays were performed on ice to reduce enzyme activity while the sandwich was being assembled, and then transferred to a heat block preset to 42 °C to initiate the transfer reaction. To assemble the sandwich on ice, a sample slide was placed on an ice platform in an ice bucket, and a drop of reaction mixture was placed on the sample slide, away from the cells bound to the slide. This kept the reaction solution cool before dragging the "skin" of the capture bead array through the reaction solution for immediate placement (beads down) directly onto the sample slide. In some cases, a cover glass or glass slide was placed on top of the capture array, and a small amount of pressure was applied. In general, this type of flexible capture array was highly successful in obtaining subcellular capture of fluorescently labeled tags released from cell-bound probes in the presence of isothermal transfer reaction components (RNAse H, Maxima RT (RNAse H minus), and dNTP cofactors in 1x RNAse H buffer). This was determined using a sample surface consisting of fixed Her2+ cells stained with an anti-Her2 antibody conjugated to fluorescent tag v6 (SEQ ID NO: 7). Images of the sample slide sandwiched between the flexible capture array showed that after a 10-minute incubation at isothermal reaction temperatures (37–42°C), nearly all of the signal obtained from the fluorescent tag was transferred from the probe (bound to cells on the sample slide) to the capture beads in the capture array.

[0215] In subsequent experiments, RNAse H was omitted from the isothermal reaction mixture used during sandwich assembly and was included only in the solution applied to the backside of the capture array. In these experiments, the addition of a trigger enzyme (in this case, RNAse H) via diffusion through the gel was observed to initiate the transfer reaction. In subsequent experiments, capture oligos containing RNA bases at their 5' ends were used under the same conditions as in the previous experiments. Significant enhancement of the signal corresponding to the release of the full-length product was detected by Q-PCR only when RNAse H was included in the solution applied to the backside of the capture array gel (Figure 12). These results suggested that not only can one or more of the components required to initiate an isothermal transfer reaction be applied to the assembled sandwich via diffusion, but the full-length product of the reaction can also be recovered through the gel by diffusion. This allows for a very simple workflow for isothermal transfer from a probed biological sample to a flexible capture array, coupled with suction-mediated recovery of the reaction product, without any additional user intervention.

[0216] Example 18: Conversion of DNA-encoded molecules for isothermal spatial encoding Here, we demonstrate the direct ligation of a T7 (RNA polymerase) promoter-containing adapter to the free end of a dsDNA fragment (mimicking a small molecule probe or library) that has a probe molecule (biotin) attached at its opposite 5' end. Following an optional gel purification step, this ligated species or library can be used in binding experiments, for example, upstream of the isothermal spatial encoding workflow described herein.

[0217] To demonstrate this, we bound adapter-modified DNA-encoded probes (biotin) to streptavidin beads, washed away unbound probes, and mixed these beads with beads containing capture oligos (COs) in the same reaction tube. In these experiments, the COs did not contain a 3'-inverted T base, but instead contained an RNA base at the 5' end of the COs. A one-pot reaction was initiated by introducing a solution containing T7 RNA polymerase and Maxima Reverse Transcriptase to generate in vitro RNA transcripts, which were then captured on capture oligos bound to capture beads. The 3' end of the COs was extended by the Maxima enzyme, combining information from both species (transcribed tag and capture oligo) into a single, contiguous DNA sequence.

[0218] It was also demonstrated that full-length double-stranded extension products can be generated by adding a DNA polymerase (Bst 2.0 or 3.0; capable of extending the 3' end of the captured RNA strand), and that these products can be released by introducing RNase H. This alternative assay chemistry can be implemented into the spatially encoded platform described herein, whereby spatially encoded capture beads are placed on a sample bound to probes. As with the other workflows described herein, the resulting contiguous sequences generated by this approach also contain information related to which probe molecules were present at a given location across the sample, since the information contained within the CO provides spatial coordinates.

[0219] Experimental Method: T7p adapter ligation: Ligation of pre-annealed dsDNA T7p adapters (T7p ligation adapter annealed to T7p ligation adapter_rc; SEQ ID NOs: 31 and 32, respectively) to pre-annealed short tag1 dsDNA oligos containing artificial A-base overhangs (short tag1 with an A-tail annealed to short tag1_rc; SEQ ID NOs: 29 and 3-, respectively) was performed using a 1 μM concentration of dsDNA T7p adapter and a 400 nM concentration of dsDNA short tag in a 200 μl reaction volume containing 1× T4 DNA ligase buffer (NEB M0202S), T4 DNA ligase (NEB M0202S) (final concentration: 30 units / μl), and T4 PNK (NEB M0201S) (final concentration: 0.25 units / μl). Ligation was performed overnight at room temperature. The next day, the ligation reaction was precipitated with EtOH, resuspended in ultrapure water, and washed with Zeba™ Spin Desalting Columns, 7K MWCO (Thermo Fisher 89882) pre-equilibrated with ultrapure water. The same procedure was performed for ligation of the dsDNA T7p adapter to the short tag1 dsDNA oligo lacking an A-base overhang (short tag1 requiring an A-tail annealed to short tag1_rc; SEQ ID NOs: 28 and 30, respectively), except that prior to the ligation step, the oligo was subjected to an A-tailing reaction using NEBNext® Ultra™ II End Repair / dA-Tailing (NEB#E7546S) to add an A-base overhang to the oligo (SEQ ID NO: 29). The A-tailing reaction was cleaned up using a QIAquick PCR Purification Kit column (Qiagen 28104), and the oligos were eluted with 30 μl of ultrapure water prior to ligation with dsDNA T7p adapters. Ligation products were analyzed on a 15% TBE-urea gel (Thermo Fisher EC6885BOX), where the formation of a 64-nt ligation product was observed after both reactions.The ligated constructs were washed via EtOH precipitation, resuspended in ultrapure water, and further washed using Zeba™ Spin Desalting Columns, 7K MWCO (Thermo Fisher 89882) pre-equilibrated with ultrapure water before being used as templates in downstream in vitro transcription reactions.

