Compositions and methods for molecule barcoding

Abstract

Described herein are nucleic acid oligonucleotides (oligo) comprising a hairpin tag nucleic acid molecule and a DNA / RNA hybrid hairpin tag nucleic acid molecule, and a capture oligo; cO) nucleic acid molecules and DNA / RNA chimeric CO nucleic acid molecules; a space code capture array; the use of the oligonucleotides in encoding and decoding features; and uses of the oligonucleotides and / or the arrays and other provided annotations, including in methods and workflows for spatially encoding and quantifying targets, such as biomarkers, in biological samples.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to and the benefit of earlier application U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023, which is incorporated by reference herein in its entirety.

[0003] Incorporation by Reference of the Sequence Listing

[0004] The computer readable text file named “O046-0080PCT.xml” was created on or about February 28, 2024, is 63,808 bytes in size, contains the sequence listing of the present application, and is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates generally to methods and systems for analyzing targets, such as biological samples and molecules. More particularly, it relates to molecular barcoding and spatially encoded analysis. BACKGROUND

[0006] Determining the identity and / or location of target molecules, such as proteins or nucleic acids, in a sample can be critical for clinical applications, diagnostics, and biomedical research. In situ hybridization (ISH), immunohistochemistry, laser capture microdissection, and these techniques enable visualization of the location of target molecules within a sample, such as a biological sample.

[0007] The identity of target molecules can also be determined using methods that label the target molecules and track them through amplification and / or sequencing methods, such as stochastic barcoding. However, there remains a continuing need for methods and systems that reliably associate the identity of target molecules with their location within a sample, such as a generally two-dimensional (2D) biological sample.

[0008] Spatial biology platforms are revolutionizing biological research and are rapidly becoming indispensable for understanding and treating cancer. However, platforms on the market now, whether for proteomic profiling or transcriptomic analysis, are expensive and cannot be scaled for widespread adoption. Current imaging-based methods for proteomic profiling can provide exquisite detail, but are slow and limited in the number of protein targets that can be analyzed on a single tissue slide. Image-free systems allow for simplified data acquisition with next generation sequencing (NGS) readouts, but these platforms struggle to achieve subcellular resolution, have high cost per analysis (due to the need to purchase specialized instrumentation), and require multiple manual intervention steps. SUMMARY

[0009] Described herein is a scalable, low-cost, imageless spatial platform with subcellular resolution suitable for use, e.g., in basic research and clinical laboratory settings. The described barcoded tags and isothermal workflow enable transfer of information to spatially encoded arrays in a single hands-off step. The workflow is non-destructive to the source material (e.g., fresh frozen or fixed tissue samples) and can function without specialized instrumentation to produce a digital "image" of the presence and abundance of components (e.g., proteins) at subcellular resolution. The described platform enables, e.g., coordinate space and architectural analysis of cellular proteomic maps for deployment into research as well as clinical laboratories. Also provided are spatial capture arrays for spatially encoding information recovered from binding probes (e.g., antibody probes) conjugated to the tags disclosed herein, and isothermal spatially encoded workflows, e.g., by NGS, to provide decoding of the recovered information that enables digital display of, e.g., protein maps in biological samples.

[0010] Provided herein are DNA / RNA hybrid tags, and workflows for spatially encoding, identifying, and / or quantifying biomarkers in biological samples.

[0011] A first embodiment is a DNA / RNA hybrid hairpin tag having a structure as illustrated or described herein. As an example, a DNA / RNA hybrid hairpin tag can include (in 5' to 3' order) a linker, a string of RNA bases, a tag PCR handle, a tag ID barcode, a tag PCR handle complement, and a string of DNA bases complementary to at least a portion of the string of RNA bases. For example, an exemplary DNA / RNA hybrid hairpin tag has a structure as shown in Figure 1A or has the sequence of tag vl, tag v2, tag v3, tag v5, or tag v6 (SEQ ID NOs: 3-7, respectively).

[0012] Also provided are released hairpin tags derived from the DNA / RNA hybrid hairpin tags described herein, including, e.g., a DNA / RNA hybrid hairpin tag having a structure as shown in Figure 1B

[0013] Another embodiment is a labeled element (structure, component, etc.) comprising an element linked (via a linker) to a DNA / RNA hybrid hairpin tag as illustrated or described herein. In an example of this embodiment, the element comprises one or more of: a biomolecule (such as a protein or nucleic acid), a cell or tissue, an affinity molecule (such as an antibody), a bead, or another addressable feature.

[0014] ​Yet another embodiment is a capture oligo (CO) having a structure as illustrated or described herein. For example, an exemplary CO includes (in 5' to 3' order) a linking moiety, a first string of DNA bases, a tag ID barcode, and a second string of DNA bases complementary to at least a portion of the first string of DNA bases. As an example, a CO can have a structure as shown in Figure 2 or Figure 3 .

[0015] Also provided is any of these capture oligos linked to a capture feature by a linking moiety.

[0016] In any embodiment of a CO or including a CO, the CO can further include a conditionally cleavable element. Optionally, this can be configured so that it allows release of the CO (or a substantial portion of the CO) from an element or surface to which the CO is linked (such as by a linking moiety).

[0017] Another embodiment is a capture element (structure, component, etc.) including an element linked to a CO (by a linking moiety). As an example, the element includes a bead or another addressable capture feature.

[0018] It is contemplated that, in embodiments, the provided capture element is one capture element within an array of different capture elements, and wherein the CO on each of the plurality of different capture elements of the array each includes a different tag ID barcode.

[0019] Also provided is a capture pair (e.g., a pair of oligos) including: a DNA / RNA hybridization hairpin tag having a structure as illustrated or described herein, or a released hairpin tag derived from a DNA / RNA hybridization hairpin tag; and a capture oligo (CO) having a structure as illustrated or described herein, wherein the sequence of the DNA / RNA hybridization hairpin tag and the sequence of the CO are at least partially complementary such that when the hairpin tag is released in the vicinity of the CO, the released hairpin tag is captured by the CO. These capture pairs are illustrated herein, for example, in the figures and corresponding descriptions, and in Appendix A included in priority U.S. provisional application no. 63 / 487,575, filed February 28, 2023.

[0020] Another embodiment is a method of detecting and / or quantifying a target in a substantially two-dimensional (2D) sample, the method as described or illustrated herein. A substantially 2D sample can be a thin tissue section, or another section of biological tissue such as an archival tissue section, a substantially 2D array of a biological sample such as can be applied to a glass slide or other surface, including for example a cell extract or synthetic mixture, and the like. A substantially 2D sample can optionally be homogeneous over its surface area with respect to content such as biological macromolecules, but more typically it is heterogeneous such that some targets are found only in some locations or have varying concentrations at some locations. Thus, embodiments of the provided methods allow for target localization within the 2D surface of the sample being analyzed.

[0021] Also provided are methods for detecting and / or quantifying and / or localizing a target in a substantially two-dimensional (2D) sample, the methods being “one-pot” methods (in which multiple chemical and / or enzymatic reactions occur simultaneously or sequentially) conducted substantially at a single temperature (i.e., isothermally).

[0022] Examples of these methods for detecting and / or quantifying and / or localizing a target in a substantially two-dimensional (2D) sample are shown (in whole or in part) in Figure 4A 、 4B , 5A, 5B, 6, 7A, 7B, 8, 9A-9E, 12, 13, or in Appendix A included in priority U.S. provisional application no. 63 / 487,575, filed February 28, 2023.

[0023] In any of the provided methods, embodiments include enzymatic activity of one or more of: RNase H, DNA polymerase, reverse transcriptase, RNA polymerase, and / or one or more restriction enzymes. Most desirably, these enzymatic activities can be conducted in a single vessel (“one-pot” method) and / or at a single temperature (isothermal method).

[0024] Any of the provided method embodiments can further include sequence analysis of the plurality of nucleic acid molecules including one or more molecular ID tags.

[0025] Another embodiment is a spatially encoded capture array, substantially as described or illustrated herein. For example, such a spatially encoded capture array can be as described or illustrated in Figure 10or Appendix A of priority U.S. provisional application no. 63 / 487,575, filed February 28, 2023. The provided spatially encoded capture arrays of embodiments include capture elements embedded in a biomolecule-permeable matrix. In examples, the capture elements include beads; the biomolecule-permeable matrix is a gel; or both. Optionally, the spatially encoded capture arrays are provided in a flexible “sticker” form that is, for example, intended for direct contact with a substantially 2D sample for analysis of targets within the sample. Examples of these analysis methods are described.

[0026] Yet another embodiment is a spatially encoded workflow substantially as described or illustrated herein. Examples of these spatially encoded workflows are described in Figure 12 or Figure 13 or Appendix A of priority U.S. provisional application no. 63 / 487,575, filed February 28, 2023.

[0027] Also described is a spatially encoded feature array having Figure 10 a “grid within a grid” arrangement as illustrated in FIGS. 1-3 and described herein.

[0028] Additional embodiments include computer-readable media or digital resources or digital databases containing spatial location information for features of spatially encoded arrays as described herein. As examples, in some cases, the computer-readable media or digital resources or digital databases contain spatial location information for substantially all features of a spatially encoded array.

[0029] Also provided is use of a computer-readable medium or digital resource or digital database for providing a user with location information for one or more targets related to spatial location information for a spatially encoded array. For example, in some cases, the relatedness is generated by use of the spatially encoded array in a workflow or method as described or illustrated herein.

[0030] Another embodiment is a method for isothermal spatial encoding of a biological sample substantially as described or illustrated herein.

[0031] Also described is use of a DNA / RNA hybridization hairpin tag as described or illustrated herein for transcriptomic analysis of a biological sample.

[0032] Also described are spatially encoded surfaces, such as capture arrays, for example, bead-based capture arrays, substantially as described or illustrated herein.

[0033] Provided herein are hairpin tag nucleic acid molecules comprising, in 5' to 3' order, portions A-B-C-D-E, wherein: the A portion comprises a cleavable site comprising one of: (1) a string of RNA bases, or (2) a string of DNA bases comprising a restriction enzyme (RE) recognition site; the B portion comprises a string of DNA bases comprising a tag PCR handle; the C portion comprises a string of DNA bases comprising a tag ID barcode, and the string of DNA bases forms part of a loop of the hairpin; the D portion comprises a string of DNA bases having reverse complementarity to the tag PCR handle, thereby forming part of a stem of the hairpin; and the E portion comprises a string of DNA bases having reverse complementarity to at least a portion of (1) the string of RNA bases of the A portion or (2) the string of DNA bases comprising the RE recognition site of the A portion, thereby forming part of the stem of the hairpin.

[0034] Also provided are groups 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 examples of these groups, the group further comprises at least one attenuated tag-like nucleic acid molecule that differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the group by lacking a functional cleavable site. As examples, provided are groups of two or more hairpin tag nucleic acid molecules and / or DNA / RNA hybrid hairpin tag nucleic acid molecules, wherein the attenuated tag-like nucleic acid molecule differs from the hairpin tag nucleic acid molecules or DNA / RNA hybrid hairpin tag nucleic acid molecules of the group by (1) replacing the RNA bases of the A portion with DNA bases, or (2) lacking the RE recognition site of the A portion.

[0035] Also provided are labeled probes comprising a probe molecule linked to a described hairpin tag nucleic acid molecule or a described DNA / RNA hybrid hairpin tag nucleic acid molecule by a linking moiety. As examples, 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 an antigen.

[0036] Another embodiment is a released hairpin tag nucleic acid molecule derived from a described hairpin tag or a described DNA / RNA hybrid hairpin tag, wherein the released hairpin tag has been separated from a linking moiety by enzymatic action of a restriction endonuclease or RNase H.

[0037] Another embodiment is a capture oligonucleotide (CO) nucleic acid molecule comprising functionally linked portions I-II-III-IV in a 5' to 3' sequence, wherein: portion I includes a cleavable site comprising one of: (1) a single RNA base or a continuous string of RNA bases, or (2) a string of DNA bases including a restriction enzyme (RE) recognition site; portion II includes a string of DNA bases containing a CO PCR stalk; portion III includes a string of DNA bases containing a spatial barcode; and portion IV includes a string of DNA bases containing a tag-capture region. Optionally, the CO nucleic acid molecule further includes a linker conjugated to the 5' end of portion I. For example, in some cases, the linker provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

[0038] Another embodiment is a group of two or more CO nucleic acid molecules provided by any one of them and / or DNA / RNA chimeric CO nucleic acid molecules provided by any one of them, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

[0039] Another provided embodiment is a spatially encoded capture feature, which includes a capture feature connected to the provided CO nucleic acid molecule or the provided DNA / RNA chimeric CO nucleic acid molecule via a linker portion.

[0040] The capture pair is also described, comprising: a provided hairpin-tagged nucleic acid molecule, or a provided DNA / RNA hybrid hairpin-tagged nucleic acid molecule, or a provided released hairpin-tagged nucleic acid molecule; and a provided capture oligonucleotide (CO) nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule, or a provided linked CO, wherein the sequences of the hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule are at least partially complementary, such that when the hairpin-tagged nucleic acid molecule is released from its linker in the vicinity of the CO nucleic acid molecule, the released hairpin-tagged nucleic acid molecule is captured by the complementary sequence bound to the 3' end of the CO nucleic acid molecule, such that the resulting complex of the released hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule is capable of undergoing a downstream extension reaction by a polymerase.

[0041] Another embodiment is a spatially encoded capture array comprising a defined array of spatially addressed capture features, wherein each capture feature includes: a spatially identifiable feature comprising: a predefined addressable location on a generally two-dimensional solid surface; or a bead or other similar individual solid capture object; and multiple copies of a provided CO nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule attached at each feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to CO nucleic acid molecules at other features in the array.

[0042] Another embodiment is a semi-ordered spatially encoded capture array comprising: a grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide being applied to the spatially addressable location by clamping together: an x-coordinate connector oligonucleotide for labeling all spatially addressable locations within a row of the grid; and a y-coordinate connector oligonucleotide for labeling all spatially addressable locations within a column of the grid.

[0043] A method for detecting and / or quantifying and / or locating targets in a substantially two-dimensional (2D) sample is also provided, the method involving: contacting a substantially 2D sample with at least one labeled probe to produce a substantially 2D stained sample, said labeled probe comprising: a hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode, or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode; and a probe molecule connected via a linker portion to the hairpin-tagged nucleic acid molecule including a tag ID barcode or the DNA / RNA hybrid hairpin-tagged nucleic acid molecule including a tag ID barcode; contacting the surface of the substantially 2D stained sample with a permeable spatially encoded capture array to form A sample-array sandwich, wherein the spatially encoded capture array comprises: a plurality of spatially identifiable features; and multiple copies of a capture oligonucleotide (CO) nucleic acid molecule or a DNA / RNA chimeric CO nucleic acid molecule linked at each spatially identifiable feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to CO nucleic acid molecules at other features in the array; a flow cell or other solution-containing cap is placed above the sample-array sandwich to form a shell containing the sample-array sandwich; a solution comprising reaction components is added to the shell to form a reaction mixture comprising: a lyase selected from RNase H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphate (rNTP) and / or deoxynucleotide triphosphate (dNTP); and Mg 2+ Ions; and a buffer; incubating a sample-array sandwich in contact with the reaction mixture at analytical temperature for 30-60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the shell; and analyzing the reaction product mixture to detect and / or quantify a target in a substantially 2D sample and / or define the location of said target. Optionally, in these methods, the shell includes a flow cell. Examples of the provided methods provide location information for more than one target within a substantially 2D sample.

[0044] Another embodiment is a spatial coding workflow comprising: contacting a hybrid RNA / DNA tag including a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; positioning a capture array including capture features in contact with the stained sample to generate a sample / array sandwich; placing a fluid-containing shell on top of the sample / array sandwich; introducing an analytical solution including active RNase H and an active polymerase into the fluid-containing shell, thereby contacting the analytical solution with the sample / array sandwich; and incubating the sample / array sandwich in the sample solution at a temperature and time sufficient to allow RNase H activity to at least partially digest the hybrid RNA / DNA tag to generate a stained sample. The process involves: cleaving the tag and releasing the cleaved tag into an analytical solution near the capture feature; allowing the cleaved tag to interact with capture oligonucleotides (COs) on the nearby capture feature to provide a cleaved tag for capture; incubating a sample / array sandwich in a sample solution at a temperature and time sufficient to allow polymerase activity to extend the captured cleaved tag using COs as a template to produce an extension product; after sufficient extension, cleaving the extension product with RNase H in the sample solution to produce a complementary RNA / DNA region based on RNA bases in COs, thereby releasing a full-length extension product; collecting at least a portion of the released full-length extension product; and amplifying and / or sequencing at least one of the released full-length extension products.

[0045] Computer-readable media or digital resources and digital databases are also provided, containing spatial location information of the features of the spatially coded array as described herein. One use of the provided computer-readable media or digital resources or digital databases is also disclosed for providing a user with location information of one or more targets related to the spatial location information of the spatially coded array. As an example, using the provided computer-readable media or digital resources or digital databases, the relevance is generated by using the spatially coded array in a workflow or method as described or illustrated herein.

[0046] Another embodiment is a kit that can be used to perform one of the methods provided herein. A representative kit includes one or more of the following: two or more described hairpin-tagged nucleic acid molecules; two or more described DNA / RNA hybrid hairpin-tagged nucleic acid molecules; a described group of two or more hairpin-tagged molecules; a group of two or more described labeled probes; two or more described capture oligonucleotide (CO) nucleic acid molecules; two or more described DNA / RNA chimeric CO nucleic acid molecules; a described group of two or more CO nucleic acid molecules; a described group of two or more DNA / RNA chimeric CO nucleic acid molecules; two or more described CO nucleic acid molecules, each linked to a capture feature; at least one described spatially encoded capture array; at least one described semi-ordered spatially encoded capture array; or a described spatially encoded surface.