[0220] In vitro transcription: In vitro transcription reactions were set up to test various T7 promoter-containing tag constructs (encoding FL T7p short tag1) generated via the three different approaches described above: (1) a fully synthesized, biotinylated, purchased construct (FL T7p short tag1 annealed to FL T7p short tag1_rc; SEQ ID NOs: 26 and 27, respectively); (2) a ligation product of an annealed T7p adapter (T7p ligation adapter annealed to T7p ligation adapter_rc; SEQ ID NOs: 31 and 32, respectively) with a short tag1 oligo containing an artificial A base overhang (short tag1 with an A tail annealed to short tag1_rc; SEQ ID NOs: 29 and 30, respectively); and (3) a short tag1 oligo without an A tail (short tag1 requiring an A tail annealed to short tag1_rc; SEQ ID NOs: The ligation products of the tag constructs described in (1) above were used to mimic the "target" to which the biotin "probe" (attached to this oligo construct) binds, prior to standard washes (described elsewhere in this application after coating streptavidin beads with biotinylated oligos) to remove unbound probe. These beads were then equilibrated with 1x transcription buffer before being subjected to in vitro transcription reactions. The remaining two tags (2 and 3) were tested for transcript generation in solution. All in vitro transcription reactions were set up using the HiScribe® T7 High Yield RNA synthesis Kit (NEB #E2040S) and incubated at 37°C for 1 hour. After this incubation, one-third of the volume of each reaction was removed and treated with RNase Cocktail™ Enzyme Mix (ThermoFisher AM2286) for 1 hour at 37°C or with TURBO™ DNase (ThermoFisher AM2239).All nuclease reactions were performed in 1x DNAse I buffer (ThermoFisher AM2239). Gel analysis of the transcription reactions was performed using a 15% TBE-urea gel (ThermoFisher EC6885BOX), which showed that approximately 10- to 20-fold amplification of RNA occurred during the reaction, based on a comparison of the intensity of the template band and the RNA band (41 bases). In all three reactions (1–3), a band of the expected size for the RNA product (41 bases) was observed, and this band completely disappeared after treatment with RNAse I; however, this band was unaffected by treatment with DNAse I.

[0221] RNA Capture and Extension: To demonstrate the capture of RNA transcribed from a dsDNA tag modified with a dsDNA T7p adapter, as well as templated extension of the RNA 3' end on a capture oligo (CO), we utilized a CO (containing 5'-RNA bases) identical to those previously described in this application, but without an inverted T (invT) base at the 3' end of the CO to enable extension of the CO after capture of the transcribed RNA. The 3' invT-free RNA CO was constructed in a similar manner to the previous CO via splint ligation between the invT-free RNA CO and CO_5_RNA (SEQ ID NOs: 35 and 15, respectively), using CO_Splint (SEQ ID NO: 17) as a template for ligation. After ligation, the oligos were PAGE purified and coupled to MyOne T1 streptavidin Dynabeads (ThermoFisher 65601) for use in downstream applications.

[0222] To test whether transcribed RNA could be captured and extended on CO2, which could be the buffer used to test this alternative one-pot isothermal reaction, transcribed RNA was spiked into a solution containing CO2 bound to beads at a concentration of 0.25 mg / ml in 1x transcription buffer from the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040S) supplemented with 5% DTT, 50 mM KCl, NTPs, and dNTPs. The reaction volume was 40 μl, to which 1 μl (8 units) of Bst 3.0 (NEB M0374S) was added, and the reaction was incubated at 37°C for 1 hour. Bst polymerase was used because it is known to be able to extend RNA primers, similar to Klenow fragments. After the reaction, the beads were pulled down into 10 mM Tris and heated to 85°C to dehybridize the extended RNA, which was then recovered from the supernatant for gel analysis. The supernatant from this reaction was analyzed on a 15% TBE-urea gel (Thermo Fisher EC6885BOX), revealing a prominent band at approximately 80 bp, corresponding to an extension product (of the RNA strand) that was efficiently completed through the DNA-templated region of the CO but not through the RNA bases templated to the 5' region of the CO. Here, if Bst 3.0 were able to efficiently extend through the RNA bases, we would expect to observe a 96-base product, thereby suggesting that Bst 3.0 possesses weak reverse transcriptase activity under these conditions. The 80-bp product corresponds to a single-stranded product containing RNA at the 5' end and the newly extended DNA bases at the 3' end; this product encompasses the primer landing sites of both CO primers DS1 and DS2. To verify that the observed approximately 80-bp product truly corresponds to the captured and extended RNA, PCR was performed to generate a 79-bp product. In the negative controls (ultrapure water, unextended RNA, or CO alone), no product was generated.