[0047] Kit examples are also provided, which further include one or more of the following: a container containing a functional RNase H; a container containing a functional polymerase; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); a container containing Mg 2+ A container for a solution of ions; or a container containing a buffer solution. Attached Figure Description

[0048] Figure 1A : Shows a hybridization hairpin tag structure with an exemplary sequence. Shows an exemplary nucleic acid tag (tag 1v6; SEQ ID NO:7) containing both RNA and DNA bases within a hairpin (stem-loop) structure. The 5' end of the tag contains a flexible linker that connects to a molecule (X in the figure) and binds to a target fixed on a surface. From 5' to 3', following the linker is an RNA base extension, a downstream constant (in the hybridization hairpin tag group) PCR stalk, 1-3G bases separating the primer landing site from the unique tag barcode, a unique tag barcode placed within the loop of the hairpin (unique within the hybridization hairpin tag group), followed by a 3' region complementary to the 5' region of the hairpin tag (i.e., its reverse complementary sequence). In the formed hairpin tag, the RNA and DNA bases of the tag produce a DNA / RNA hybridization region (part of the hairpin stem) that can be cleaved by RNase H.

[0049] Figure 1B Once cleaved by RNase H, the tag is released from the molecule (X). Digestion of the RNA region of the tag by RNase H (to produce, for example, SEQ ID NO:8) exposes the ssDNA base region within the hairpin stem designed to capture onto the capture oligonucleotide (CO).

[0050] Figure 2 : Captures cleaved hairpin tags by capturing capture oligonucleotides (COs) on the capture beads. The 3' end of the capture oligonucleotide (an oligonucleotide designed to capture the released hairpin tag) contains a capture region that, after digestion with RNase H, is complementary to the exposed DNA bases on the released tag (see See...). Figures 1A-1B The sequence shown is in italics, depicting an exemplary capture region bound to the capture hairpin tag (SEQ ID NO:6). In this embodiment, the capture oligonucleotide is attached to the capture feature (shown here as a bead) at its 5' end.

[0051] Figure 3 Capture beads with a capture oligonucleotide (CO) structure. An exemplary single-stranded CO structure is shown, containing a flexible linker at its 5' end for attachment to a capture feature (shown herein as a bead), followed by a 5' PCR stem, a spatial barcode, and a 3' capture region. The CO may also contain a unique molecular identifier (UMI), for example as a base extension randomly or dispersed within the spatial barcode region. The linker between the capture oligonucleotide and the capture feature (shown herein as a bead) is conditionally cleavable, for example by including a lightly or chemically cleavable linker, or by including an RNA base or a half-dsDNA restriction enzyme site, as described herein. The 2D position of each capture feature is predetermined (e.g., within an array), and this information is recorded within the spatial barcode region of the CO. All COs within / located at a capture feature share a unique spatial barcode corresponding to the position of the capture feature to which they are attached (e.g., a bead, an addressable region of a solid surface, etc.).

[0052] Figure 4A The hairpin tag is released near the capture feature. A pool of molecules (such as probe groups or libraries) is incubated with the sample immobilized on the surface, whereby each unique molecule in the pool is encoded with a DNA barcode sequence (within the tag loop region) that recognizes the specific molecule. The sample is then washed to remove any unbound or non-specifically bound molecules. Subsequently, one or more enzymes with overall RNase H and polymerase activity are introduced with appropriate cofactors (such as dNTPs, Mg2+, etc.). 2+ In a common buffer solution, the target molecule (shown as X in the figure) is released from the surface, thus releasing the tag (molecule ID, "released tag", "hairpin tag"). This release occurs near the CO attached to the capture feature (shown as a bead).

[0053] Figure 4BHairpin tag capture and extension. In an isothermal one-pot reaction, the hairpin tag is released from the sample surface, captures onto nearby CO (based on the sequence of the capture region of CO and the proximity of the CO to the point of tag release), 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. The inclusion of a DNA polymerase within the one-pot reaction prevents the tag from "jumping" to other nearby capture features because the melting temperature of the interaction (between the captured hairpin tag and the capture sequence of the captured oligonucleotide) is significantly increased by the extension reaction. If such jumping occurs, it can lead to a loss of positional fidelity in the analysis (e.g., despite increased diffusion).

[0054] Figure 5A Regarding compatibility, alternative tag construction is possible. Alternative tags can be constructed to ensure compatibility with the described isothermal one-pot reaction. As shown in this figure, a compatible and cleavable tag can be produced by simply changing the base composition of the tag to include RNA bases between the tag's linker region and the molecular ID region. In this way, other tag types can be used in the same isothermal one-pot reaction workflow, such as... Figure 4B As described in the text.

[0055] Figure 5B Alternative tag capture and extension. Figure 5A The alternative tag designs outlined in [the document] can capture CO near a capture feature (such as beads) and are extended by a polymerase included in an isothermal one-pot reaction. This is similar to hairpin oligonucleotide capture (…). Figures 4A-4B As shown in the diagram, when capturing labels with alternative designs, the 3' end of CO can be closed or extended during a one-pot reaction.

[0056] Figure 6 Alternative tag designs for compatibility and isothermal amplification. Another type of tag design is also considered for similar isothermal one-pot reactions. As shown, promoter regions recognized by RNA polymerases (e.g., T7 polymerase) can be designed for incorporation into the tag sequence at one or both ends of the tag. The dsDNA tag carries two molecules (X and Y), thereby encoding (labeling, recognizing) each molecule within different strands of the dsDNA tag, as shown. Here, opposing T7 promoter regions are installed to generate an amplifiable template from both strands of the tag. These probes can bind to a sample immobilized on a surface, followed by washing the surface to remove unbound probes and exposing the washed surface to a solution containing T7 polymerase and cofactors suitable for RNA transcription. This reaction can be carried out in an isothermal one-pot reaction containing reverse transcriptase (RT) and cofactors suitable for DNA elongation, near a capture feature containing CO (e.g., as provided herein).

[0057] Figure 7A : ssDNA tag conversion for compatibility and isothermal amplification. For compatibility with the isothermal information transfer methods described herein, tags for existing DNA-tagged molecules can be converted by installing a promoter region recognized by an RNA polymerase (e.g., T7 polymerase). A common ssDNA tag structure is shown, containing two constant regions (typically used as PCR handles) flanked by a sequence (molecular ID) encoding (labeling, recognizing) a molecule (X), which is linked to the end of the tag (5' shown here) via a linker. This linker can be installed before or after the labeled probe is incubated with the target sample (containing the target molecule). Similarly, extension reactions to generate dsDNA tags via extension of the 3' end of the linker can be performed before or after the probe is incubated with the sample. After probe binding, unbound probes are 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 performed in an isothermal one-pot reaction containing reverse transcriptase (RT) and cofactors suitable for DNA extension, near a CO-containing capture feature.

[0058] Figure 7B dsDNA tag conversion for compatibility and isothermal amplification. For compatibility with isothermal information transfer, tags of existing DNA-tagged molecules can be converted by installing a promoter region recognized by an RNA polymerase (e.g., T7 polymerase). A common dsDNA tag structure is shown, containing two constant regions (typically used as PCR handles) flanked by a sequence (molecular ID) encoding (labeling, recognizing) a molecule (X), which is linked to the end of the tag (5' shown here) via a linker. This linker can be installed before or after the labeled probe is incubated with the sample. After probe binding, unbound probes are 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 performed in an isothermal one-pot reaction containing RT enzyme and cofactors suitable for DNA extension, near a CO-containing capture feature.

[0059] Figure 8: Capturing RNA generated from a tag template. When the transcribed RNA is released into solution, it is captured to the 3' end of a capture oligonucleotide linked to a nearby capture feature (shown as beads), and the 3' end is immediately extended by an RT enzyme present in the same reaction mixture. Once the RNA strand has been copied to the 3' end of the capture oligonucleotide through the extension reaction, it is then destroyed by RNase H activity (either contained within the RT enzyme or by means of an enzyme containing RNase H activity, such as E. coli RNase H). The resulting extension product contains spatial information provided by a spatial barcode, tag information, and 3'- and 5'-PCR stems for downstream amplification. Spatial-encoded products can be generated in various ways (such as... Figures 9A-9E (As outlined in the document) Release from the captured features.

[0060] Figure 9A The double-stranded product is released from the capture feature via RNase H. After the tag on the capture oligonucleotide extends to the 3' end to produce a spatially encoded dsDNA product, these spatially encoded products can be released into the surrounding solution within the same isothermal one-pot information transfer reaction workflow. Here, the RNA base is included within the 5' end of the capture oligonucleotide, such that it can be released into the surrounding solution via DNA polymerase (specifically, RNA and DNA-dependent DNA polymerases; e.g., Maxima, available from Thermo Scientific). TM After extension, a DNA / RNA hybridization region is generated, which becomes the 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.

[0061] Figure 9B The double-stranded product is released from the capture feature via a nuclease. An alternative method for releasing the spatially encoded dsDNA extension product from the capture feature is to include a half-dsDNA restriction enzyme (RE) site within the 5' end of the capture oligonucleotide. Following extension from the 3' end of the tag on the capture oligonucleotide, a fully formed dsDNA RE site is generated. The homologous RE can be introduced during the isothermal information transfer reaction or after the reaction is complete. Upon introduction, this RE will cleave the spatially encoded dsDNA product from the capture feature, and it will be released into the reaction solution.

[0062] Figure 9C Release of double-stranded products from the capture feature; photochemical or chemical. An alternative method for releasing spatially encoded dsDNA extension products from the capture feature is to include a photochemically cleavable portion within the linker that ligates the capture oligonucleotide to the capture feature. Here, the cleavage agent is introduced after the one-pot isothermal information transfer reaction is complete. This method can be combined with other methods that release dsDNA capture oligonucleotides in a known percentage (e.g., via RNase H or RE activity) for a variety of specialized applications.

[0063] Figure 9D Release of single-stranded products from the capture feature; photochemical or chemical. Following the capture and elongation of the transcription product, a spatially encoded single-stranded DNA product is produced. These products can be released from the capture feature by including a photochemically cleavable portion within a linker that ligates the capture oligonucleotide to the capture feature. Here, the cleavage agent is introduced after the one-pot isothermal information transfer reaction is complete. This method can be combined with other methods that release ssDNA capture oligonucleotides in a known percentage (e.g., via RNase H or RE activity after conversion to dsDNA, as outlined below) for a variety of specialized applications.

[0064] Figure 9E This is used for the conversion of single-stranded products released from the capture feature. Following capture and extension of the transcription product, a spatially encoded single-stranded DNA product is generated. Constant primers anneal to the 3' end of these oligonucleotides, and DNA polymerase extension converts them onto the capture feature to form spatially encoded dsDNA products. Including an RNA base or a half-dsDNA RE site at the 5' end of the capture oligonucleotide will enable the enzymatic release of these products from the capture feature after their conversion to dsDNA. Extension of the tag's 3' end on the capture oligonucleotide will produce a fully formed dsDNA RE site, or a DNA / RNA hybridization region. Upon exposure to a suitable enzyme, the product is released into the reaction solution.

[0065] Figure 10 : Exemplary spatially encoded bead arrays. A major problem with trap arrays consisting of small (1-10 μm) compactly packed trap beads (or high-density monodisperse beads) is the number of different beads that must be used to generate trap arrays with large surface areas. For example, to construct a 1.75 cm × 1.75 cm trap array consisting of stacked 1 μm beads, approximately 10⁻⁶ B (1 × 10⁻⁶) of beads are required. 10 Individual 1μm beads are used to completely cover the surface area. Furthermore, there can be very little redundancy (ideally none) in the spatial barcodes of the beads in the capture array; in this example, this would require at least 10 16The capture size of the bead library for different beads is used to reduce the probability of spatial barcode redundancy to approximately 1 / 10,000. Furthermore, bead libraries designed for the visual encoding and decoding methods described herein (e.g., orthogonal cleavage sequencing (OCS) as described in WO 2022 / 187719, and other libraries constructed using splitting and merging methods) are not ideally designed to achieve this level of diversity. The co-construction of NGS barcodes and visual barcodes as described herein requires the use of constant bases for splice bonding (e.g., as described by Kershaw and O'Keiffe, Methods Mol Biol. 941:257-269, 2012), which increases the length of the NGS barcode with each round of splitting and merging (due to the increased diversity within the library). A representative application of this technology will use large spatially encoded arrays (e.g., >1cm × 1cm, and at most 10cm × 10cm), and therefore this problem needs to be addressed. To reduce the number of capture beads required to generate a large capture array surface, a random bead array can be patterned within a grid. The grid configuration allows the random bead array to be subdivided into smaller features (shown as 35-100μm features). When using 35μm grid features and 1μm diameter beads, each grid feature will contain approximately 1,250 individual beads. Similarly, a 100μm grid feature containing stacked 3μm beads will have approximately 1,000 individual beads in each feature. After the beads are attached to the surface (loaded grid), each feature of the grid accommodates a unique spatial address. To address each feature of the grid spatially, x and y coordinate connectors are added to the grid, for example, via droplet printing. In simple terms, x-coordinate connectants (a unique connectant sequence for each row of the grid) are deposited horizontally in the bead array by droplet printing onto features of the grid. These connectants are included in a solution containing ligase and constant nucleic acid clips for clip-on bonding of constant regions of the NGS barcodes attached to the beads. Similarly, y-coordinate connectants (a unique connectant sequence for each column of the grid) are deposited vertically in the bead array by droplet printing onto features of the grid. These connectants are included in a solution containing ligase and constant nucleic acid clips for clip-on bonding of constant regions of the x-coordinate connectants already attached to the NGS barcodes attached to the beads. The y-coordinate connectants also contain a 3' capture region for capturing probe tags released from the sample. This allows for unique addressing of each feature of the grid, and the process can be scalable to produce very large capture arrays containing high-density stacked or monodisperse 1 μm beads.To avoid significant redundancy of spatial barcodes in the array in this configuration, a bead library diversity of approximately 1M can be used, even when generating a capture array surface area of ​​10cm × 10cm or larger.

[0066] Figure 11A : Bead trapping array; trapping beads embedded in a macromolecularly permeable material (e.g., gel). After reading the bead positions within the trapping array grid (e.g., using OCS or another method), the position of each bead in the grid is recorded, for example, in a software package. The NGS trapping oligonucleotides on the beads contain operatively coupled to a visual barcode sequence and (in the examples) x and y coordinate connectors for position addressing via droplet printing (see Figure 10 The information is then presented. The beads can then be embedded within a gel or other material that is permeable to macromolecules but rigid enough to retain the beads within the matrix after casting or hardening. After embedding the beads, the bead-containing matrix can be removed from the semi-patterned bead array, and this product can be used as a spatial recognition trap array. As an example, in various embodiments, the "sticker" (cast gel / matrix) thickness is between 100 μm and 250 μm, or (e.g., when casting) as thin as 50 μm. In the described embodiments, the gel is a 0.5%–2% agarose gel cast using a 10 mM Tris pH 7.5 buffer, in which agarose powder with a high melting temperature (e.g., from Thermo Technologies, such as 16500100) is melted. Other permeable matrices considered include polyacrylamide (e.g., 4% polyacrylamide, which has been shown to allow sufficient diffusion of macromolecules) or hydrogels of varying compositions. Optionally, the permeable matrix (gel) may include functionalizations for attaching and fixing features (such as beads) within the matrix (gel).

[0067] Figure 11BThe capture of oligonucleotide information is copied and retained within the gel. To generate numerous capture array gels from a single bead array, the extension products generated as copies of NGS capture oligonucleotides can be retained within the casting gel. Simply put, the NGS capture oligonucleotides on the beads are constructed with a reverse complementary orientation, such that the sequence encoding the 3' capture region is closest to the bead orientation, and the 5' PCR stem is furthest from the bead orientation. These reverse complementary NGS barcodes can be primed using a common primer that anneals to the constant 5' region on all capture oligonucleotides, and this primer can be extended by DNA polymerase to copy the NGS capture oligonucleotide information (spatial barcode). The common primer used for priming and extending the DNA will contain a portion for polymerization within a macromolecularly permeable material (such as a gel) (e.g., using a primer modified with 5' acrydite for polymerization within a polyacrylamide gel). After the extension reaction, a suitable unpolymerized (or uncured) gel can be cast onto the beads for copolymerization, or the 5' chemical portion of the extension product can be incorporated into the gel in other ways. The gel now contains NGS-captured oligonucleotides with the correct orientation (copy), which are constructed in reverse complementarity and retain their relative positions. The gel without beads can then be removed, and more gels can be cast using a semi-patterned array of beads through repeated annealing, stretching, and gel casting procedures.