[0223] To test whether CO could be extended via the captured transcribed RNA, 1 μl of Maxima (ThermoFisher EP0741), a reverse transcriptase containing RNAse H activity (RNAse H+), was added to a solution containing CO immobilized on beads along with RNA transcribed from the T7p FL short tag. This step was performed in the same buffer (1X transcription buffer) as described above, and the reaction was incubated at 37°C for 1 hour. The beads were then pulled down with a magnet and suspended in ultrapure water. The beads were then heated to 85°C to disrupt the biotin / streptavidin interaction between the biotin-conjugated CO and the streptavidin beads. After heating, the beads were pulled down with a magnet, and the supernatant was analyzed on a 15% TBE-urea gel (ThermoFisher EC6885BOX). A prominent band was observed at approximately 100 bases, corresponding to the expected size of fully extended CO (templated by the captured RNA strand). To demonstrate that this observed approximately 100-base product truly corresponds to extended CO, as described above, PCR was performed using CO primer DS1 and CO primer DS2 primers to generate a 100-bp product as determined by gel analysis. While it was possible to amplify the expected product, PCR using the same primers failed to amplify a negative control (CO coated beads + transcribed RNA, but without Maxima enzyme), which was subjected to the same heating procedure used to disrupt the oligo attachment (of the biotin moiety from the streptavidin beads).

[0224] Alternative one-pot isothermal reaction: Streptavidin beads coated with biotinylated FL T7p short tags (described above) were subjected to an in vitro transcription reaction supplemented with 1 μl of T7 RNA polymerase (MO255AVial), Bst 3.0 (8 units, NEB M0374S), and Maxima reverse transcriptase (200 units, ThermoFisher EP0741) at a final concentration of 0.05 mg / ml, similar to streptavidin beads coated with CO (described above). This experiment was performed to mimic signal transfer from a surface-bound T7p adapter-modified tag to another surface containing CO. Here, the reaction proceeded for 1 hour at 37°C. RNAse H (5 units, NEB M0297S) was optionally introduced into the reaction after the described in vitro transcription-mediated signal transfer reaction. In both cases, the magnetic beads were pulled down onto a magnet at room temperature, and the product was collected in the supernatant. These two supernatants were then run on a 15% TBU gel. In both cases, a band at the expected size of extended CO (approximately 100 bases) was detected, but this band was more prevalent in the reaction that had been further treated with RNAse H. In both cases, a 79 bp product was amplified via CO primer DS 1 and CO primer DS 2 primers, indicating that this approximately 100 base band was indeed extended CO.

[0225] The above results provide supporting data for Figures 7B and 8.

[0226] Restriction enzyme-mediated release of extension products from capture beads: To demonstrate restriction enzyme (RE)-mediated release of full-length extension products generated via the alternative one-pot isothermal signaling reaction described above, a new capture oligo (biotin TEG-DNA CO without invT; SEQ ID NO: 36) containing an XhoI restriction site was synthesized. This oligo was coupled to MyOne T1 streptavidin Dynabeads (ThermoFisher 65601) as described above for COs containing RNA bases at their 5' ends. Following the in vitro transcription-mediated signaling reaction, half of the reaction was aspirated using a pipette and placed into the same tube. Both reactions were then placed under a magnet to pellet the beads, the supernatant removed, and the beads resuspended in 1x rCutSmart™ buffer (NEB B6004S). 60 units of XhoI (NEB R0146S) was added to one tube (+ XhoI) and then mixed. Both tubes were incubated at 37°C for 15 minutes. After this incubation, both reactions (with and without XhoI) were placed on a magnet, and the supernatants were collected from each tube. These supernatants were used in PCR reactions using CO primers DS 1 and DS 2 (SEQ ID NOs: 33 and 34, respectively), followed by gel analysis. A band of the expected size, 59 bp, was detected in the reaction containing the supernatant from the sample treated with XhoI, but this band was absent in the reaction containing the supernatant from the sample not treated with XhoI enzyme. This result demonstrates that the transferred information (transcribed RNA) was captured by CO and then extended to include both the probe tag and the capture oligo (full-length extension product), and that the introduction of XhoI enzyme subsequently released this information into solution. The above results provide supporting data for Figures 9E and 9B.

[0227] List of oligos used in Example 18 FL T7p Short Tag 1: PAGE purified, T7p, capture region, CO DS Primer 1 landing site (SEQ ID NO: 26) FL T7p Short Tag1_Biotin_rc:PAGE Purified (SEQ ID NO: 27) Short tag 1 with A tail: PAGE purified (SEQ ID NO: 28) Short tag 1 with A tail: PAGE purified (SEQ ID NO: 29) Short tag1_rc: PAGE purified (SEQ ID NO: 30) T7p Ligation Adapter: HPLC Purified (SEQ ID NO: 31) T7p ligation adapter_rc: HPLC purified (SEQ ID NO: 32) CO DS Primer 1: Standard desalted Tm=47-51 (SEQ ID NO: 33) CO DS Primer 2: Standard desalted Tm=47-51 (SEQ ID NO: 34) RNA CO:Page purified without invT (SEQ ID NO: 35) CO_5_RNA (SEQ ID NO: 15) CO_3_invT (SEQ ID NO: 16) CO_Splint(SEQ ID NO: 17) Biotin-TEG-DNA CO without invT: HPLC purified, XhoI restriction site (SEQ ID NO: 36)