[0068] Figure 12 : Demonstration of spatially encoded workflow. Isothermal information transfer reactions can be carried out in close proximity to capture beads embedded in a macromolecularly permeable material (such as a gel) (e.g., placed in direct contact with it). Using a macromolecularly permeable material (such as a gel) for the capture array allows for the introduction of spatially encoded components (enzymes and cofactors) and the release of the spatially encoded product by diffusion directly through the material (gel). As shown, the anti-Her2 antibody probe with tag v6 (SEQ ID NO:7; Figure 1AThe sample was conjugated (as shown) and used to detect Her2+ cells (SKBR3 cells) immobilized on a glass slide. After probe incubation, the slide was washed, and a thin layer of a solution containing DNA polymerase (Maxima RT) and dNTPs in 1×RT buffer (approximately 20 μl) was placed on the sample. The capture bead array gel (“sticker”) was then “attached” (placed, laid out) onto the sample (i.e., placed very close to the sample, e.g., in direct contact). In this example, the capture oligonucleotides contained RNA bases in their 5' region. Subsequently, a solution containing dNTPs (to avoid diluting these desired small molecular weight cofactors) and 1×RT buffer (approximately 40 μl) was placed on the back side of the gel (i.e., the side away from the biological sample). After incubation at 40°C for 20 minutes, this solution was removed and kept as a negative control. After removing the negative control solution, a solution containing *E. coli* RNase H and dNTPs (to avoid diluting these desired small molecular weight cofactors) in 1× buffer was placed on the back side of the gel matrix. After incubation at 40°C for 20 minutes, this solution was removed and used in qPCR experiments alongside the negative control sample to quantify the level of extension products present in each solution. This figure shows that approximately 130-fold more extension products were recovered in the RNase H+ sample compared to the negative control (RNase H-) sample. This indicates that, under these conditions, the enzyme RNase H, required to initiate the isothermal information transfer reaction, was able to diffuse across / through the gel to the sample surface. This enzyme releases the information contained within the probe tag to capture it onto the capture beads, thereby enabling the DNA polymerase to extend the 3' end of the captured tag by capturing the RNA bases of the oligonucleotide, allowing the release of the extension product through the gel. The released product was then harvested by removing the solution from the back side of the gel, allowing for analysis.

[0069] Figure 13 This illustrates a simple spatial encoding device and workflow. The capabilities described herein, including the ability to perform isothermal information transfer reactions and the ability to release extended products from the trap beads into solution, enable... Figure 13The present invention outlines a simple and straightforward spatial coding workflow. After adding probes to a sample fixed to a surface (tissue section on a slide shown here), the sample surface is washed to remove unbound probes, and a macromolecularly permeable spatial coding capture array (gel sticker) is placed on the sample. Optionally, a flow cell sample cap may be placed above the coding surface. This type of device applies gentle pressure to the coding surface to ensure and / or increase the proximity of the capture features (within the macromolecularly permeable spatial coding capture array) to the sample. Once assembled, the device allows a solution to flow over the capture array placed on the tissue / sample, thereby introducing one or more of the spatially encoded reaction components (enzymes and / or cofactors required for one-pot isothermal information transfer and release of spatially encoded products into solution, as illustrated herein) into the macromolecularly permeable gel, and releasing the spatially encoded products through the gel and into the solution contained within the flow cell. After incubation at an isothermal reaction temperature (e.g., 37-42°C), the spatially encoded product in the analytical solution is recovered (e.g., by aspiration), and the spatially encoded product contained in the recovered solution is analyzed. In an example, this involves amplification (e.g., by PCR) for use in next-generation sequencing library preparation.

[0070] Figure 14 "One-pot" isothermal reaction. This diagram is in... Figure 12 The above extends and illustrates aspects of an embodiment of the analysis. According to... Figure 13As shown, tissue samples (such as freshly frozen or formalin-fixed paraffin-embedded (FFPE) tissue sections on glass slides or other surfaces) are stained with conjugated antibody probes (i.e., antibodies conjugated to DNA / RNA hybridization hairpin tags). A capture array (containing spatially defined capture features linked to capture oligonucleotides, each of which includes a 5'-PCR stalk, a spatial barcode, a UMI, and a probe capture region; four capture features are shown) is placed on top of the stained tissue. A flow cell is placed on top of the tissue / array sandwich, and an analytical solution (containing RNase H and polymerase) is introduced. The action of RNase H releases the hairpin tag barcode from the bound probe. The released tag diffuses out of the tissue and (A) interacts with spatially encoded array features (i.e., the released hairpin tag barcode is captured on the nearest spatially defined feature carrying the barcoded capture oligonucleotide), wherein (B) it is extended by polymerase in analytical solution, thereby copying the spatially encoded information (i.e., the polymerase extends the 3' end of each probe, thereby copying the UMI, spatial barcode, and 5'-PCR stem of the capture oligonucleotide). After extension is complete, complementary RNA / DNA regions are generated, which are subsequently (C) cleaved by RNase H in analytical solution (isothermal release of the probe and template oligonucleotide from the spatially defined capture features by RNase H enables Illumina library preparation based on PCR amplicon). Subsequently, the fully extended released tag diffuses through a permeable gel matrix and is collected for further analysis, such as amplification and NGS (see [link to relevant documentation]). Figure 12 The figure in the image shows that cleavage and release do not occur in the absence of RNase H.

[0071] Figure 15A An anti-Her2 antibody tagged with HPv6 was applied to MCF7 cells (which were Her2-negative). This was performed to test for background staining issues with hairpin-tagged antibodies. The Her2-HPv6 antibody was observed using secondary anti-mouse Alexa555 to assess background markers. The ability of three separate blocking / washing conditions (DMSO, Denhardt's, and salmon sperm DNA) to eliminate background staining was tested. The image is quantified in the imaging software, and the data is input into GraphPad Prism to generate a graph. (**=p<.001).

[0072] Figure 15B For example, regarding Figure 15AThe experimental conditions, quantification, and plotting are described. In this experiment, anti-ER-HPv6 antibody was tested on SKBR3 cells (ER-negative) to evaluate background staining and blocking methods.

[0073] Figures 16A-16B Representation of all 256 ring barcodes. Figure 16A This graph was generated from 1 million (randomly selected) reads out of 26,000,000 paired-end (PE) reads aligned to a reference genome library; the percentage of each barcode's representation was then plotted. This was performed four times, each time randomly selecting 1 million reads to allow for error calculation. The percentage of reads obtained for each of the 256 barcodes was then plotted using GraphPad Prism. The corresponding raw data for the reads are provided in Annex B to Priority Application No. 63 / 487,575, filed February 28, 2023. Figure 16B The logo shown is generated by uploading 10,000 reads (which have been successfully aligned with the reference genome) to WebLogo (available online at weblogo.berkeley.edu / logo.cgi).

[0074] Figures 17A-17C Extending the spatially encoded capture array surface area using a dual-coordinate oligonucleotide system: Further expansion of the spatially encoded array surface area can be achieved by droplet printing different combinations of two barcode types. Figure 17A The diagram shows a grid pattern of subarrays, outlining a physical map of different combinations of oligonucleotides (x and y coordinates shown here), which can optionally be directly droplet-printed onto the subarray features, whereby each subarray contains multiple capture features (e.g., beads, each coated with a unique capture oligonucleotide (CO)). Exemplary subarray features are 100 μm × 100 μm in size and contain approximately 1,000 3 μm beads or approximately 10,000 1 μm beads tightly packed together but randomly arranged within the subarray. Within each subarray, each bead contains multiple copies of a unique visual barcode operatively coupled to a DNA barcode sequence (VBC) contained within the linked and corresponding capture oligonucleotide nucleic acid, also represented in multiple copies. The region of the capturing oligonucleotide corresponding to a unique, operatively coupled visual barcode is referred to as the VBC in the figure; this region can be further addressed spatially by droplet printing of a unique first oligonucleotide barcode (shown here as the x-coordinate barcode oligonucleotide (x-BC)), which is then attached to the VBC using a splint oligonucleotide. This step is in Figure 17BThe diagram further illustrates the addition of two distinct x-BC oligonucleotides (x1 and x2 oligonucleotides) to two adjacent subarrays containing bead-capturing features of VBC regions carrying only the capturing oligonucleotides via droplet printing. This step is ideally performed in the presence of RNA-clamp oligonucleotides (which may contain a single DNA base at both the 3' and 5' ends to improve stability) and SplintR ligase (available from NEB M0375S), which recognizes RNA / DNA hybrids for conjugation to adjacent DNA strands templated by the RNA oligonucleotides. Following a washing step, droplets of a second spatial coordinate oligonucleotide are printed onto each subarray for conjugation to the first spatial coordinate oligonucleotide. Figure 17C The second oligonucleotide type, y-coordinate barcode (y-BC) oligonucleotide, is added to each subarray in an orientation perpendicular to the addition of the x-coordinate oligonucleotide, which is also coupled for connection. This results in a unique combination of two different types of coordinate oligonucleotides that can be added to and coupled to the VBC oligonucleotide to create a spatially addressed, scalable surface region for producing a large capture array. Here, the y-BC oligonucleotide also contains capture regions for capturing nucleic acids and / or tagged oligonucleotides from the stained sample.

[0075] sequence reference

[0076] The nucleic acid and / or amino acid sequences described herein are shown using standard letter abbreviations as defined in 37 C. FR § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand should be understood to be included in the appropriate examples. All oligonucleotides are shown in 5' to 3' orientation. Symbol explanation: 5AmMC6 = 5' flexible 6-carbon linker carrying a reactive primary amine (IDT: / 5AmMC6 / ); 5AmMC12 = 5' flexible 12-carbon linker carrying a reactive primary amine (IDT: / 5AmMC12 / ); 5Phos = 5' phosphorylation, which, for example, makes the oligonucleotide a substrate for T4 DNA ligase; 3InvdT = 3' reverse dT modification, producing a 3'-3' bond that inhibits both 3' exonuclease degradation and DNA polymerase elongation; 5BioTEG = 5' modification including biotin and triethylene glycol (TEG) spacers, which increases the oligonucleotide-biotin distance to 15 atoms; 5Biosg = 5' biotin, which allows the oligonucleotide to be captured by binding to avidin; "r" indicates that the following base is RNA rather than DNA; + indicates that the following base is a locked nucleic acid (LNA) base.

[0077] In the sequence list:

[0078]

[0079]

[0080] Detailed Implementation

[0081] The hybridization tag structure disclosed herein contains both DNA and RNA bases and can be linked to solid vectors or various macromolecular probes to enable molecular coding (recognition, labeling). The tag is designed as a hairpin, keeping the extension of DNA bases complementary to the RNA bases in the tag nearby to create a stable RNA / DNA hybridization region; the RNA bases within the tag are laterally attached to DNA bases on both sides. These features protect the RNA bases within the tag from degradation (primarily by ribonucleases lacking RNase H activity). This structure allows the tag to be released from its linking site upon introduction of RNase H. Furthermore, the hairpin constitutes a self-contained coding element that can be used without recognition strand hybridization (required by current DNA paint workflows and other cyclic immunofluorescence (IF) and digital coding workflows utilizing nucleic acid-labeled probes).

[0082] The exemplary label design and experimental examples shown herein involve the use of hybridization hairpin tags for encoding antibody probes, which can be used, for example, in workflows for detecting and quantifying protein targets in tissue sections or other generally two-dimensional (2D) sample arrangements. Embodiments of this workflow also utilize previously described types of visual molecular barcodes that enable the identification of numerous different features (beads) fixed to a surface (e.g., as disclosed 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 (located, reliably addressed), and the surface containing the addressed (or located) features can be used for spatially resolved analysis. In embodiments, the visual barcode associated with each feature corresponds (functionally coupled to) sequence information within a capture oligonucleotide, which is retained throughout the next-generation sequencing (NGS) workflow: each bead (or other addressable fixed feature) contains at least one visual barcode and at least one corresponding capture oligonucleotide that is unique to that bead.

[0083] In some embodiments, more than one unique visual barcode corresponds to more than one unique capture oligonucleotide.

[0084] In the analysis described herein, tissue samples (such as those used in standard IHC assays) are prepared and exposed to a mixture or group of antibody-DNA / RNA hybridization tag-conjugated probes, washed to remove unbound probes, and incubated in a compatibility solution for downstream steps (transfer buffer). At low temperatures (below room temperature, e.g., on ice), a surface containing spatially resolved beads (or other features) is generated, positioned, and coated with a solution containing (in representative examples) transfer buffer, RNase H, and DNA polymerase. The array of spatially resolved features (e.g., beads) is then positioned “bead-side down” such that the features (beads) are in direct contact with the (tissue) sample. As an example, the (tissue) sample is mounted on / supported by a solid surface (such as a standard microscope slide). Subsequently, the sandwich (bead array / tissue slide) is incubated at high temperatures (e.g., room temperature to 42°C) to isothermally transfer tags, resulting from the cleavage of probes bound to the tissue sample (by RNase H, in an exemplary embodiment), to spatially encoded capture oligonucleotides, which are immobilized on features (e.g., beads) of the spatially encoded (bead) array. In this manner, hybrid DNA / RNA hairpin tags can be released from tissue-bound probes and directly captured onto the nearest bead / feature to each released tag.

[0085] Because DNA polymerase is also present in the isothermal lysis / capture solution in this embodiment, each tag will extend its 3' end once captured onto the nearest bead in the array, effectively copying spatial information from the bead to the tag via DNA extension. The 3' end of the capturing oligonucleotide is closed, and therefore cannot be extended. This exemplary "downward contact" extension reaction stabilizes tag capture and is expected to increase analytical resolution by reducing lateral diffusion of released tags. This extension step will address or significantly reduce the problem of tags "jumping" to and from nearby beads (capture feature) after release from the probe, because after extension, during the extension reaction, as the length of the newly synthesized DNA / DNA double helix interaction increases, the melting temperature (Tm) of the interaction (between the tag oligonucleotide and the capturing oligonucleotide) will increase with each base added to the 3' end of each captured tag.

[0086] The released tag contains (from 5' to 3'): (1) a sequence that acts as a constant 3' PCR handle; (2) a short sequence corresponding to the probe type (in this example, an antibody that recognizes the barcode); and (3) a constant capture region that is exposed as single-stranded DNA after digestion with RNase H. The capture oligonucleotide on each bead of the spatially encoded feature (bead) array is linked (fixed) to the fixed feature (bead) through its 5' end by a standard linker linker chemistry (here, biotin-streptavidin), and from 5' to 3' includes (in the examples): (1) a short extension of 3 DNA bases; (2) eight RNA bases (but this can vary, as described herein); (3) a constant 5' PCR handle; (4) a spatial barcode corresponding to a visual barcode on the bead; (5) optionally, an extension of randomized bases that produces a unique molecular identifier (UMI); and (6) a constant 3' capture region for capturing the released tag.

[0087] In this example, the extension reaction copies a unique molecular identifier (UMI), a barcode region recognizing the bead-specific location, and a 5' constant PCR stalk onto the probe to produce a continuous sequence containing both the 3' and 5' constant PCR stalks. Throughout this article, "constant" means that the region / element is common to all probes and capture oligonucleotides in the analysis.

[0088] Because the capturing oligonucleotide also contains RNA bases at its 5' end, after the polymerase extends through the DNA template bases of the capturing oligonucleotide, it will extend through the RNA bases of the capturing oligonucleotide, thus producing a DNA / RNA hybrid. Since RNase H is also (already) present in the isothermal transfer reaction solution, this newly synthesized DNA / RNA hybrid is cleaved by RNase H, thereby releasing the capturing oligonucleotide from the immobilization tag (bead). After release, the released capturing oligonucleotide still hybridizes with the full-length extension tag.

[0089] Examples of this overall process allow for: 1) isothermal lysis of the tag from the probe bound to the tissue / sample being analyzed; 2) capture of the released tag onto a spatially encoded array (such as a spatially encoded bead array); 3) extension of spatial coordinates onto the tag; and 4) release of a spatially encoded full-length tag containing both the 5' and 3' PCR stems (constant regions) for recovery and PCR-based amplification of the full-length tag alone using a common set of primers. The amplification primers may, for example, contain an inmena flow cell adaptor sequence, enabling the spatially encoded biomarker analysis to be resolved and quantified via next-generation sequencing. More generally, step 3) can be viewed as combining tag information with spatial barcode information via an extension reaction, encompassing embodiments described in addition to those specified above. An option may be included to close the 3' end of the tag, which will not be extended during the extension step, as only the CO will be extended. In such embodiments, the extension product release step is a separate step because the method relies on the presence of a second RNA / DNA hybrid generated during the extension step for releasing the product via a one-pot reaction.

[0090] Methods for controlling or influencing the direction of the elongation step include: 1) blocking the 3' end of the capture oligonucleotide to prevent CO elongation (see example...). Figure 4B (as shown herein), which only allows the tag to extend (as illustrated herein); or 2) closing the 3' end of the tag so that it cannot extend when released as a hairpin, which would only allow CO extension. For the illustrated product release step, the method relies on generating an extension product that creates a DNA / RNA hybridization region by mounting RNA bases in CO, and therefore the release step is optionally not part of an isothermal reaction. One option described herein is light release of these, which is illustrated herein.

[0091] In the RNA capture example (amplified via the T7 promoter region), elongation occurs via reverse transcription, which elongates the CO rather than the 3' end of the captured RNA. Although not in hairpin format and actually from the captured RNA, the elongation direction is from the 3' end of the CO.

[0092] The diagram provided in this article illustrates a representative overall workflow scheme comprising the following components: encoding and decoding beads carrying spatially defined barcodes (e.g., using visual molecular barcodes, such as those described in WO 2022 / 187719 and similar), to generate a spatially encoded array; a probe-tag composition (RNA / DNA hybridization hairpin) that enables isothermal information transfer steps; generation and cleavage of prototype tags; and a one-pot reaction showing cleavage of the tags from one surface (bead) to another surface (bead) carrying the captured oligonucleotides, resulting in extension and release of the full-length extended product from the captured beads into the supernatant.

[0093] The workflow described herein consists of two key elements: “stickers” (generally, spatially encoded, thin (<250 μm), flexible 3D macromolecular-permeable matrices containing 2D arrays of capture features at predetermined or “addressed” locations) and DNA / RNA hybridization tag design. When combined with spatially encoded systems (such as spatially encoded beads as described in WO 2022 / 187719), the workflow provides a one-step isothermal lysis, extension, and release within a sandwich structure. This workflow is extremely user-friendly. This platform does not require custom instrumentation because pre-reading of the spatial bead array can be performed offline. Due to the nature of visual barcodes, the user cannot reposition the array after reading (reading would destroy the visual barcode). In an embodiment, the consumer receives the spatially encoded bead array and uses it in combination with reagents, for example, in kit form, for spatially resolved biomarker expression in an tissue sample. The array is a single-use consumer product.

[0094] Spatially encoded bead arrays (such as those described herein) and their uses are also provided.

[0095] Spatial-encoded bead-capturing arrays have a variety of other applications; this would be the cheapest way to prepare spatially-encoded bead arrays (to read bead positions by sequencing, which is much more expensive, or to prepare microarrays to encode positions), whereby each bead has an NGS-capturing oligonucleotide containing a spatial barcode that corresponds to (operably coupled to) a visual bead barcode on the bead.

[0096] This document provides a hairpin-tagged nucleic acid molecule comprising functionally linked portions ABCDE in a 5' to 3' sequence, wherein: portion A comprises a cleavable site comprising one of: (1) an RNA base string or (2) a DNA base string comprising a restriction enzyme (RE) recognition site; portion B comprises a DNA base string containing a tag PCR handle; portion C comprises a DNA base string containing a tag ID barcode, and the DNA base string forms part of a loop of the hairpin; portion D comprises a DNA base string having an inverse complementary sequence to the tag PCR handle, thereby forming part of a stem of the hairpin; and portion E comprises a DNA base string having an inverse complementary sequence to at least a portion of the RNA base string of portion (1) A or the DNA base string comprising a RE recognition site, thereby forming part of a stem of the hairpin.

[0097] Optionally, these hairpin-tagged nucleic acid molecules may further include one or more of the following: a linker attached to the 5' end of part A (in embodiments, the linker provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity); a linker between the linker and part A (in embodiments, the linker includes PEG(n), where n = 1-20); a tag ID barcode that is at least 4 bases long, or in embodiments, the tag ID barcode is 4, 5, 6, 7, 8 or more bases long; and / or the tag PCR stalk in part B and its reverse complementary sequence in part D, each of which is at least 10 bases long (in embodiments, the tag PCR stalk in part B and its reverse complementary sequence in part D are each 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 20 bases long).

[0098] The hairpin-tagged nucleic acid molecules presented in this article can optionally be constructed, at least in part, using a templated conjugation reaction.

[0099] In a representative hairpin-tagged nucleic acid molecule embodiment, portion A comprises a DNA base string containing a RE recognition site, and the RE recognition site is at least 4 bases long. For example, the RE recognition site may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 bases long. Optionally, the RE recognition site within any hairpin-tagged nucleic acid molecule may be an IIS-type restriction enzyme recognition site, a site that generates a 3' overhang, or both.

[0100] Other representative hairpin-tagged nucleic acid molecules are DNA / RNA hybrid hairpin-tagged nucleic acid molecules, where part A includes a single RNA base or a string of RNA bases, and the RNA base string in part A is at least 5 bases long. Therefore, consider DNA / RNA hybrid hairpin-tagged nucleic acid molecules where the RNA base string in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases long.

[0101] Groups of two or more described hairpin-tagged nucleic acid molecules and / or described DNA / RNA hybrid hairpin-tagged nucleic acid molecules are also provided, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence. In examples of these groups, the group further includes at least one attenuated tag-like nucleic acid molecule that differs from the hairpin-tagged nucleic acid molecules or DNA / RNA hybrid hairpin-tagged nucleic acid molecules of the group in that it lacks a functional cleavable site. As an example, groups of two or more hairpin-tagged nucleic acid molecules and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules are provided, wherein the attenuated tag-like nucleic acid molecule differs from the hairpin-tagged nucleic acid molecules or DNA / RNA hybrid hairpin-tagged nucleic acid molecules of the group in that it (1) replaces the RNA bases of part A with DNA bases, or (2) lacks the RE recognition site of part A.

[0102] Labeled probes are also provided, comprising probe molecules connected via a linker portion to a described hairpin-tagged nucleic acid molecule or a described DNA / RNA hybrid hairpin-tagged nucleic acid molecule. As an example, the probe molecule comprises an affinity molecule having binding affinity to a target molecule. Optionally, the affinity molecule comprises an antibody-binding domain having affinity to an antigen, and the target molecule comprises an antigen.

[0103] Labeled probes are also provided, wherein the probe molecules include one or more of the following: antibodies or their binding fragments, nucleic acids, small molecules, organic or inorganic chemical substances, putative drug targets, identified drugs, or biomacromolecule complexes. For example, a probe molecule may be one of a group of probe molecules, each of which includes multiple members of: small molecule libraries, drug target libraries, bioaffinity molecule libraries, natural product libraries, bioactive compound libraries, genomic libraries, transcriptomic libraries, metabolomic libraries, or drug screening libraries.

[0104] In other embodiments of the labeled probe, the target molecule includes a biomolecule, an inorganic object, or an addressable feature of an array. For example, the target molecule may include a biomolecule, and the biomolecule includes one or more of the following: proteins, lipids, carbohydrates, nucleic acid molecules, or combinations of proteins, lipids, carbohydrates, and / or nucleic acid molecules. Optionally, in some cases, the target molecule is one of a plurality of molecules constituting a complex, and the complex is located outside or inside one or more cells in a tissue sample.

[0105] In any labeled probe embodiment, the labeled probe may further include an amplified sequence containing a polymerase promoter sequence. As an example, the amplified sequence includes a T7 promoter sequence, such as a T7 promoter adaptor.

[0106] Another embodiment is a released hairpin-tagged nucleic acid molecule derived from the described hairpin tag or the described DNA / RNA hybrid hairpin tag, wherein the released hairpin tag has been separated from the linker by the enzymatic action of a restriction endonuclease or RNase H.

[0107] Another embodiment is a capture oligonucleotide (CO) nucleic acid molecule comprising functionally linked portions I-II-III-IV in a 5' to 3' sequence, wherein: portion I includes a cleavable site comprising one of: (1) a single RNA base or a continuous string of RNA bases, or (2) a string of DNA bases including a restriction enzyme (RE) recognition site; portion II includes a string of DNA bases containing a CO PCR stalk; portion III includes a string of DNA bases containing a spatial barcode; and portion IV includes a string of DNA bases containing a tag-capture region. Optionally, the CO nucleic acid molecule further includes a linker conjugated to the 5' end of portion I. For example, in some cases, the linker provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

[0108] In other options, the CO nucleic acid molecule includes a linker between the linker portion and the I portion. For example, an exemplary linker includes PEG(n), where n = 1-20.

[0109] In other CO nucleic acid molecule embodiments, the spatial barcode is at least 4 bases long. For example, the spatial barcode can be 4, 5, 6, 7, 8 or more bases long.

[0110] Optionally, in the examples of CO nucleic acid molecules, the CO PCR stalk in part II is at least 5 bases long. For example, the CO PCR stalk in part II may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases long.

[0111] CO nucleic acid molecules that further include a unique molecular identifier (UMI) were also considered.

[0112] In any of the CO nucleic acid molecule examples, the following example is considered: the sequence has no more than two consecutive self-complementary internal sequences. As an example, the CO nucleic acid molecule sequence has no more than three, four, five, six, seven, eight, nine, ten, eleven, or twelve consecutive self-complementary internal sequences.

[0113] In any of the CO nucleic acid molecule examples, the instance was constructed using at least in part a templated conjugation reaction.

[0114] In any of the CO nucleic acid molecule examples, optionally, part I comprises a DNA base string containing a RE recognition site, and the RE recognition site is at least 4 bases long. For example, the RE recognition site may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more bases long. Optionally, the RE recognition site is an IIS-type restriction enzyme recognition site, a site with a 3' overhang, or both.

[0115] CO nucleic acid molecules as DNA / RNA chimeric CO nucleic acid molecules are also considered, wherein part I comprises a single RNA base. DNA / RNA chimeric CO nucleic acid molecules are also provided, wherein part I comprises a continuous string of RNA bases. As an example, these DNA / RNA chimeric CO nucleic acid molecules may contain a continuous string of RNA bases in part I, said continuous string of RNA bases being 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases long or longer.

[0116] Another embodiment is a group of two or more CO nucleic acid molecules provided by any one of them and / or DNA / RNA chimeric CO nucleic acid molecules provided by any one of them, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

[0117] CO nucleic acid molecules or DNA / RNA chimeric CO nucleic acid molecules are also provided, each of which is linked to a capture feature via a linker portion of the nucleic acid molecule. As examples, in various embodiments, the capture feature is a bead, a chemically functionalized point on a glass surface, a chemically functionalized and defined region of a 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 linked to the gel. Linked CO nucleic acid molecules are also considered, wherein the capture feature is a spatially addressable feature in an array (such as a microarray, e.g., a microarray having at least 100 addressable capture features).

[0118] Another provided embodiment is a spatially encoded capture feature, which includes a capture feature connected to the provided CO nucleic acid molecule or the provided DNA / RNA chimeric CO nucleic acid molecule via a connecting portion. As an example, the capture feature includes an addressable location on a bead or generally two-dimensional surface. For instance, a representative spatially encoded capture feature embodiment includes a spatially encoded capture feature within an array of at least 100 different spatially encoded capture features, wherein the CO nucleic acid molecule on each of the at least 100 different spatially encoded capture features in the array each includes a different spatial barcode.

[0119] The capture pair is also described, comprising: a provided hairpin-tagged nucleic acid molecule, or a provided DNA / RNA hybrid hairpin-tagged nucleic acid molecule, or a provided released hairpin-tagged nucleic acid molecule; and a provided capture oligonucleotide (CO) nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule, or a provided linked CO, wherein the sequences of the hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule are at least partially complementary, such that when the hairpin-tagged nucleic acid molecule is released from its linker in the vicinity of the CO nucleic acid molecule, the released hairpin-tagged nucleic acid molecule is captured by the complementary sequence bound to the 3' end of the CO nucleic acid molecule, such that the resulting complex of the released hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule is capable of undergoing a downstream extension reaction by a polymerase.

[0120] Another embodiment is a spatially encoded capture array comprising a defined array of spatially addressed capture features, wherein each capture feature includes: a spatially identifiable feature comprising: a predefined addressable location on a generally two-dimensional solid surface; or a bead or other similar individual solid capture object; and multiple copies of a provided CO nucleic acid molecule or a provided DNA / RNA chimeric CO nucleic acid molecule attached at each feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to CO nucleic acid molecules at other features in the array. In an example, one or more CO nucleic acid molecules are applied to the spatially addressed capture features in the array by: printing CO droplets onto a predefined addressable location on a generally two-dimensional solid surface, or attaching CO to beads via a connecting portion.

[0121] Optionally, the spatially encoded capture array includes beads, each of which includes a visual barcode operatively coupled to the CO. For example, an instance of a spatially encoded capture array includes a visual barcode that enables the beads to be assigned to positions within the capture array.

[0122] Examples of spatially encoded trapping arrays are also considered, in which beads or other similar individual trapping objects are embedded in a biomolecularly permeable matrix. For example, the trapping object may include beads; the biomolecularly permeable matrix may include a gel; or both. In a specific example, the biomolecularly permeable matrix, including a gel, is formatted as a flexible sticker.

[0123] Other exemplary spatially encoded trapping arrays include a biomolecularly permeable matrix that is structurally stable at a selected temperature between 4-45°C; permeable to proteins such as functional RNase H and polymerases; permeable to ribonucleoside triphosphates (rNTPs) and / or deoxyribonucleotide triphosphates (dNTPs); and permeable to Mg. 2+The matrix is ​​permeable to ions; generally inert to biomolecules; and flexible enough to allow relatively thin matrix layers to be applied directly to generally two-dimensional samples or surfaces. For example, spatially encoded trapping arrays can be configured into three-dimensional thin-layer gels with widths and lengths significantly greater than their thickness, wherein spatially identifiable trapping features are generally arranged in a single plane on the gel surface defined by its length and width. In some cases, at least the first of the spatially identifiable trapping features is directly connected to and / or in contact with the second.

[0124] In any of the provided spatially encoded capture arrays, the matrix may comprise a hydrogel or a polyacrylamide gel. As an example, the thickness of the matrix or gel is no more than about 2 mm. In specific cases, the thickness of the matrix or gel is no more than about 1 mm, no more than 500 μm, no more than 250 μm, no more than 200 μm, no more than 150 μm, no more than 125 μm, no more than 100 μm, or less than 100 μm. Advantageously, in some cases, the thickness of the matrix or gel is 100-200 μm, 100-150 μm, or about 125 μm.

[0125] In any case of the provided spatial coding capture array, the array may be reinforced by an inert net or other support structure.

[0126] In the provided examples of spatially encoded capture arrays, predefined addressable locations on a generally two-dimensional solid surface have a surface area of ​​no more than about 1 μm × 1 μm; or the diameter of a bead or other similar individual solid capture object does not exceed about 20 μm.

[0127] In the provided examples of spatially encoded trapping arrays, the diameter of the bead or other similar individual solid trapping object does not exceed 18 μm, 15 μm, 12 μm, 10 μm, 8 μm, 5 μm, 3 μm, 1 μm, or about 100 nm. For example, the diameter of the bead or other similar individual solid trapping object may be between 1 and 3 μm.

[0128] Another embodiment is a semi-ordered spatially encoded capture array comprising: a grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide being applied to the spatially addressable location by clamping together: an x-coordinate connective oligonucleotide for labeling all spatially addressable locations within a row of the grid; and a y-coordinate connective oligonucleotide for labeling all spatially addressable locations within a column of the grid. Examples of these semi-ordered spatially encoded capture arrays at least partially utilize... Figures 17A-17CThe method provided is used for construction. In an embodiment, the semi-ordered spatially encoded capture array has an in-mesh grid format, such as... Figure 10 As shown in the image.

[0129] A semi-ordered spatially encoded capture array is also provided, comprising: an array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray comprising uniquely identifiable capture features, the location of which is at least partially specified by identification of the subarrays within the semi-ordered spatially encoded capture array.

[0130] Another example is a semi-ordered spatially encoded capture array comprising: a group of two or more subarrays, each subarray including multiple capture features, wherein each capture feature within each subarray is connected to a capture oligonucleotide including a unique position tag, the capture features within each subarray are randomly arranged, and each capture feature within each subarray further includes an oligonucleotide tag that identifies the subarray, the oligonucleotide tag that identifies the subarray being attached to the capture oligonucleotide of each feature in the subarray via a clamping connection.

[0131] A method for detecting and / or quantifying and / or locating targets in a substantially two-dimensional (2D) sample is also provided, the method involving: contacting a substantially 2D sample with at least one labeled probe to produce a substantially 2D stained sample, the labeled probe comprising: a hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode, or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode; and a probe molecule connected via a linker portion to the hairpin-tagged nucleic acid molecule including a tag ID barcode or the DNA / RNA hybrid hairpin-tagged nucleic acid molecule including a tag ID barcode; contacting the surface of the substantially 2D stained sample with a permeable spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: Multiple spatially identifiable features; and multiple copies of a capture oligonucleotide (CO) nucleic acid molecule or a DNA / RNA chimeric CO nucleic acid molecule linked at each spatially identifiable feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to CO nucleic acid molecules at other features in the array; placing a flow cell or other solution-containing cap above the sample-array sandwich to form a shell containing the sample-array sandwich; adding a solution comprising reaction components to the shell to form a reaction mixture, said components comprising: a lyase selected from RNase H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); Mg 2+Ions; and a buffer; incubating a sample-array sandwich in contact with the reaction mixture at analytical temperature for 30-60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the shell; and analyzing the reaction product mixture to detect and / or quantify a target in a substantially 2D sample and / or define the location of said target. Optionally, in these methods, the shell includes a flow cell. Examples of the provided methods provide location information for more than one target within a substantially 2D sample.

[0132] Embodiments of the method are performed at a single temperature (isothermal) or within a range of about 5°C of a single temperature. As an example, the analysis temperature ranges from 20-55°C or 37-42°C.

[0133] As an example, in any of the method embodiments, at least one polymerase provides the enzymatic activity of DNA polymerase, reverse transcriptase, or RNA polymerase.

[0134] As an example, in any of the method embodiments, analyzing the reaction product mixture includes sequence analysis of multiple nucleic acid molecules containing spatial barcodes and tag ID barcodes. For example, in some cases, analyzing the reaction product mixture includes next-generation sequencing (NGS) of multiple nucleic acid molecules containing spatial barcodes and tag ID barcodes. For example, in some methods, the multiple nucleic acid molecules containing spatial barcodes and tag ID barcodes are fully extended products released from spatially identifiable features by cleavage at CO cleavage sites (e.g., cleavage based on RNase H enzyme activity or restriction endonuclease activity). In examples of the methods, the analysis includes allocating the spatial location of at least one nucleic acid molecule containing spatial barcodes and tag ID barcodes within a substantially 2D sample.

[0135] Methods for isothermal spatial coding of biological samples are also provided, including methods for detecting and / or quantifying and / or locating targets in generally two-dimensional (2D) samples, wherein the generally 2D sample is a biological sample.

[0136] Another embodiment is the use of a set of hairpin-tagged nucleic acid molecules as provided herein, or a set of capture oligonucleotide (CO) nucleic acid molecules as provided herein, or both, for transcriptomic analysis of biological samples.