[0228] Selected literature Fu et al., Cell 185:4621-4633, 2022, doi.org / 10.1061 / j.cell.2022.10.021 Rodrigues et al., Science 363:1463-1467, 2019 Troll et al., BMC Genomics. 20:1023, 2019, doi.org / 10.1186 / s12864-019-6355-0 Yang et al., Chembiochem 16(9):1365-1370, 2015 US Patent No. 10,002,316 “Spatially addressable molecular barcoding: US 2022 / 0033802 “Method and apparatus for encoding cellular spatial position information” US 2020 / 0190583 “Methods and Compositions for Sequentially Detecting Targets” WO 2018 / 087539 “Tagless encoding chemical library”

[0229] Conclusion As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particularly specified elements, steps, ingredients, or components. Thus, the terms "include" or "including" should be interpreted as stating: "comprise, consist of, or consist essentially of." The transitional terms "comprises" or "comprises" mean having, but are not limited to, and allow for the inclusion of even greater amounts of unspecified elements, steps, ingredients, or components. The transitional phrase "consisting of" excludes all unspecified elements, steps, ingredients, and components. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment.

[0230] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and appended claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the appended claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. For further clarification, the term "about," when used in conjunction with a stated numerical value or range, has the meaning that would reasonably be regarded as such by one of ordinary skill in the art, i.e., some more or some less than the stated numerical value or range, up to within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0231] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0232] As used in the context of describing the present invention (particularly in the context of the appended claims), the terms "a," "an," "the," and similar referents should be considered to cover both the singular and the plural unless otherwise indicated herein and unless clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not otherwise pose a limitation on the scope of the claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0233] Groupings of alternative elements or aspects of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion is made, the specification shall be deemed to include the group as modified so as to satisfy all Markush group descriptions used in the appended claims.

[0234] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will occur to those skilled in the art, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0235] Additionally, throughout this specification, numerous references are made to patents, printed publications, journal articles, other writings, and website content ("References Therein"). Each reference is individually incorporated herein by reference in its entirety for the referenced teachings as of the filing date of the first application in the priority chain in which the particular reference is included. For example, with respect to chemical compounds, nucleic acid, and amino acid sequences referred to herein that are available in public databases, the database entry information is incorporated herein by reference as of the filing date of the first application in the priority chain in which a database identifier for that compound or sequence is first included in the text.

[0236] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0237] The specific details shown in this specification are presented for purposes of illustration and exemplary discussion of preferred embodiments of the present invention only, and for the purpose of providing what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the invention, and the description and / or examples accompanying the drawings will make clear to those skilled in the art how several forms of the present invention may be embodied in practice.

[0238] The definitions and explanations used in this disclosure are meant and intended to be controlling in any future interpretations unless clearly and unambiguously modified in the examples, or unless application of a meaning would make any interpretation meaningless or essentially meaningless, in which case the definition will be applied as provided in Webster's Dictionary, 11th Edition, or the Oxford Dictionary of Biochemistry and Molecular Biology, 2002. nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8 th The word "language" should be cited from a dictionary known to those skilled in the art, such as the English Dictionary of Linguistic Terms (Eds. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

1. A hairpin tag nucleic acid molecule comprising parts A, B, C, D, and E operably linked in 5' to 3' order, Part A is (1) a stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site a cleavable site comprising any of: Part B contains a stretch of DNA bases that includes a tag PCR handle; part C comprises a stretch of DNA bases comprising a tag ID barcode, and said stretch of DNA bases forms part of the loop of said hairpin; Part D comprises a stretch of DNA bases having the reverse complement of the tag PCR handle, thereby forming part of the stem of the hairpin; and Part E is (1) the stretch of RNA bases in part A, or (2) the stretch of DNA bases containing the RE recognition site and a stretch of DNA bases having the reverse complement of at least a portion of any of the following sequences, thereby forming part of the stem of the hairpin: The hairpin tag nucleic acid molecule.

2. 2. The hairpin tag nucleic acid molecule of claim 1, further comprising an attachment moiety conjugated to the 5' end of part A.

3. 3. The hairpin tag nucleic acid molecule of claim 2, wherein the attachment moiety provides amine-reactive crosslinker activity or thiol-reactive crosslinker activity.

4. 3. The hairpin tag nucleic acid molecule of claim 2, further comprising a linker between the attachment moiety and part A.

5. The hairpin tag nucleic acid molecule of claim 4, wherein the linker comprises PEG(n), where n=1 to 20.

6. 2. The hairpin tag nucleic acid molecule of claim 1, wherein the tag ID barcode is at least 4 bases in length.

7. 7. The hairpin tag nucleic acid molecule of claim 6, wherein the tag ID barcode is 4, 5, 6, 7, 8, or more than 8 bases in length.

8. 2. The hairpin tag nucleic acid molecule of claim 1, wherein the tag PCR handle in part B and its reverse complement in part D are each at least 10 bases in length.

9. 9. The hairpin tag nucleic acid molecule of claim 8, wherein the tag PCR handle in part B and its reverse complement in part D are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length, respectively.

10. 10. The hairpin tag nucleic acid molecule of any one of claims 1 to 9, constructed at least in part using a templated ligation reaction.

11. The hairpin tag nucleic acid molecule of any one of claims 1 to 10, wherein part A comprises the stretch of DNA bases including the RE recognition site, and the RE recognition site is at least 4 bases in length.