[0137] Spatial coding surfaces, such as trapping arrays, for example bead-based trapping arrays, are also described, generally as described or illustrated herein.

[0138] Another embodiment is a spatial coding workflow comprising: contacting a hybrid RNA / DNA tag including a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to generate a stained sample; positioning a capture array including capture features in contact with the stained sample to generate a sample / array sandwich; placing a fluid-containing shell on top of the sample / array sandwich; introducing an analytical solution including active RNase H and an active polymerase into the fluid-containing shell, thereby contacting the analytical solution with the sample / array sandwich; and incubating the sample / array sandwich in the sample solution at a temperature and time sufficient to allow RNase H activity to at least partially digest the hybrid RNA / DNA tag to generate a stained sample. The process involves: cleaving the tag and releasing the cleaved tag into an analytical solution near the capture feature; allowing the cleaved tag to interact with capture oligonucleotides (COs) on the nearby capture feature to provide a cleaved tag for capture; incubating a sample / array sandwich in a sample solution at a temperature and time sufficient to allow polymerase activity to extend the captured cleaved tag using COs as a template to produce an extension product; after sufficient extension, cleaving the extension product with RNase H in the sample solution to produce a complementary RNA / DNA region based on RNA bases in COs, thereby releasing a full-length extension product; collecting at least a portion of the released full-length extension product; and amplifying and / or sequencing at least one of the released full-length extension products.

[0139] Computer-readable media or digital resources and digital databases are also provided, which contain spatial location information of the features of the spatially coded array as described herein. As an example, in some cases, the computer-readable media or digital resources or digital databases contain spatial location information of substantially all the features of the spatially coded array.

[0140] The document also discloses an application of the provided computer-readable medium, digital resource, or digital database for providing a user with location information of one or more targets related to the spatial location information of a spatially coded array. As an example, using the provided computer-readable medium, digital resource, or digital database, the relevance is generated by using a spatially coded array in a workflow or method as described or illustrated herein.

[0141] Another embodiment is a kit that can be used to perform one of the methods provided herein. A representative kit includes one or more of the following: two or more described hairpin-tagged nucleic acid molecules; two or more described DNA / RNA hybrid hairpin-tagged nucleic acid molecules; a described group of two or more hairpin-tagged molecules; a group of two or more described labeled probes; two or more described capture oligonucleotide (CO) nucleic acid molecules; two or more described DNA / RNA chimeric CO nucleic acid molecules; a described group of two or more CO nucleic acid molecules; a described group of two or more DNA / RNA chimeric CO nucleic acid molecules; two or more described CO nucleic acid molecules, each linked to a capture feature; at least one described spatially encoded capture array; at least one described semi-ordered spatially encoded capture array; or a described spatially encoded surface.

[0142] In examples of these kits, spatially encoded capture arrays, semi-ordered spatially encoded capture arrays, or spatially encoded surfaces are in the form of flexible stickers.

[0143] Kit examples are also provided, which further include one or more of the following: a container containing a functional RNase H; a container containing a functional polymerase; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); a container containing Mg 2+ A container for a solution of ions; or a container containing a buffer solution. Optionally, in these kits, at least one of the containers contains at least two of the following: RNase H, polymerase, restriction endonuclease, rNTPs and / or dNTPs, Mg... 2+ Ionic or buffer solutions. Optionally, the complete analytical solution is provided in a single container.

[0144] The kit examples may also optionally include one or more of the following: components that can be used to prepare samples for analysis using the methods provided herein; a cover containing a solution adapted to be placed over a sample-array sandwich on a glass slide to form a fluid-containing shell for the sample-array sandwich; a flow cell cover; or a glass slide or other surface adapted to accommodate a generally two-dimensional sample.

[0145] Various aspects of this disclosure are now described using other details and options.

[0146] Methods and systems for isothermal spatial coding of biological samples, and analysis

[0147] Isothermal method: isothermal information transfer, spatial encoding, and release of the spatially encoded product into solution. As described herein, a novel nucleic acid tag is used to encode molecules or probes, the tag comprising a hairpin structure, thereby chimerating RNA / DNA molecules to generate RNA / DNA hybridization regions through base complementarity. This unit is extremely stable, resistant to RNA exonuclease activity, and can contain specific barcodes within the loop region of the hairpin (an example is provided with a tetrabase extension having consecutive unpaired bases, but longer lengths are covered) to distinguish different probes from each other in multiplex analysis. The hairpin tag sequence also contains the first of two PCR handles for downstream amplification of the spatially encoded product. Methods for constructing different types of this hairpin tag are provided, and its utility in workflows when conjugated to antibody probes (e.g.) is demonstrated.

[0148] After the probe, conjugated to a hairpin tag containing an RNA / DNA hybrid, binds to the surface and unbound conjugates are washed away, the hairpin tag can be released from the probe as a smaller whole-DNA hairpin (the released tag) while simultaneously binding to the sample surface using an enzyme containing RNase H activity. In an embodiment, a spatially encoded capture array is placed near (e.g., in direct contact) the sample surface containing the bound probe conjugates before its release by RNase H. As described herein, in an embodiment, this capture array surface is an array of 1 μm beads coated with capture oligonucleotides. It has also been demonstrated here that this capture surface may comprise 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 a matrix that is flexible enough to allow for direct contact application with the sample surface being analyzed.

[0149] The following embodiment is also considered, in which a number of different tag barcodes are included on the same antibody, making it possible to analyze the normal distribution of the tags after amplification (e.g., where they are all released from the same antibody type and should be in equimolar ratios). This embodiment is generally outside the normal working workflow but can be used, for example, as an internal control or calibration.

[0150] In this embodiment, the capturing oligonucleotide binds to a capturing feature (such as a bead) at its 5' end and contains a 3' capturing region designed to be complementary to the 3' end-specific bases of the released DNA hairpin, as well as a position barcode, and the second of the two PCR stalks is used for the downstream amplification of the spatially encoded product. The capturing oligonucleotide may also contain a 5' RNA base extension located between the second PCR stalk and the 5' bead connection portion.

[0151] The location of the bead / feature within the capture array is predetermined, and this bead location information is contained within the sequence (spatial or positional barcode region) of the capture oligonucleotide bound to the capture bead or feature. Each capture bead or feature contains a unique spatial barcode, which is common to all capture oligonucleotides on the bead or within the address of the feature. The location of the bead or feature within the capture array can be uploaded and stored in a software package or database, for example, in the form of a DNA sequence (positional or spatial barcode) corresponding to a specific location of each bead or feature.

[0152] Once placed nearby and after the introduction of RNase H, information from the sample is transferred (from the probe releasing the hairpin) to the (nearest) spatially encoded capture bead or feature through specific hybridization with the capture region contained within the 3' sequence of the capture oligonucleotide bound to the capture bead. In the same reaction mixture are polymerase and an essential cofactor (Mg) for extending the 3' end of the hairpin by DNA polymerase. 2+ The extension (dNTPs) is achieved by templated capture oligonucleotides, which copy positional information and a second PCR handle to the released hairpin oligonucleotide. Optionally, a 5' RNA base is placed within the capture oligonucleotide to generate a second substrate for recognition and cleavage by RNase H activity present in a one-pot isothermal reaction mixture. Including an RNA base at the 5' end of the capture oligonucleotide allows a spatially encoded probe to be released from the capture bead and into the surrounding solution.

[0153] When using a capture array in which capture oligonucleotides (or beads) are embedded within a macromolecularly permeable surface or gel, the capture surface can be placed near the sample to which the probe binds, followed by the introduction of enzymes and other reaction components that diffuse through the permeable surface / gel. A flexible encoding surface can be advantageous in conforming to the sample surface, placing the spatially encoded capture features (beads, as illustrated in the examples herein) very close to the sample. The provided "one-pot" isothermal encoding reaction can then be initiated by immersing the one-pot encoding reaction solution in the gel while incubating at the reaction temperature (e.g., 37–42 °C). During the reaction, information from the sample is transferred via RNase H digestion of the hairpin tag, followed by capture of the released hairpin tag near the site of binding to the sample, spatial encoding via an extension reaction using a polymerase, and optionally release into the surrounding solution after spatial encoding. Applying a coding, macromolecularly permeable capture surface or gel directly to a sample allows one or more of the enzymes (such as at least the triggering enzyme E. coli RNase H, as shown in this paper) and / or cofactors required for isothermal reactions to diffuse through the coding (recognition space) surface and come into contact with the appropriate substrate without requiring the capture surface to move relative to the sample.

[0154] Following the reaction, the spatially encoded reaction products diffuse out of the gel (or other permeable matrix) and into the surrounding solution, where they can be recovered by aspiration. A sufficient volume of solution is required to cover the spatially encoded gel (capture array), but optionally a flow cell covering the entire area of ​​the spatially encoded gel may exist, which will subsequently define the solution volume. Ideally, this is a low-volume solution, so that the concentration of the diffused products remains high. However, in embodiments employing the described optional capture step, volume is less critical.

[0155] In the illustrated embodiment, the solution volume is approximately 40 μl. If this volume is significantly higher, an additional (bead) capture step can be performed to capture the diffused, spatially encoded product onto, for example, beads. This can be achieved by the fact that, after an extension step that generates new DNA / RNA hybrid regions, the spatially encoded product can be released from the capture beads. The DNA regions exposed after the removal of RNA regions (following the extension and exposure to RNase H activity in the reaction solution) can be captured using beads (or another capture surface) containing fixed oligonucleotides designed to contain complementary regions for capturing the extended and released products. After capture in a 2× capture buffer (added 1:1 by volume to the aspirate solution containing spatially encoded and released extension products) containing 20 mM Tris pH 7.5 and 1 M NaCl, optional washing can be performed in a 1× buffer, followed by reducing the beads to a low volume (e.g., by magnetic or centrifugal aggregation) for resuspending in a consistent low volume (less than 25 μl) of elution solution or buffer containing low to no salt (e.g., water or 10 mM Tris pH 7.5). Higher salt in the capture step will increase the tendency to capture spatially encoded extension products by hybridization with the oligonucleotides immobilized in the beads. Lower salt in the elution buffer will increase the tendency to dehybridize captured products from the oligonucleotides immobilized in the beads when exposed to heat (>75°C). Lower elution volumes will provide consistency between experiments and will allow for the recovery of all products that diffuse from the gel during isothermal reactions.

[0156] These elution products can be further processed, for example, by capture and / or by PCR amplification. These amplified products can then be prepared for next-generation sequencing using known amplicon-based or conjugation-based sequencing library technologies. See, for example, information available online at illumina.com / techniques / sequencing / ngs-library-prep.html (which describes various types of library preparation for Imina sequencing, including both amplicon-based and conjugation-based preparation). The resulting sequencing data library contains consecutive reads (counts) containing individual probe hairpin sequences fused with positional information contained within capture oligonucleotides bound to beads on which the hairpins are captured. A digital image is then reconstructed using a predetermined map of bead positions (barcodes), e.g., accessible via a license or subscription service, by overlaying the counts of each probe obtained from sequencing onto a spatially encoded array at the location where they were captured. Thus, this workflow enables digital image reconstruction using next-generation sequencing.

[0157] The hairpin nucleic acid tag described in this article contains both RNA and DNA to produce a substrate for cleavage by RNase H (see [link to article]). Figures 1A-1B The benefits of this design include: the ability to expose rationally designed DNA sequence extensions via RNase H digestion for optimal capture and release of hairpins at isothermal temperatures; the ability to capture such sequences onto and extend capture oligonucleotides that bind to capture features contained within an array of spatially assigned capture features; optimization of the hairpin capture region Tm under isothermal reaction conditions; and the ability of the method to operate without impairing antibody recognition of the target in the analyzed sample.

[0158] As described in this article, the hairpins provided have been shown to be stable for at least 6 weeks when conjugated to antibodies and stored at 4°C in phosphate-buffered saline (PBS) + bovine serum albumin (BSA).

[0159] This article describes the use of a hairpin tag containing both RNA and DNA for isothermal release of information from a DNA-encoded molecule bound to a surface following a selective binding event. The resulting benefits include: the ability to “trigger” hairpin release upon contact with an enzyme containing RNase H activity; and the ability to trigger hairpin release at temperatures compatible with capturing the released tag onto a capturing oligonucleotide.

[0160] It also provides the use of conjugated capture oligonucleotides (structure in) Figure 3As shown in Figure 4, following a selective binding event, the DNA-encoded molecule bound to the surface is captured via an extension reaction utilizing a polymerase included in the same reaction mixture. Figure 2 The resulting benefits include: reduced probe lateral diffusion or the ability of probes to "jump" to different capture oligonucleotides, as the extension reaction immediately "locks" each hairpin onto the first capture oligonucleotide (or one of the first capture oligonucleotides) that it contacts via extension using polymerase; and the ability to copy positional information from highly proximate capture oligonucleotides to released DNA hairpin oligonucleotides within the same reaction mixture (and vice versa), where the hairpin oligonucleotide is released from the sample surface via templated extension of the 3' end of the captured oligonucleotide (and the capture oligonucleotide is of one type; when the 3' end of the capture oligonucleotide is not blocked).

[0161] When using a polymerase containing reverse transcriptase activity (e.g., RNA-dependent DNA polymerase activity), it is possible to read RNA bases contained within the captured oligonucleotide template during the extension reaction. Here, a polymerase containing both RNA and DNA-dependent DNA polymerase activity is used, which enables the incorporation of deoxyribonucleotide triphosphate (dNTP) bases in response to DNA or RNA bases in the captured oligonucleotide template. The data described herein demonstrate: PCR amplification, which is used for hairpin capture and extension on capture beads followed by recovery of full-length products; qPCR results of + / - experiments; gelation of the amplified full-length products; and Sanger sequencing traces for the preparation of full-length extension products for NGS (see Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023).

[0162] Release the spatially encoded product from the spatial capture array ( Figures 9A-9E (This can be done in multiple ways) within the same isothermal reaction mixture. This can be achieved by including RNA bases in the 5' region of the captured oligonucleotide, allowing polymerase to extend this region to generate new DNA / RNA hybrids, which then become substrates for RNase H already included in the one-pot isothermal signal transfer reaction. Figure 9A and 9E This can also be achieved by including an encoded restriction enzyme site (at the 5' end of a single-stranded capture oligonucleotide), such that dsDNA restriction sites are generated by polymerase elongation, said dsDNA restriction sites being recognized and cleaved by the corresponding restriction enzyme during a one-pot isothermal information transfer reaction, or after the elongation step has been performed. Figure 9B and 9EA third mechanism, including photo- or chemically cleavable linkers, is used to attach the 5' end of a capture oligonucleotide to a capture feature that enables the release of a spatially encoded product. Figure 9C and 9D ).

[0163] Another benefit is that product recovery does not require user disposal. In previously known workflows, after capture, the two surfaces are separated, the capture array is washed, and the captured probe is eluted from the capture array at high temperature. The method disclosed herein avoids the need for any of these steps.

[0164] Not all capture oligonucleotides can be modified to contain 5'-RNA bases for spontaneous release of the product into solution after spatial encoding. Instead, a certain percentage of capture oligonucleotides at each capture feature (here, on each bead) may contain 5'-RNA bases for spontaneous product release, while a certain percentage of these capture oligonucleotides contain whole DNA bases but are linked to the capture feature via photolytically cleavable linkers. This embodiment describes an extremely useful capability. This allows the user to later return to the sample of interest, screened with a relatively "shallow" read depth (i.e., not all capture information is released during the isothermal reaction, but the resulting library is sequenced, and a reconstructed digital image is read from this portion), and select the region of interest (ROI) via software that displays the digital image. Subsequently, it is possible to employ an instrument for defined site-specific photorelease of all remaining spatially encoded products within the selected ROI. This has been achieved, for example, by using a digital micromirror array to focus UV light wavelengths at a defined location on the surface, and this type of array can be manufactured or commercially available with a resolution of 10 μm or higher. Additional data can then be obtained from the remaining sample.

[0165] Furthermore, compatibility with the isothermal space coding system described herein is also considered as a way to extend the application of the techniques described herein. To this end, the molecular ID tag can be designed to include RNA bases therein. Figure 5A This will work seamlessly with the one-pot isothermal information transfer reaction described in this paper. Figure 5BIn one example, this is achieved by including a 5'-RNA base extension within a linear (non-hairpin) tag linked to the encoded molecular library. From the 5' to the 3' end of the tag, this tag type contains: a 5'-linker for linking the tag to the molecular library; followed by a 5'-RNA base extension encoding the reverse complementary sequence of the sequence to be captured (a constant region on all tags); followed by a variable region encoding the individual molecule linked to the tag; and then a second constant region consisting of DNA bases. The dsDNA region can be generated by hybridization of complementary oligonucleotides with the 3'-constant region of the tag, or installed as a constant hairpin structure, before or after binding analysis (screening or selection). This constant "primer" or hairpin structure at the 3' end is preferably installed before binding analysis and can be extended using an extension reaction with a polymerase containing both RNA- and DNA-dependent DNA polymerases for extension via both base types contained within the tag, to generate a DNA / RNA hybridization region near the 5' end of the linked molecular library. The extension reaction can be performed before or after binding analysis. After binding, the methods described above can be used to spatially resolve labeled probes or molecular libraries. Upon introduction of an enzyme containing RNase H activity, these tags will be released from the sample surface in the same manner as described above, exposing the 3' end of the extension product (the constant capture region on all tags), and thus allowing inclusion within the same isothermal information transfer workflow described herein. Other sequences can also be designed to be included in the tag sequence, such as... Figure 6 As shown, it has many additional benefits, as outlined below. Figure 6 In this case, using two opposing RNA polymerase promoters will be optimal.

[0166] Other methods of achieving this involve adding adaptor molecules to other DNA tags to make them compatible with one-pot isothermal information transfer reactions. These various adaptor molecules can appear in different forms, examples of which are described below:

[0167] The first form of the adaptor is simply an RNA / DNA hybridization hairpin tag attached to the free end of the DNA encoding the probe, as described in this paper (Figure 1). This type of adaptor can be fused with other DNA tags as a continuous polymer or chemically linked to other DNA tags or molecules via a compatible covalent reaction. In this case, the tag barcode will now be encoded within the loop of the hairpin attached to the DNA-encoding probe. This will work for converting and encoding antibodies or other smaller groups of probes (no more than tens of thousands of probes in the mixture), including reagents from other companies (such as SomaLogic) or specific transcriptomics probes. This form of adaptor has been shown to work well within one-pot isothermal information transfer reactions.

[0168] Amplification:

[0169] The second type of adaptor imparts amplification characteristics within the method. Here, an adaptor containing a polymerase promoter sequence (such as T7 or S6) is attached to the DNA strand of a DNA-encoded molecule via a templated conjugation reaction. Figures 7A-7B To achieve the binding of the adaptor to the ssDNA tag (). Figure 7A For conjugation, the sequence information must be known because the conjugation reaction must be templated using a splint sequence. Fortunately, most encoded molecular libraries contain two constant regions within their DNA tag structure (for downstream recovery after screening or selection via PCR amplification), and these constant regions are flanked by information corresponding to the encoded library members. One of these constant regions (region 1) can be used to conjugate the adaptor, and the other constant sequence (region 2) can be used for capture. For conjugating this adaptor type to a dsDNA tag ( Figure 7B Sequence information of the constant region is not required. Once conjugated, this promoter sequence is capable of initiating transcription of the sequence encoding the probe upon introduction of an appropriate polymerase. For the T7 adaptor example, this adaptor may consist of a double-stranded DNA sequence encoding the T7 polymerase promoter, with prominent compatibility "sticky ends" for conjugation to the coding DNA strand of the molecular library. Alternatively, this adaptor may consist of a single continuous DNA strand within a hairpin structure, where the stem (dsDNA region) of the hairpin encodes the T7 polymerase promoter sequence, and the prominent compatibility "sticky ends" for conjugation to the coding DNA strand of the molecular library are available for ligation. The same structure can be used to encode any polymerase promoter sequence. This type of adaptor can conjugate to the coding DNA strand of the molecular library before or after the probe binds to the sample surface. After washing unbound probes from the sample, the same type of capture surface used in the previous examples can be placed near the sample being probed.

[0170] To initiate these warm information transfer reactions, a suitable polymerase (in this case, T7 RNA polymerase) and appropriate cofactors (NTPs and Mg2+ for transcription in this case) are introduced. 2+ (etc.), and it is estimated that at most 1,000 RNA copies are generated from each adaptor-modified DNA tag that binds to the surface (via its associated probe). In this case, the 3'-capture region of the capture oligonucleotide within the capture array is designed to specifically capture the RNA generated from the coding tag via a second constant region not used for adaptor binding. Figure 8In the presence of a polymerase containing reverse transcriptase activity, the RNA produced by this reaction is proximally captured onto a spatially encoded capture feature. Upon contact (capturing the RNA onto the capture oligonucleotide), the reverse transcriptase extends the information contained within the released RNA to the capture oligonucleotide of the capture array by extending the 3' end of the capture oligonucleotide (CO). This process produces a series of DNA molecules containing (amplified) information (probe or molecule ID barcode) released from the adaptor-modified probe, as well as information from the spatially encoded CO molecule located closest to the sample surface. Subsequently, the constant region of the encoding tag used for conjugation becomes one of two PCR stalks for library amplification, while the other PCR stalk is provided on the capture oligonucleotide. These consecutive molecules constitute a spatially encoded library and can be released from the capture array into solution by chemical cleavage, photolysis, RNase H, or restriction enzyme cleavage. Figure 9D and 9E Subsequently, as described above, the spatially encoded library products are recovered by aspirating the solution for downstream NGS library preparation and sequencing.

[0171] The application of methods includes transcriptomics.

[0172] This general first approach (DNA / RNA hybridization hairpin tagging method) is scalable to include probe transcriptomics within the described spatial coding workflow. This method consists of probe pairs using a neighboring RNA sequence that recognizes the target RNA (this has been accomplished otherwise). Once the target RNA sequence is identified, each probe pair is designed to produce a hairpin structure of the described type only when it binds to its nearby target; the probe-bound RNA generates a template for proximal conjugation of the matching probe pair to produce the disclosed hairpin structure. When multiplexed, each hairpin formed by proximal-initiated probe pair conjugation contains a barcode corresponding to the probed RNA in the loop region. To accommodate a large library of tens of thousands of probes, the barcode in the hairpin loop can be extended to 8-10 bp. The isothermal information transfer reaction is initiated in the same manner as with antibody probes labeled with DNA / RNA hybridization hairpin oligonucleotide tags. This ultimately enables multi-omics analyses that can be performed simultaneously on the same sample surface using the described isothermal information transfer reaction. If not performed simultaneously, this method can be performed in tandem (first probe RNA, followed by protein from the same sample, or vice versa) or on sequential slices for tissue analysis (if needed or necessary) for some sample types.

[0173] Other types of tags for generating RNA / DNA hybrid species through proximity have also been designed and demonstrated. When used within similar workflows, these tag types can also be used to probe nucleic acid sequences in a sample and / or to determine the interaction between labeled molecules and spatial resolution in a sample. These can consist of two complementary regions forced together by proximity, designed so that they do not produce complementarity at the temperature of the hybridization step (these complementary regions are less than 10 bp in length, so that one of the two complementary regions consists of RNA bases and the other consists of DNA bases within the complementary region). Therefore, when used in this workflow, proximity is generated by probe specificity to the corresponding target of each probe, and if the two tags are close enough to generate RNA / DNA species through proximity, information about their proximity can be released upon contact with an enzyme containing RNase H activity, and this information can be spatially encoded by capturing the released information using downstream steps of the isothermal spatial encoding workflow described above.

[0174] Spatial Coding Surface

[0175] As described herein, in some cases, spatially encoded capture surfaces are characterized arrays, each comprising bound capture oligonucleotides. The arrays may be characterized by high-density stacked, randomly arranged beads or spotted microarrays, or by using both techniques for total surface expansion (described below). The capture oligonucleotides (COs) at each feature of the capture array contain a capture region (CR) for capturing information released from the sample via base complementarity. This CR may be common to all COs contained within the spatially encoded capture surface. Each CO also contains a spatial barcode (SB) (common to each CO within the CO layer at each feature location), which is specific to each individual feature and is used to assign the capture feature location by one or more forms of sequencing. Each CO also contains one of two constant regions (CRs) common to each CO in the array, which can serve as a PCR handle (PH) required for the recovery and / or amplification of the spatially encoded product after the spatially encoded reaction. CO may also contain continuous or discontinuous extensions of randomized bases, including unique molecular identifiers (UMIs) that can be used to eliminate amplification biases generated by PCR (well known in the art; see, for example, Kivioja et al., Nature Methods 9:72-74, 2012, doi.org / 10.1038 / nmeth.1778). A preferred structural orientation of the nucleic acid elements of CO within the capture array, from 5' to 3', is: a 5'-linker for attaching CO to the surface (feature); followed by an optional 4-8 5'-RNA base extension; followed by one of two pH regions; followed by an SB region; followed by a CR for capturing information released from the sample. In this way, information released from the sample during isothermal spatial encoding reactions via RNase H or via RNA amplification can be captured onto CO at each spatially resolved capture feature.

[0176] Although examples using eight (8) RNA bases are provided herein, this number can be reduced, possibly down to the length that RNase H can cleave (which in some cases is as short as a single RNA base from the hybrid strand). Examples in which the number of RNA bases is increased beyond 8 are also considered to expose more (captureable) bases after elongation and release the space-encoded elongation product (as described herein).

[0177] In the case of using RNase H, the 3' end of the oligonucleotide captured from the sample can be extended by polymerase to produce a product consisting of a continuous sequence containing information about the released probe and positional information provided by the capture features. Additionally, both pH values ​​are required for downstream recovery and / or amplification of spatially encoded products.

[0178] In cases where tag information amplification is achieved by attaching an adaptor region to a probe nucleic acid tag containing a competent promoter region (as described above, in 5, under amplification, this is T7 RNA polymerase), the information released from the sample is captured by a novel synthetic sequence common to all probe tags. The COs in the capture array contain a common 3'CR for capturing the 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 CO to contain information templated by the RNA product released from the bound probe. These fully extended COs that have already encountered the released RNA sequence cannot be extended again. Furthermore, the RNase H activity within the reverse transcriptase will destroy the released RNA after its copying through the extension reaction, and therefore should be destroyed, which reduces its chance of being used as a template for subsequent extension reactions. This effectively results in one sequencing count for each released RNA.

[0179] In cases where the extension of 4-8 RNA bases is included within the 5' end of the CO of the capture array, a polymerase containing both RNA- and DNA-dependent DNA polymerase activity (or two polymerases containing these activities together) is required for the capture of oligonucleotides via the CO region consisting of two base types from the CO template. Preferably, an enzyme containing both activities (such as Maxima) is used under conditions compatible with other enzymes used in the isothermal reaction mixture. TM RT polymerase).

[0180] Optionally, some percentage or all of the CO contains this 5'-RNA base extension for spontaneous release of the space-encoded product into solution by an enzyme containing RNase H activity.

[0181] Optionally, some percentage or all of the CO is connected to features of the trapping array via photosplittable linkers, and these extended and spatially encoded products can be released after the isothermal spatially encoded reaction is complete.

[0182] Optionally, some percentage or all of the CO contains a 5' restriction enzyme (RE) site, which is formed after extension by a polymerase (which does not necessarily need to have any RNA-dependent DNA polymerase activity, only DNA-dependent DNA polymerase activity), thereby releasing the spatially encoded product that can be recognized by the RE newly formed at this site after polymerase extension. This RE may be included in the same reaction mixture as the isothermal information transfer reaction, or introduced after the isothermal spatial encoding reaction is complete.

[0183] Different forms of direct visual sequencing of spatial barcodes contained within the features of a capture array, which can be used to determine the positions of randomly arranged capture features, may include those known in the art (sequencing by synthesis (SBS), sequencing by binding (SBB), or orthogonal cleavage sequencing (OCS), such as those described in WO 2022 / 187719). This allows for the identification of each individual feature position within the capture array, and the coordinates of these positions are recorded as a unique, continuous DNA sequence within a package that generates a physical map of the feature positions (no different from that of a microarray). This product can be used as a capture surface, but preferably, the capture array is subsequently modified. Determining the positions of randomly arranged capture features (such as beads coated with capture oligonucleotides, which are randomly arranged on the surface) is not required for microarrays because the positions of the capture features are predetermined.

[0184] For direct sequencing using SBS or SBB methods, positional barcodes within captured oligonucleotides can comprise 30 or fewer consecutive bases. This is because positional barcodes within captured oligonucleotides can be directly sequenced using visual decoding methods (next-generation sequencing). However, the construction of visual barcodes for decoding via OCS experiments, along with spatial barcodes of captured oligonucleotides, is coordinately constructed in a bead-based library after three rounds of splitting and merging. This method was first described in WO 2022 / 187719; its use in encoding captured oligonucleotides for next-generation sequencing applications is further described herein (see Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023). In simple terms, barcode segments consisting of dsDNA containing designed cleavage sites (for RE recognition) are linked to one of five detectable markers (recognizers) and arranged in a plate or tube, whereby the recognizer segments of the five different markers are mixed in a 1:1 ratio without redundancy. For example, instead of mixing it with more AF405-marked Id1, the Id1 of an AF488-marked, AF550-marked, AF647-marked, or AF750-marked Id1 is mixed. This improves visual detection during OCS experiments. This is done for each different-marked segment in the chain to provide a total of 10 equally mixed two-color options at each segment. Here, 6 segments of this type will be used to generate a combined library of 1M beads. This bead library will be generated within 3 rounds of splitting and merging, rather than 6 rounds (for 6 recognizer segments), because segments Id1 and Id2 will be pre-joined to form 100 options for recognizer joining in round 1 (Id1+2), segments Id3 and Id4 will be pre-joined to form 100 options for recognizer joining in round 2 (Id3+4), and segments Id5 and Id6 will be pre-joined to form 100 options for recognizer joining in round 3 (Id5+6). This will produce a total library diversity of 1M distinctly labeled beads after only 3 rounds of splitting and merging. Here, each cycle removes two distinct labels from each bead, thus achieving robust results for the OCS experiment. Before, during, or after the first round of recognition binding to the bead, 100 different single-stranded oligonucleotides are bound to the bead (using the same or unique / orthogonal / non-competitive binding chemicals) to distinguish each of the 100 different options (Id1+2) by a unique nucleic acid sequence. This single-stranded nucleic acid sequence represents the first segment of the oligonucleotide captured by NGS, which will be co-constructed with the visual recognition barcode.The first segment (NGS1) of the NGS-captured oligonucleotide contains: a 5' linker chemical compatible with the bead linker, optionally a photolytically cleavable linker; subsequently, optionally, an RNA base (a constant region composed of RNA or DNA) is included within the 5' end of the oligonucleotide; followed by one of the two PCR handles required for downstream PCR (the constant region); followed by a 5-base sequence encoding each of 100 recognizant combinations (Id1+2); followed by a common 4-base site for all 100 different NGS1 variants, designed for orthogonal binding to the subsequent NGS barcode segment. After the first coordinated (commonly encoded) recognizant segment (Id1+2) is linked to its corresponding NGS segment (NGS1), the beads are washed, incorporated into a mixture, and split into the next set of 100 different options. Because the enzymatic binding of the subsequent recognizants to the NGS barcode segments is completely orthogonal, these reactions can occur simultaneously within the same reaction mixture. For example, the recognizer segment contains sticky ends for conjugation, which have a different length and base composition than the sticky ends produced for conjugation of the NGS segment. This is done not only by carefully designing the sticky ends of each conjugation (recognizer and NGS segment) to be orthogonal according to base composition and length, but also by using RNA clipping to template NGS segment conjugation. This allows the use of T4 DNA ligase (whose preferred substrate is the dsDNA of the recognizer segment) with SplintR enzyme (whose preferred substrate is RNA clipped DNA-DNA conjugation ligation). This should further improve the specificity of the reaction occurring for both segments when performed in tandem or simultaneously within the same mixture. Thus, orthogonal enzymatic conjugation of Id3+4 with NGS2 can be performed simultaneously in the same well, where Id3+4 consists of 100 different options encoded within the spatial barcode region of NGS2. NGS2 contains: a 5' region complementary to the constant RNA splint, which will be used for templated conjugation of NGS2 and NGS1 on the bead; and a region encoding each of 100 different options corresponding to Id3+4; followed by a 3' end, which is used for templated conjugation of the next NGS segment in the conjugation strand. The beads are then washed, incorporated into the mixture, and split into a final 100 distinct wells for orthogonal conjugation of Id5+6 and NGS3 segments. NGS3 contains: a 5' region complementary to the constant RNA splint, which will be used for templated conjugation of NGS3 and NGS2 on the bead; and a 5-base region encoding each of 100 different options corresponding to Id5+6; followed by a 3' end, which is used for capture or for templated conjugation of the next NGS segment in the conjugation strand (described below under the capture array).Subsequently, the beads were washed and merged to form a bead library with 1M diversity, wherein each bead type in the library contains a unique sequence of visual markers and coordinate-based NGS-captured oligonucleotides containing DNA sequence information corresponding to color codes (identifier strands).

[0185] Benefits include a significant reduction in bead reading time and cost compared to SBB or SBS (next-generation sequencing). This is because the OCS workflow required for reading the recognition strand sequence on the beads involves introducing different unique / orthogonal / non-competitive restriction endonucleases (REs) in each cycle, followed by imaging. These REs are much cheaper than the components required for next-generation sequencing. Furthermore, all the lysis reagents required for bead reading via the OCS workflow described here are available in the same reaction buffer (from NEB). It operates in a buffer solution. Furthermore, the entire OCS bead decoding workflow occurs at a relatively low isothermal reaction temperature (37°C), and therefore no high temperature or any temperature changes are required during decoding. Finally, the cycle time for decoding this type of recognition chain is approximately 5 minutes per cycle; decoding a recognition chain of the six segments described herein using the required OCS workflow will take approximately 30 minutes. After decoding the recognition chain on the bead to determine the bead position on the surface, the NGS-captured oligonucleotides containing information about the recognition chain on the bead (color code) remain intact for subsequent capture of nucleic acids (e.g., during the isothermal spatial encoding workflow described herein).

[0186] Alternatively, conventional NGS reagents, as mentioned above, can be used to determine bead positions. Conventional NGS sequencing experiments require expensive reagents to accurately determine DNA sequences, including reversibly capped and / or labeled nucleotides. High temperatures (typically 65°C) are required during decoding, and multiple different reagents are introduced in each decoding cycle. These reagents cannot be mixed (e.g., lysis solutions cannot be mixed with incorporation solutions), necessitating more complex fluid dynamics, and each NGS cycle takes significantly longer than 5 minutes. To read a large surface area of ​​1 μm beads by NGS, such as a 1.75 cm × 1.75 cm surface of stacked 1 μm beads, where approximately 10B of individual 1 μm beads will exist, the length of the NGS position barcode must exceed 24 bp to achieve at least 200T diversity of different beads (possible position barcode combinations) in the library to reduce the chance of barcode redundancy in a large surface area. This ultimately requires well over 6 decoding cycles.