12. 12. The hairpin tag nucleic acid molecule of claim 11, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length.

13. 12. The hairpin tag nucleic acid molecule of claim 11, wherein the RE recognition site is a type IIS restriction enzyme recognition site, a site for an RE that generates a 3' overhang, or both.

14. a DNA / RNA hybrid hairpin tag nucleic acid molecule, Part A comprises the stretch of RNA bases, and the stretch of RNA bases in part A is at least 5 bases long. A hairpin tag nucleic acid molecule according to any one of claims 1 to 10.

15. 15. The DNA / RNA hybrid hairpin tag nucleic acid molecule of claim 14, wherein the stretch of RNA bases in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length.

16. A set of two or more hairpin tag nucleic acid molecules of any one of claims 1 to 13 and / or DNA / RNA hybrid hairpin tag nucleic acid molecules of claim 14 or claim 15, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence.

17. 17. A set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules according to claim 16, further comprising at least one attenuation tag-like nucleic acid molecule, wherein the attenuation tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by the lack of a functional cleavable site.

18. 18. The set of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules of claim 17, wherein the attenuation tag-like nucleic acid molecules differ from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the set by (1) having DNA bases instead of RNA bases of part A, or (2) lacking an RE recognition site of part A.

19. A tagged probe comprising a probe molecule attached to a hairpin tag nucleic acid molecule of any one of claims 2 to 13 or to a DNA / RNA hybrid hairpin tag nucleic acid molecule of claim 14 or claim 15 via the attachment moiety.

20. 20. The tagged probe of claim 19, wherein the probe molecule comprises an affinity molecule having binding affinity for a target molecule.

21. 21. The tagged probe of claim 20, wherein the affinity molecule comprises an antibody binding domain having affinity for an antigen, and the target molecule comprises the antigen.

22. 20. The tagged probe of claim 19, wherein the probe molecule comprises one or more of an antibody or binding fragment thereof, a nucleic acid, a small molecule, an organic or inorganic chemical, a putative drug target, an identified pharmaceutical drug, or a biological macromolecular complex.

23. 20. The tagged probe of claim 19, wherein the probe molecule is one of a set of probe molecules, each of which comprises one of a plurality of members of a small molecule library, a drug target library, a biological affinity molecule library, a natural product library, a bioactive compound library, a genome library, a transcriptome library, a metabolomics library, or a drug screening library.

24. 21. The tagged probe of claim 20, wherein the target molecule comprises a biological molecule, an inorganic object, or an addressable feature of an array.

25. 25. The tagged probe of claim 24, wherein the target molecule comprises a biological molecule, the biological molecule comprising one or more proteins, lipids, carbohydrates, nucleic acid molecules, or a combination of proteins, lipids, carbohydrates, and / or nucleic acid molecules.

26. 26. The tagged probe of claim 25, wherein the target molecule is one of a plurality of molecules that constitute a complex, and the complex is located outside or inside one or more cells in a tissue sample.

27. 27. The tagged probe of any one of claims 19 to 26, further comprising an amplification sequence comprising a polymerase promoter sequence.

28. 28. The tagged probe of claim 27, wherein the amplified sequence comprises a T7 promoter sequence, such as a T7 promoter adapter.

29. 16. A released hairpin tag nucleic acid molecule derived from a hairpin tag according to any one of claims 2 to 13 or from a DNA / RNA hybrid hairpin tag according to claim 14 or claim 15, wherein the released hairpin tag has been separated from the attachment moiety by the enzymatic action of a restriction endonuclease or RNase H enzyme.

30. A capture oligo(CO) nucleic acid molecule comprising parts I-II-III-IV operably linked in 5' to 3' order, Part I is (1) a single RNA base or a continuous stretch of RNA bases, or (2) A stretch of DNA bases containing a restriction enzyme (RE) recognition site a cleavable site comprising any of: Part II comprises a stretch of DNA bases including a CO PCR handle; Part III comprises a stretch of DNA bases comprising a spatial barcode; and Part IV comprises a stretch of DNA bases comprising a tag capture region; the capture oligo(CO) nucleic acid molecule;

31. 31. The CO nucleic acid molecule of claim 30, further comprising an attachment moiety conjugated to the 5' end of part I.

32. 32. The CO nucleic acid molecule of claim 31, wherein the attachment moiety provides an amine-reactive crosslinker activity or a thiol-reactive crosslinker activity.

33. 32. The CO nucleic acid molecule of claim 31, further comprising a linker between the attachment moiety and part I.

34. 34. The CO nucleic acid molecule of claim 33, wherein the linker comprises PEG(n), where n=1 to 20.

35. 31. The CO nucleic acid molecule of claim 30, wherein the spatial barcode is at least 4 bases in length.

36. 36. The CO nucleic acid molecule of claim 35, wherein the spatial barcode is 4, 5, 6, 7, 8, or more than 8 bases in length.

37. 31. The CO nucleic acid molecule of claim 30, wherein the CO PCR handle in part II is at least 5 bases in length.

38. 38. The CO nucleic acid molecule of claim 37, wherein the CO PCR handle in part II is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length.

39. 39. The CO nucleic acid molecule of any one of claims 30 to 38, further comprising a unique molecular identifier (UMI).

40. 39. The CO nucleic acid molecule of any one of claims 30 to 38, wherein the sequence of the CO nucleic acid molecule has no more than two consecutive bases with internal sequence self-complementarity.

41. 41. The CO nucleic acid molecule of claim 40, wherein the sequence of the CO nucleic acid molecule has no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, or no more than 12 consecutive bases with internal sequence self-complementarity.

42. 42. The CO nucleic acid molecule of any one of claims 30 to 41, constructed at least in part using a templated ligation reaction.

43. 43. The CO nucleic acid molecule of any one of claims 30 to 42, wherein part I comprises the stretch of DNA bases comprising the RE recognition site, and the RE recognition site is at least 4 bases in length.

44. 44. The CO nucleic acid molecule of claim 43, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases in length.

45. 44. The CO nucleic acid molecule of claim 43, wherein the RE recognition site is a type IIS restriction enzyme recognition site, a site for an RE that leaves a 3' overhang, or both.

46. a DNA / RNA chimeric CO nucleic acid molecule, Part I comprises a single RNA base; A CO nucleic acid molecule according to any one of claims 30 to 42.

47. a DNA / RNA chimeric CO nucleic acid molecule, Part I comprises the contiguous stretch of RNA bases; A CO nucleic acid molecule according to any one of claims 30 to 42.

48. 48. The DNA / RNA chimeric CO nucleic acid molecule of claim 47, wherein the contiguous stretch of RNA bases in part I is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bases in length.

49. A set of two or more CO nucleic acid molecules described in any one of claims 30 to 45 and / or DNA / RNA chimeric CO nucleic acid molecules described in any one of claims 46 to 48, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

50. A CO nucleic acid molecule according to any one of claims 31 to 45 or a DNA / RNA chimeric CO nucleic acid molecule according to any one of claims 46 to 48, attached to a capture feature via the attachment moiety.

51. 51. The CO nucleic acid molecule of claim 50, wherein the capture feature is one of a bead, a chemically functionalized spot on a glass surface, a defined region of a chemically functionalized permeable gel, beads or other inorganic objects embedded within or on the surface of a permeable gel, or a series of spatially defined objects attached to a gel.

52. 51. The CO nucleic acid molecule of claim 50, wherein the capture feature is a spatially addressable feature in an array.

53. 53. The CO nucleic acid molecule of claim 52, wherein the array is a microarray having at least 100 addressable capture features.

54. A spatially encoded capture feature comprising a capture feature to which a CO nucleic acid molecule described in any one of claims 31 to 42 or a DNA / RNA chimeric CO nucleic acid molecule described in any one of claims 43 to 48 is attached via the attachment moiety.

55. 55. The spatially encoded capture feature of claim 54, wherein the capture feature comprises a bead or an addressable location on a substantially two-dimensional surface.

56. a spatially encoded capture feature in an array of at least 100 different spatially encoded capture features; each of the CO nucleic acid molecules on each of the at least 100 different spatially encoded capture features of the array comprises a different spatial barcode; 56. A spatially encoded capture feature according to claim 54 or claim 55.

57. A hairpin tag nucleic acid molecule according to any one of claims 2 to 13, or a DNA / RNA hybrid hairpin tag nucleic acid molecule according to claim 14 or claim 15, or a released hairpin tag nucleic acid molecule according to claim 29; and A capture oligo (CO) nucleic acid molecule according to any one of claims 31 to 42, or a DNA / RNA chimeric CO nucleic acid molecule according to any one of claims 43 to 48, or an attached CO according to any one of claims 50 to 73. a capture pair comprising: the sequence of the hairpin tag nucleic acid molecule and the sequence of the CO nucleic acid molecule are at least partially complementary such that when the hairpin tag nucleic acid molecule is released from its attachment moiety in the vicinity of the CO nucleic acid molecule, the released hairpin tag nucleic acid molecule is captured by sequence-complementary bonding at its 3' end to the CO nucleic acid molecule, such that the resulting complex of the released hairpin tag nucleic acid molecule and the CO nucleic acid molecule is competent for a downstream extension reaction by a polymerase enzyme; said capture pair.

58. A spatially coded capture array comprising a defined array of spatially addressed capture features, each capture feature comprising: Predefined, addressable locations on a substantially two-dimensional solid surface, or Beads or other similar discrete solid capture objects spatially distinguishable features, including: Multiple copies of the CO nucleic acid molecule of any one of claims 31 to 42 or the DNA / RNA chimeric CO nucleic acid molecule of any one of claims 43 to 48 attached at each feature. Including, CO nucleic acid molecules in each feature have a unique spatial barcode sequence compared to CO nucleic acid molecules in other features within the array. The spatially encoded capture array.

59. one or more CO nucleic acid molecules, droplet printing of the CO onto the predefined addressable locations on the substantially two-dimensional solid surface; or Attachment of the CO onto beads through the attachment moiety.

59. The spatially coded capture array of claim 58, wherein the signal is applied to spatially addressed capture features within the array by

60. 60. The spatially coded capture array of claim 58 or claim 59, wherein the array comprises beads each comprising a visual barcode operatively linked to the CO.

61. 61. The spatially coded capture array of claim 60, wherein the visual barcode allows beads to be assigned to locations within the capture array.

62. 59. The spatially encoded capture array of claim 58, wherein the beads or other similar discrete capture entities are embedded in a biomolecule-permeable matrix.

63. the capture object comprises beads; the biomolecule-permeable matrix comprises a gel; or It's both.