[0187] In exemplary prior art, all beads are labeled with different combinations of fluorophores (e.g., as described in WO2022 / 187719) before decoding via OCS. Therefore, high dynamic range (HDR) imaging should be used for accurate decoding when determining the identifier chain sequence via the OCS workflow. In the case of the described identifier chain and OCS workflow, the bead library diversity is only 1M. Therefore, how can a 1.75cm × 1.75cm surface area be covered with a capture array in such cases of low bead diversity? This is achieved using a semi-patterned bead array, where x and y coordinate oligonucleotide barcodes are directly printed onto randomly arranged beads within each feature of the semi-patterned bead array. These x and y coordinate barcodes are directly attached to the bead-capturing oligonucleotides and impart additional positional information to the beads within each feature of the semi-patterned bead array. This is more clearly outlined in the capture array section below. The problem with this method is that each randomly arranged bead-capturing array must be read individually, and this is better suited for creating many arrays by copying the information contained in the bead-capturing oligonucleotide array to the acceptor array. This is described in more detail below.

[0188] Capture Array:

[0189] Using one of many different linking chemistry methods (covalent, non-covalent), a coordinated identifier / NGS bead library can be immobilized on a surface at a desired density for downstream visual encoding and decoding experiments, such as those using OCS. Preferably, the beads are arranged randomly in a semi-patterned manner, whereby the patterned features of the array consist of defined regions for bead immobilization. These bead immobilization features can consist of squares or other shapes of defined sizes (e.g., 100 μm × 100 μm squares, thereby randomly arranging approximately 1,000 stacked 3 μm beads; or 35 μm × 35 μm squares, thereby randomly arranging approximately 1,200 1 μm beads). Once randomly arranged within the squares, a droplet printer can add additional oligonucleotides to the array using x and y coordinates. These x and y coordinate oligonucleotides bind directly to the NGS barcode on the beads to impart additional positional information. For example, an array of 35μm×35μm squares arranged in a grid of 500 squares × 500 squares with a spacing of 1-3μm will cover a surface area larger than 1.75cm × 1.75cm. Figure 10Each square of the grid will accept a unique combination of oligonucleotides, whereby the x-coordinate oligonucleotide is spliced ​​to an NGS barcode containing identification body chain information via a splice. First, the x-coordinate oligonucleotide is added to the grid row and contains: a 5' region for templated splice attachment to the NGS 3 barcode (3' acceptor region); a 5' base region corresponding to the grid row; and a 3' region for templated splice attachment to the y-coordinate oligonucleotide. After the x-coordinate oligonucleotide has been attached to the bead, the y-coordinate oligonucleotide is added to the grid and contains: a 5' region for splice attachment to the x-coordinate oligonucleotide on the bead; a 5' base region corresponding to the grid column; and a 3' capture region for capturing the oligonucleotide (e.g., during the isothermal information transfer reaction described herein). Thus, by using the additional spatial information provided by the x and y coordinate oligonucleotides deposited in the squares, and by reading a relatively low diversity library (1M as described herein) of the differently barcoded beads, a very large surface area can be covered without the risk of bead redundancy. The square grid can be expanded to increase the total surface area of ​​the capture array. This overall approach (a square grid containing randomly arranged beads) reduces the overall bead diversity required to produce capture arrays with large surface areas and significantly reduces the time required to read each capture array.

[0190] Reading the location of the bead encoded by the identifier / NGS coordinates: The identifier chain on the bead can be distinguished by imaging before and after exposure to a unique / orthogonal / non-competitive RE in a series of cycles (described in WO2022 / 187719 and briefly described herein). Importantly, the lysis reagent used (such as the RE) will not cleave the NGS barcode. Therefore, after the OCS experiment used to read the bead location, the corresponding NGS-captured oligonucleotide encoding the bead location will remain intact and capable of capturing the information released during the one-pot isothermal information transfer / spatial encoding reaction.

[0191] Use of Capture Arrays: There are several ways to make spatially encoded capture arrays work. After reading the bead positions within a semi-patterned random bead array, the beads can be embedded in a gel, peeled off from the surface where their positions are determined (as in the example shown in this paper), and used directly as a capture array. Figure 11ARigid surfaces (such as patterned silicon wafers, glass, or plastic) are not well-suited for information transfer reactions. This is because these rigid surfaces will not conform well to the sample, as they are not flexible (like gels or membranes). Furthermore, it is difficult to maintain bead fixation on rigid surfaces during the transfer reaction, even when the beads are covalently bound to the trapping array surface. This is because, to achieve subcellular resolution using any information transfer method, the trapping array must be extremely close to (within only a few micrometers or in contact with) the sample. This physical contact could literally wipe the fixed beads off the rigid surface. However, when beads are embedded in a flexible matrix (such as a gel) and subsequently peeled from their initially fixed surface, the beads tend to remain within their initial relative positions during the transfer reaction. Moreover, semi-permeable gels can be used throughout the reaction due to their isothermal properties and potential integrity (in one form, the release of spatially encoded products from the trapping beads during the isothermal reaction). In this way, reagents (enzymes and / or their essential cofactors) required to initiate and / or carry out a complete isothermal information transfer reaction can be introduced into the semi-permeable gel matrix via diffusion. The reaction proceeds by simply cultivating the array and sample (sandwich) in an isothermal information transfer reaction solution at isothermal temperatures (37-42°C), and the product can be released from the captured feature into the surrounding solution, allowing it to diffuse through the gel and subsequently be recovered by aspirating the solution. Figure 12 See also Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023, which claims priority.

[0192] Following aspiration, additional analysis can be as simple as adding isothermal reaction components directly to beads containing linked RNA / DNA hybridization hairpin tags or to samples with bound antibody-tag conjugates, incubating at an isothermal reaction temperature for a period of time, and then pulling the solution from the sample surface (or from the beads via a magnet), capturing the tags released in the aspiration solution onto capture beads containing capture oligonucleotides added to the aspiration solution. These tags are captured at the reaction temperature in the presence of all isothermal reaction components, thus extending the tags on the capture beads to contain encoded spatial barcode regions and PCR stems within the capture oligonucleotides. Subsequently, the full-length extension products (containing 5'-RNA bases, allowing this to be achieved after the extension reaction) are cleaved from the beads by RNase H activity, and these products are suitable for amplification using designed primers.

[0193] There are other ways to improve the stability of beads within a gel matrix (the degree to which they remain bound) beyond those shown in this paper. Exemplary methods are described below.

[0194] In the examples shown in this paper, the spatially encoded surface consists of 1 μm beads containing a single type of capture oligonucleotide (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 prepare robust, spatially encoded capture arrays, their production can be industrialized, requiring the development of software components for reading bead positions and reconstructing digital images using next-generation sequencing counts.

[0195] Capture oligonucleotides by printing x and y coordinates

[0196] As described elsewhere (in this document, but also in the OCS PCT application), visual barcodes can be operatively coupled to capture oligonucleotides on the same beads, thereby encoding the visual code within the DNA sequence of the corresponding capture oligonucleotide. To extend the surface area of ​​capture arrays containing monodisperse, randomly arranged capture beads of low diversity (e.g., less than 10M different beads), it is possible to generate bead arrays for further spatial addressing (as described elsewhere). Figures 17A-17C(See illustration). Subarrays of a defined size can be generated using photolithography techniques known in the art, using substrates ranging from silicon wafers to glass, and alternative methods can be used to fabricate subarrays using PDMS. Further spatial addressing can be achieved by printing droplets of oligonucleotides with x and y coordinates into known locations, whereby unique combinations of oligonucleotides with x and y coordinates correspond to specific subarrays. In this example, the visual barcode (VBC) region capturing the oligonucleotides will contain (from 5' to 3'): a linker and a linker; an RNA base extension; a 5' PCR stalk; a variable region (DNA base extension) specific to the visual barcode on the bead; followed by a region (DNA base extension) complementary to the x-splice oligonucleotide used to bind the x-coordinate barcode (x-BC) oligonucleotide. After a solution containing a ligase (preferably SplintR) and a unique x-BC oligonucleotide is droplet-printed in one direction into a subarray, this oligonucleotide is clamped to the VBC oligonucleotide via base complementarity between the 3' end of a clamping oligonucleotide (preferably an RNA clamping oligonucleotide) and the VBC oligonucleotide. The x-BC oligonucleotide will contain (from 5' to 3'): a DNA base extension complementary to the x-clamping oligonucleotide; a unique x-coordinate oligonucleotide barcode region (different between each x-BC oligonucleotide); and a DNA base extension complementary to the y-clamping oligonucleotide. Each row (if printed horizontally) or column (if printed vertically) of the subarray will receive a unique x-BC oligonucleotide for clamping the VBC oligonucleotide attached to the bead. After clamping, the array (containing all subarrays) is then washed and prepared to print the unique y-coordinate barcode (y-BC) oligonucleotide into each subarray. Subsequently, y-BC oligonucleotides are printed, for example, in the same manner as x-BC oligonucleotides (using a ligase in buffer solution), but perpendicular to the orientation in which the x-BC oligonucleotides are printed. This will produce a unique combination of x-BC and y-BC oligonucleotides in each subarray. The y-BC oligonucleotides will contain (from 5' to 3'): a DNA base extension complementary to the y-splice oligonucleotide; a unique y-coordinate oligonucleotide barcode region (different between each y-BC oligonucleotide); and a DNA base extension complementary to the released hairpin tag (capture region). After conjugation, the array (containing all subarrays) is washed and prepared for optional splint removal. If the splint oligonucleotides are composed of RNA and the conjugation step is performed using SplintR enzyme, then the remaining splint oligonucleotides can be removed using RNase or a denaturation step to peel the splint from the captured oligonucleotides. The visual barcode can be decoded before or after the conjugation of the x and y coordinate oligonucleotides to impart additional spatial information to the visually barcoded beads.

[0197] The weakening of the "high" abundance signal

[0198] This article also considers attenuated tag-like nucleic acid molecules and their use in attenuating signals generated by high-abundance targets in the analysis. Generally, these attenuated tag-like nucleic acid molecules differ from hairpin-tagged nucleic acid molecules or DNA / RNA hybrid hairpin-tagged nucleic acid molecules in that they lack a functional cleavable site, i.e., the RNA bases that can be cleaved / released by RNase H are replaced, or the restriction endonuclease recognition site that acts as the tag is omitted / modified.

[0199] When multiple probes or conjugates are used for spatial coding readout, this type of attenuation is used to "see" (detect, read out) all tag sequences, whether they are high or low abundance. This type of probe attenuation has been previously confirmed, but has never been confirmed in systems such as those described in this paper.

[0200] For example, the analysis described herein can be used in a weakened form by including “whole DNA bases” mixed with hairpin DNA / RNA hybridization tags (e.g., in a predetermined ratio or percentage), such that these conjugates behave identically in the analysis (having similar size and structure, including any hairpin structure), but weakening the signal from high-abundance targets. Similarly, the signal from hairpin tags that depend on DNA base strings containing restriction enzyme (RE) recognition sites for cleavage can be weakened by including similar tags that do not include RE recognition sites.

[0201] Expanding probe content to a large number of differently encoded probes carries risks. Because the described system is capable of unprecedented expansion, it is important to consider highly expressed biomarkers so as not to “drows over” readings of low-expression biomarkers. For example, attenuation of probes with extremely high counts can be achieved by mixing two conjugate types (one type conjugated to a hairpin tag containing full DNA bases, and the other type conjugated to a hairpin tag containing an RNA / DNA hybridization region) at a known ratio, which can be reverse-calculated later during analysis.

[0202] Improving bead stability (immobilization) within the gel matrix: By adding components to the gel that are specifically bound to the various parts of the beads, the beads can be made more stable within the gel matrix. For example, beads can be made multifunctional by chemical modification to contain a variety of different unique / orthogonal / non-competitive reactive chemicals available in the solvent. Trifunctional beads may contain: a first reactive chemical stalk for attaching to the recognition chain; a second (unique / orthogonal / non-competitive) reactive stalk for attaching to the NGS barcode; and a third (orthogonal, i.e., unique and different from the other two) reactive stalk for covalent or non-covalent immobilization within the gel matrix.

[0203] Preparation of the capture array: Advantageously, each time a capture array is prepared, it is not necessary to prepare a new bead array and read it via OCS. A preferred method would involve: generating a bead library as described above; immobilizing the beads as described above for reading their positions within the semi-patterned array; but instead of embedding the beads in a gel to physically transfer the beads within the gel (the capture array previously described above), copying the information contained within the NGS-captured oligonucleotides into the gel. Figure 11B This can be achieved by generating NGS barcodes with reverse complementary orientation, where the capture region and the PCR stalk of the capture oligonucleotide are switched relative to each other. In this configuration, the capture region (reverse complementary) is oriented at the 5' end, closest to the bead, and the constant PCR stalk is oriented at the 3' end, furthest from the bead. A constant initiating oligonucleotide complementary to the 3' end of the reverse complementary NGS barcode oligonucleotide can hybridize with all beads on the surface. This initiating oligonucleotide may contain chemical modifications for polymerization into a gel (such as polyacrylamide). Subsequently, an extension reaction converts all reverse complementary NGS barcode oligonucleotides into dsDNA. The full-length NGS barcode (correctly complementary) is then ligated by hybridization with the reverse complementary NGS barcode oligonucleotides on the beads. Primers modified with phosphorus acrylate for the extension reaction (this modification is available from commercial oligonucleotide suppliers such as IDT) can be directly polymerized into polyacrylamide gels cast in various percentages. A 4% polyacrylamide gel should allow for macromolecular permeability during isothermal information transfer reactions. These gels can be cast to extremely thin layers (<200 μm thickness), and NGS barcodes (location and information) can be directly polymerized into the gel. The casting gel can be peeled from the beads without removing the beads from the array surface (in the presence of a dsDNA denaturing agent, such as >50% formamide, >3M urea, or 0.1M sodium hydroxide). This allows for gentle removal of the gel from the bead bed, enabling the casting of numerous gels from the same bead array through repeated extension and gel casting steps. Therefore, the ideal capture array does not contain beads but contains information from the beads, which is enzymatically copied and subsequently transferred to the gel via polymerization. Figure 11B To impart rigidity to a 4% polyacrylamide gel or other gels that can ultimately be used, a semi-permeable membrane can be placed on top of the gel before polymerization or gel hardening to create a layered “sticker” that can be used as a trapping array. The rigid matrix can be composed of a bio-inert material with defined pore sizes, such as cellulose, different webs made of suitable plastics, or, for example, PTFE.

[0204] Instrumentation. General instrumentation requirements for reading the features described herein (e.g., beads) are typically conventional, including those previously described in WO 2022 / 187719, as well as descriptions of other DNA sequencing technologies. In embodiments, it may be advantageous for the instrument to use high dynamic range (HDR) imaging to scan large surface areas filled with 1 μm beads (up to 10 cm × 10 cm due to the described grid system) to resolve fixed barcodes, capable of referencing, for example, at least five or more colors (in some embodiments, such as standard fluorophores), or even more colors in embodiments employing quantum dots with appropriate filters during each decoding cycle. Solution circulation is performed under automated microfluidics, regulated by instrument control software also used for timing image acquisition.

[0205] Software. It should be recognized that the methods, systems, and workflows described herein are supported, for example, by: instrument control software (including, as described in the literature, modified to adapt to the encoding and decoding loops for features described herein), software for decoding the image analysis of beads, software for generating 2D maps of bead arrays in the form of DNA sequences (spatial barcode sequences) at each feature / bead location, software for comparing NGS reads to generate a list based on the spatial barcode sequence (e.g., allowing tag counts to be attributed to physical locations in the 2D feature array map), and software for displaying digital images by superimposing tag read counts onto physical locations in the 2D feature (bead) array map to generate digital images.

[0206] The term "orthogonal" refers to a multicomponent system in which a component is chemically reactive with a specific reagent under a particular set of reaction conditions, while at least one other component in the multicomponent system has limited or no reactivity with said reagent, even if all components in the multicomponent system are present in the same environment. Additional terms used include "unique" (e.g., reactivity acting on different targets, such as REs that cleave at distinguishable nucleic acid sites) and "non-competitive" (e.g., reactivity not acting in overlapping or competitive (for components, such as reaction sites) reactions, although they may occur under the same conditions). Homo-cleaving restriction endonucleases are enzymes (usually a pair) that are unique (in terms of enzyme structure) but have the same target sequence recognition and cleavage activity. Thus, two homo-cleaving restriction endonucleases have the same biological activity but are (structurally) unique enzymes. Homo-cleaving restriction endonucleases are not considered "non-competitive" because they cleave at the same target sequence, and therefore may not be suitable for a single workflow as described herein.

[0207] Similarly, the phrase "orthogonal reactivity" refers to a multi-component system in which a component is chemically reactive with a specific reagent under a particular set of reaction conditions, while at least one or more components in the system are not chemically reactive, even if all components in the system exist in the same environment.

[0208] The following exemplary embodiments and examples are included to illustrate specific embodiments of this disclosure. Based on this disclosure, those skilled in the art will recognize that many changes can be made to the specific embodiments disclosed herein without departing from the spirit and scope of this disclosure and still obtaining the same or similar results.

[0209] Exemplary Example Group 1:

[0210] 1. A DNA / RNA hybridization hairpin tag having the structure described or illustrated herein.

[0211] 2. The DNA / RNA hybridization hairpin tag according to Example 1, comprising (in 5' to 3' order) a linker portion, an RNA base string, a tag PCR handle, a tag ID barcode, a tag PCR handle complementary sequence, and a DNA base string complementary to at least a portion of the RNA base string.