63. The spatially encoded capture array of claim 62.

64. 64. The spatially coded capture array of claim 63, wherein the biomolecule-permeable matrix comprising the gel is formatted as a flexible sticker.

65. The biomolecule-permeable matrix is Structurally stable at selected temperatures between 4 and 45°C; permeable to proteins such as functional RNAse H and polymerase; permeable to ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ is permeable to ions; is substantially inert to biological molecules; and be sufficiently flexible to allow for the direct application of a relatively thin layer of said matrix to a substantially two-dimensional sample or surface; 63. The spatially encoded capture array of claim 62.

66. 66. The spatially coded capture array of claim 65, constructed as a three-dimensional thin gel, the width and length of which are substantially greater than the thickness of the gel, and the spatially distinguishable capture features are arranged in a substantially single plane across the entire surface of the gel defined by the length and width of the gel.

67. 67. The spatially encoded capture array of claim 66, wherein at least a first of said spatially distinguishable capture features is directly attached to and / or in contact with a second of said spatially distinguishable capture features.

68. 68. The spatially encoded capture array of any one of claims 62 to 67, wherein the matrix comprises a hydrogel or a polyacrylamide gel.

69. 69. The spatially encoded capture array of claim 68, wherein the thickness of the matrix or gel is about 2 mm or less.

70. 70. The spatially coded capture array of claim 69, wherein the thickness of the matrix or gel is about 1 mm or less, 500 μm or less, 250 μm or less, 200 μm or less, 150 μm or less, 125 μm or less, 100 μm or less, or less than 100 μm.

71. 71. The spatially coded capture array of claim 70, wherein the thickness of the matrix or gel is 100-200 μm, 100-150 μm, or about 125 μm.

72. 72. The spatially encoded capture array of any one of claims 58 to 71, wherein the array is reinforced by an inert mesh or other support structure.

73. the predefined addressable locations on a substantially two-dimensional solid surface have a surface area of ​​about 1 μm×1 μm or less; or the beads or other similar discrete solid capture objects have a diameter of about 20 μm or less; 73. A spatially encoded capture array according to any one of claims 58 to 72.

74. 74. The spatially coded capture array of claim 73, wherein the beads or other similar discrete solid capture objects have a diameter of 18 μm or less, 15 μm or less, 12 μm or less, 10 μm or less, 8 μm or less, 5 μm or less, 3 μm or less, 1 μm or less, or about 100 nm.

75. 75. The spatially encoded capture array of claim 74, wherein said beads or other similar discrete solid capture objects have a diameter of 1 to 3 μm.

76. A semi-ordered spatially coded capture array, including: A grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide comprising: an x-coordinate adaptor oligonucleotide used to label all spatially addressable locations within a row of the grid; and y-coordinate adapter oligonucleotides used to label all spatially addressable locations within a column of the grid the grid being applied to the spatially addressable locations by splint ligation of

77. 77. The semi-aligned spatially encoded capture array of claim 76, constructed at least in part using the method provided in Figures 17A-17C.

78. 77. The semi-aligned spatially encoded capture array of claim 76 having a grid-within-a-grid format as shown in FIG.

10.

79. A semi-aligned spatially coded capture array comprising: An array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray comprising a uniquely identifiable capture feature, the location of which is determined at least in part by the identity of the subarray within the semi-aligned spatially coded capture array.

80. A semi-aligned spatially coded capture array comprising: A set of two or more subarrays, each containing a plurality of capture features, wherein each capture feature in each subarray is attached to a capture oligo containing a unique position tag, the capture features in each subarray are randomly arranged, and the capture features in each subarray further contain a subarray identifying oligo tag attached by splint ligation to the capture oligo on each feature in the subarray.

81. 1. A method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample, comprising: contacting the substantially 2D sample with at least one tagged probe to generate a substantially 2D stained sample, the tagged probe comprising: a hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode, or a DNA / RNA hybrid hairpin tag nucleic acid molecule comprising an attachment moiety and a tag ID barcode; and a probe molecule attached via the attachment moiety to the hairpin tag nucleic acid molecule containing a tag ID barcode or the DNA / RNA hybrid hairpin tag nucleic acid molecule containing a tag ID barcode. a process comprising: contacting the surface of the substantially 2D stained sample with a transparent, spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: a plurality of spatially distinguishable features; and Multiple copies of capture oligo (CO) nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules attached at each spatially distinguishable feature wherein the CO nucleic acid molecule in each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules in other features in the array; placing a flow cell or other solution-containing cover over the sample-array sandwich to form an enclosure containing the sample-array sandwich; adding a solution containing reaction components to the enclosure to form a reaction mixture, the components comprising: a cleaving enzyme selected from RNAse H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ ions; and buffer a process comprising: incubating the sample-array sandwich contacted with the reaction mixture at an assay temperature for 30 to 60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the enclosure; and Analyzing the reaction product mixture to detect and / or quantify and / or define the location of the target in the substantially 2D sample.

82. 82. A method for the detection and / or quantification and / or localization of a target in a substantially two-dimensional (2D) sample according to claim 81, wherein the enclosure comprises a flow cell.

83. 83. The method of claim 81 or claim 82, carried out at a single temperature (isothermally) or within a range of about 5°C within a single temperature.

84. 84. The method of any one of claims 81 to 83, wherein the assay temperature has a range of 20 to 55°C or 37 to 42°C.

85. 85. The method of any one of claims 81 to 84, wherein the method provides location information for more than one target within a substantially 2D sample.

86. 86. The method of any one of claims 81 to 85, wherein the at least one polymerase provides a DNA polymerase, reverse transcriptase, or RNA polymerase enzymatic activity.

87. 87. The method of any one of Claims 81-86, wherein analyzing the reaction product mixture comprises sequence analysis of a plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes.

88. 88. The method of Claim 87, wherein analyzing the reaction product mixture comprises next generation sequencing (NGS) of a plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes.

89. 89. The method of Claim 87 or Claim 88, wherein said plurality of nucleic acid molecules comprising spatial barcodes and tag ID barcodes are full extension products released from spatially distinguishable features by cleavage at the cleavage site of a CO.

90. 90. The method of claim 89, wherein said cleavage comprises RNAse H enzymatic activity or restriction endonuclease activity.

91. 91. The method of any one of Claims 81-90, wherein said analyzing step comprises assigning a spatial location of at least one nucleic acid molecule comprising a spatial barcode and a tag ID barcode within the substantially 2D sample.

92. 92. A method for isothermal spatial encoding of a biological sample, comprising the method of any one of claims 81 to 91, wherein said substantially 2D sample is a biological sample.

93. Use of a set of hairpin tag nucleic acid molecules according to any one of claims 16 to 18, or a set of capture oligo (CO) nucleic acid molecules according to claim 49, or both, for transcriptome analysis of a biological sample.

94. 10. A spatially encoded surface, such as a capture array, e.g., a bead-based capture array, substantially as described or shown herein.

95. Spatial coding workflows, including: contacting a hybrid RNA / DNA tag containing a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; placing a capture array containing capture features in contact with the stained sample to create a sample / array sandwich; placing a fluid containment enclosure on top of the sample / array sandwich; introducing an assay solution containing active RNAse H and an active polymerase into the fluid containment enclosure, thereby contacting the assay solution with the sample / array sandwich; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient for RNAse H activity to at least partially digest the hybrid RNA / DNA tag to generate a cleaved tag, thereby releasing the cleaved tag into the assay solution in the vicinity of the capture feature; allowing the cleavable tag to interact with a capture oligo(CO) on the adjacent capture feature to provide a captured cleavable tag; incubating the sample / array sandwich in the sample solution at a temperature and for a period of time sufficient to allow polymerase activity to extend the captured cleavable tags using CO as a template to generate extension products; cleaving the extension product with RNAse H in the sample solution once it has been extended sufficiently to generate a complementary RNA / DNA region based on the RNA bases in the CO, thereby releasing a full-length extension product; recovering at least a portion of the released full-length extension products; and amplifying and / or sequencing at least one of the released full-length extension products.

96. 81. A computer readable medium or digital resource, or digital database, comprising spatial location information for features of a spatially encoded array according to any one of claims 58 to 80.

97. 97. The computer readable medium or digital resource, or digital database of claim 96, containing spatial location information for substantially all features of the spatially encoded array.

98. Use of a computer-readable medium or digital resource, or digital database, as described in claim 96 or claim 97, to provide a user with location information about one or more targets that correlates with spatial location information of a spatially coded array.

99. 99. The method of claim 98, wherein the correlation occurs through the use of a spatially encoded array in a workflow or method described or illustrated herein.

100. A kit comprising one or more of the following: two or more hairpin tag nucleic acid molecules of any one of claims 1 to 13; two or more DNA / RNA hybrid hairpin tag nucleic acid molecules of claim 14 or claim 15; A set of two or more hairpin tags according to any one of claims 16 to 18; a set of two or more tagged probes according to any one of claims 19 to 28; two or more capture oligo(CO) nucleic acid molecules according to any one of claims 30 to 46; two or more DNA / RNA chimeric CO nucleic acid molecules of claim 47 or claim 48; A set of two or more CO nucleic acid molecules according to any one of claims 30 to 45; A set of two or more chimeric CO nucleic acid molecules according to any one of claims 46 to 48; two or more attached CO nucleic acid molecules according to any one of claims 50 to 53; at least one spatially encoded capture array according to any one of claims 58 to 75; At least one semi-aligned spatially coded capture array according to any one of claims 76 to 80; or 95. The spatially coded surface of claim 94.

101. 101. The kit of claim 100, wherein the spatially coded capture array, semi-aligned spatially coded capture array, or spatially coded surface is in the form of a flexible sticker.

102. 101. The kit of claim 100, further comprising one or more of the following: a container containing a functional RNAse H enzyme; a container containing a functional polymerase enzyme; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphates (rNTPs) and / or deoxynucleotide triphosphates (dNTPs); Mg 2+ a container containing a solution containing ions; or A container containing a buffer solution.

103. At least one of the containers contains an RNAse H enzyme, a polymerase enzyme, a restriction endonuclease enzyme, rNTPs and / or dNTPs, Mg 2+ 102. The kit of claim 101, comprising at least two of: a soluble ion;

104. The kit of any one of claims 100 to 103, further comprising one or more of the following: Components useful for preparing samples for analysis using the methods provided herein; a solution-containing cover suitable for placement over the sample-array sandwich on the slide to form a fluid-containing enclosure for the sample-array sandwich; or A flow cell cover, glass slide, or other surface suitable for receiving a substantially two-dimensional sample.

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