[0212] 3. The DNA / RNA hybridization hairpin tag according to Example 1 or Example 2 has the following characteristics: Figure 1A The structure shown is a sequence with labels v1, v2, v3, v5, or v6.

[0213] 4. A hairpin tag derived from the release of a DNA / RNA hybridization hairpin tag according to any one of Examples 1 to 3, for example, such as Figure 1B As shown in the image.

[0214] 5. A labeled element comprising an element connected via a connecting portion to a DNA / RNA hybridization hairpin tag according to Example 2 or 3.

[0215] 6. The labeled components according to Example 5, wherein the elements include one or more of the following: biomolecules (such as proteins or nucleic acids), cells or tissues, affinity molecules (such as antibodies), beads, or another addressable feature.

[0216] 7. A capture oligonucleotide (CO) having a structure as illustrated or described herein.

[0217] 8. The CO according to Example 7, comprising (in 5' to 3' order) a linker portion, a first DNA base string, a tag ID barcode, and a second DNA base string complementary to at least a portion of the first DNA base string.

[0218] 9. The CO according to Example 7 or Example 8, which has the following characteristics: Figure 2 or Figure 3 The structure shown.

[0219] 10. The CO according to Embodiment 8 or Embodiment 9, which is connected to the capture feature via the connection portion.

[0220] 11. The CO according to any one of Examples 7 to 10, further comprising a conditionally decomposable element.

[0221] 12. A capturing element comprising an element connected via a connecting portion to a CO according to any one of embodiments 8 to 11.

[0222] 13. The capture element according to embodiment 12, wherein the element comprises a bead or another addressable capture feature.

[0223] 14. The capture element according to Embodiment 12 or Embodiment 13 is one capture element within an array of different capture elements, wherein the CO on each of the plurality of different capture elements in the array comprises a different tag ID barcode.

[0224] 15. A capture pair comprising: a DNA / RNA hybridization hairpin tag having a structure as illustrated or described herein, or a hairpin tag derived from the release of said DNA / RNA hybridization hairpin tag; and a capture oligonucleotide (CO) having a structure as illustrated or described herein.

[0225] The sequence of the DNA / RNA hybrid hairpin tag is at least partially complementary to the sequence of the CO, such that when the hairpin tag is released in the vicinity of the CO, the released hairpin tag is captured by the CO.

[0226] 16. A method for detecting and / or quantifying a target in a substantially two-dimensional (2D) sample, as described or illustrated herein.

[0227] 17. The method according to Example 16 is a “one-pot” method carried out essentially at a single temperature (i.e., isothermal).

[0228] 18. The method according to embodiment 16 or 17, wherein... Figure 4A , 4B The priority is shown in any one of the appendices (in whole or in part) to U.S. Provisional Application No. 63 / 487,575 filed on February 28, 2023, or 5A, 5B, 6, 7A, 7B, 8, 9A-9E, 12, 1 ...13, filed on February 28, 2023.

[0229] 19. The method according to any one of the embodiments of Examples 16 to 18, wherein the method further provides location information of one or more targets within the generally 2D sample.

[0230] 20. The method according to any of the embodiments of Examples 16 to 19, comprising one or more of the following enzyme activities: RNase H, DNA polymerase, reverse transcriptase, RNA polymerase and / or one or more restriction enzymes.

[0231] 21. The method according to any of the embodiments of Examples 16 to 20, further comprising sequence analysis of a plurality of nucleic acid molecules containing one or more molecular ID tags.

[0232] 22. A spatially encoded capture array, generally as described or illustrated herein.

[0233] 23. The spatially encoded capture array according to Embodiment 22, which is as follows: Figure 10 Or as shown in Figure 11, or in Appendix A of U.S. Provisional Application No. 63 / 487,575 filed on February 28, 2023.

[0234] 24. The spatially encoded capture array according to Example 22 or 23, comprising a capture element embedded in a biomolecularly permeable matrix.

[0235] 25. The spatially encoded capture array according to Example 24, wherein: the capture element comprises beads; the biomolecule-permeable matrix is ​​a gel; or both.

[0236] 26. A spatially encoded capture array according to Example 24 or Example 25, which is in the form of a flexible “sticker” intended for direct contact with a substantially 2D sample for analysis of a target within the sample.

[0237] 27. A spatial coding workflow, which is substantially as described or illustrated herein.

[0238] 28. The spatial coding workflow according to Example 27, which is as follows: Figure 12 or Figure 13 The priority is shown in Appendix A of U.S. Provisional Application No. 63 / 487,575, filed on February 28, 2023.

[0239] 29. A spatial coding feature array, which has Figure 10 The grid arrangement within the grid shown and described herein.

[0240] 30. A computer-readable medium or digital resource or digital database containing spatial location information of features of a spatially coded array as described herein.

[0241] 31. The computer-readable medium, digital resource, or digital database according to embodiment 30, which contains spatial location information of substantially all of the features of the spatial coding array.

[0242] 32. An application of a computer-readable medium or digital resource or digital database according to embodiment 30 or 31, for providing a user with location information of one or more targets related to the spatial location information of the spatially coded array.

[0243] 33. According to the use described in embodiment 32, the correlation is generated by using the spatial coding array in a workflow or method as described or illustrated herein.

[0244] 34. A method for isothermal spatial coding of biological samples, generally as described or illustrated herein.

[0245] 35. The use of a DNA / RNA hybrid hairpin tag as described or illustrated herein for transcriptomic analysis of biological samples.

[0246] 36. A spatially encoded surface, such as a trapping array, for example a bead-based trapping array, generally as described or illustrated herein.

[0247] Exemplary Implementation Example Group 2:

[0248] 1. A hairpin-tagged nucleic acid molecule comprising functionally linked portions ABCDE in a 5' to 3' sequence, wherein: portion A includes a cleavable site comprising one of: (1) an RNA base string, or (2) a DNA base string including a restriction enzyme (RE) recognition site; portion B includes a DNA base string containing a tag PCR handle; portion C includes a DNA base string containing a tag ID barcode, and the DNA base string forms part of a loop of the hairpin; portion D includes a DNA base string having an inverse complementary sequence of the tag PCR handle, thereby forming part of a stem of the hairpin; and portion E includes a DNA base string having (1) the RNA base string of portion A or (2) an inverse complementary sequence of at least a portion of the DNA base string including the RE recognition site, thereby forming part of the stem of the hairpin.

[0249] 2. The hairpin-tagged nucleic acid molecule according to Example 1, further comprising a linker portion attached to the 5' end of the A portion.

[0250] 3. The hairpin-tagged nucleic acid molecule according to Example 2, wherein the linker portion provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

[0251] 4. The hairpin-tagged nucleic acid molecule according to Example 2, further comprising a linker between the linker portion and portion A.

[0252] 5. The hairpin-tagged nucleic acid molecule according to Example 4, wherein the linker comprises PEG(n), where n = 1-20.

[0253] 6. The hairpin-tagged nucleic acid molecule according to Example 1, wherein the tag ID barcode is at least 4 bases long.

[0254] 7. The hairpin-tagged nucleic acid molecule according to Example 6, wherein the tag ID barcode is 4, 5, 6, 7, 8 or more bases long.

[0255] 8. The hairpin-tagged nucleic acid molecule according to Example 1, wherein the tag PCR handle in part B and the reverse complementary sequence in part D are each at least 10 bases long.

[0256] 9. The hairpin-tagged nucleic acid molecule according to Example 8, wherein the tag PCR handle in part B and the reverse complementary sequence in part D are each 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length.

[0257] 10. The hairpin-tagged nucleic acid molecule according to any one of Examples 1 to 9, which is constructed at least in part using a templated conjugation reaction.

[0258] 11. The hairpin-tagged nucleic acid molecule according to any one of Examples 1 to 10, wherein part A includes the DNA base string containing the RE recognition site, and the RE recognition site is at least 4 bases long.

[0259] 12. The hairpin-tagged nucleic acid molecule according to Example 11, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more bases in length.

[0260] 13. The hairpin-tagged nucleic acid molecule according to Example 11, wherein the RE recognition site is an IIS-type restriction enzyme recognition site, a site that generates a 3' protrusion of the RE, or both.

[0261] 14. The hairpin-tagged nucleic acid molecule according to any one of Examples 1 to 10 is a DNA / RNA hybrid hairpin-tagged nucleic acid molecule, wherein part A includes the RNA base string, and the RNA base string in part A is at least 5 bases long.

[0262] 15. The DNA / RNA hybrid hairpin-tagged nucleic acid molecule according to Example 14, wherein the RNA base string in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length.

[0263] 16. A group of two or more hairpin-tagged nucleic acid molecules according to any one of Examples 1 to 13 and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules according to Example 14 or Example 15, wherein each of the two or more nucleic acid molecules has a unique tag ID barcode sequence.

[0264] 17. A group of two or more hairpin-tagged nucleic acid molecules and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules according to Example 16, further comprising at least one attenuated tag-like nucleic acid molecule, said attenuated tag-like nucleic acid molecule differing from the hairpin-tagged nucleic acid molecules or DNA / RNA hybrid hairpin-tagged nucleic acid molecules of the group in that it lacks a functional cleavable site.

[0265] 18. A group of two or more hairpin-tagged nucleic acid molecules and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules according to Example 17, wherein the weakened tag-like nucleic acid molecule differs from the hairpin-tagged nucleic acid molecule or DNA / RNA hybrid hairpin-tagged nucleic acid molecule of the group in that (1) the RNA base of part A is replaced with a DNA base, or (2) the RE recognition site of part A is lacking.

[0266] 19. A labeled probe comprising a probe molecule connected via the connecting portion to a hairpin-tagged nucleic acid molecule according to any one of Examples 2 to 13 or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule according to Example 14 or Example 15.

[0267] 20. The labeled probe according to Example 19, wherein the probe molecule comprises an affinity molecule having binding affinity to the target molecule.

[0268] 21. The labeled probe according to Example 20, wherein the affinity molecule includes an antibody-binding domain having affinity for the antigen, and the target molecule includes the antigen.

[0269] 22. The labeled probe according to Example 19, wherein the probe molecule comprises one or more of the following: antibody or its binding fragment, nucleic acid, small molecule, organic or inorganic chemical substance, putative drug target, identified drug or biomacromolecule complex.

[0270] 23. The labeled probe according to Example 19, wherein the probe molecule is one of a group of probe molecules, each of which includes one of a plurality of members of: small molecule library, drug target library, bioaffinity molecule library, natural product library, bioactive compound library, genomic library, transcriptome library, metabolome library, or drug screening library.

[0271] 24. The labeled probe according to Example 20, wherein the target molecule comprises a biomolecule, an inorganic object, or an addressable feature of an array.

[0272] 25. The labeled probe according to Example 24, wherein the target molecule comprises a biomolecule, and the biomolecule comprises one or more of the following: protein, lipid, carbohydrate, nucleic acid molecule, or a combination of protein, lipid, carbohydrate and / or nucleic acid molecule.

[0273] 26. The labeled probe according to Example 25, wherein the target molecule is one of a plurality of molecules constituting a complex, and the complex is located outside or inside one or more cells in a tissue sample.

[0274] 27. The labeled probe according to any one of Examples 19 to 26, further comprising an amplified sequence containing a polymerase promoter sequence.

[0275] 28. The labeled probe according to Example 27, wherein the amplification sequence includes a T7 promoter sequence, such as a T7 promoter adaptor.

[0276] 29. A released hairpin-tagged nucleic acid molecule derived from a hairpin tag according to any one of Examples 2 to 13 or a DNA / RNA hybridization hairpin tag according to Example 14 or Example 15, wherein the released hairpin tag has been separated from the linker by enzymatic action of a restriction endonuclease or RNase H.

[0277] 30. A capture oligonucleotide (CO) nucleic acid molecule comprising functionally linked portions I-II-III-IV in a 5' to 3' sequence, wherein: portion I comprises a cleavable site containing one of: (1) a single RNA base or a continuous string of RNA bases, or (2) a string of DNA bases including a restriction enzyme (RE) recognition site; portion II comprises a string of DNA bases containing a CO PCR stalk; portion III comprises a string of DNA bases containing a spatial barcode; and portion IV comprises a string of DNA bases containing a tag capture region.

[0278] 31. The CO nucleic acid molecule according to Example 30, further comprising a linker portion conjugated to the 5' end of the I portion.

[0279] 32. The CO nucleic acid molecule according to Example 31, wherein the linker portion provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

[0280] 33. The CO nucleic acid molecule according to Example 31, further comprising a linker between the linker portion and the I portion.

[0281] 34. The CO nucleic acid molecule according to Example 33, wherein the linker comprises PEG(n), where n = 1-20.

[0282] 35. The CO nucleic acid molecule according to Example 30, wherein the spatial barcode is at least 4 bases long.

[0283] 36. The CO nucleic acid molecule according to Example 35, wherein the spatial barcode is 4, 5, 6, 7, 8 or more bases in length.

[0284] 37. The CO nucleic acid molecule according to Example 30, wherein the CO PCR stem in Part II is at least 5 bases long.

[0285] 38. The CO nucleic acid molecule according to Example 37, wherein the CO PCR stem in Part II is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length.

[0286] 39. The CO nucleic acid molecule according to any one of Examples 30 to 38, further comprising a unique molecular identifier (UMI).

[0287] 40. The CO nucleic acid molecule according to any one of Examples 30 to 38 has a sequence having no more than two internal sequence self-complementary consecutive bases.

[0288] 41. The CO nucleic acid molecule according to Example 40 has a sequence having 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 self-complementary consecutive bases.

[0289] 42. The CO nucleic acid molecule according to any of Examples 30 to 41 is constructed at least in part using a templated conjugation reaction.

[0290] 43. The CO nucleic acid molecule according to any one of Examples 30 to 42, wherein part I includes the DNA base string containing the RE recognition site, and the RE recognition site is at least 4 bases long.

[0291] 44. The CO nucleic acid molecule according to Example 43, wherein the RE recognition site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more bases in length.

[0292] 45. The CO nucleic acid molecule according to Example 43, wherein the RE recognition site is an IIS type restriction enzyme recognition site, a site with a 3' overhang of the RE, or both.

[0293] 46. ​​The CO nucleic acid molecule according to any one of Examples 30 to 42 is a DNA / RNA chimeric CO nucleic acid molecule, wherein part I includes the single RNA base.

[0294] 47. The CO nucleic acid molecule according to any one of Examples 30 to 42, wherein it is a DNA / RNA chimeric CO nucleic acid molecule, and wherein part I includes the continuous RNA base string.

[0295] 48. The DNA / RNA chimeric CO nucleic acid molecule according to Example 47, wherein the continuous RNA base string in part I is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases long or longer than 20 bases.

[0296] 49. A group of two or more CO nucleic acid molecules according to any one of Examples 30 to 45 and / or DNA / RNA chimeric CO nucleic acid molecules according to any one of Examples 46 to 48, wherein each of the two or more nucleic acid molecules has a unique spatial barcode sequence.

[0297] 50. A CO nucleic acid molecule according to any one of Examples 31 to 45 or a DNA / RNA chimeric CO nucleic acid molecule according to any one of Examples 46 to 48, which is connected to the capture feature via the connecting portion.

[0298] 51. The CO nucleic acid molecule according to Example 50, wherein the capture feature is one of the following: a bead, a chemically functionalized point on a glass surface, a chemically functionalized and defined region of a permeable gel, a bead or other inorganic object embedded in or on the surface of a permeable gel, or a series of spatially defined objects attached to the gel.

[0299] 52. The CO nucleic acid molecule according to Example 50, wherein the capture feature is a spatially addressable feature in an array.

[0300] 53. The CO nucleic acid molecule according to Example 52, wherein the array is a microarray having at least 100 addressable capture features.

[0301] 54. A spatially encoded capture feature comprising a capture feature connected via a linker portion to a CO nucleic acid molecule according to any one of Examples 31 to 42 or a DNA / RNA chimeric CO nucleic acid molecule according to any one of Examples 43 to 48.

[0302] 55. The spatially encoded capture feature according to Example 54, wherein the capture feature includes addressable locations on a bead or generally two-dimensional surface.

[0303] 56. The spatially encoded capture feature according to Example 54 or Example 55, which is one spatially encoded capture feature within an array of at least 100 different spatially encoded capture features, wherein the CO nucleic acid molecule on each of the at least 100 different spatially encoded capture features in the array comprises a different spatial barcode.

[0304] 57. A capture pair comprising: a hairpin-tagged nucleic acid molecule according to any one of Examples 2 to 13, or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule according to Example 14 or Example 15, or a released hairpin-tagged nucleic acid molecule according to Example 29; and a capture oligonucleotide (CO) nucleic acid molecule according to any one of Examples 31 to 42, or a DNA / RNA chimeric CO nucleic acid molecule according to any one of Examples 43 to 48, or a linked CO according to any one of Examples 50 to 73, wherein the sequence of the hairpin-tagged nucleic acid molecule and the sequence of the CO nucleic acid molecule are at least partially complementary, such that when the hairpin-tagged nucleic acid molecule is released from its linker portion near the CO nucleic acid molecule, the released hairpin-tagged nucleic acid molecule is captured by a complementary sequence bound to the 3' end of the CO nucleic acid molecule, such that the resulting complex of the released hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule is capable of undergoing a downstream extension reaction by a polymerase.

[0305] 58. A spatially encoded capture array comprising a defined array of spatially addressed capture features, wherein each capture feature includes: a spatially identifiable feature comprising: a predefined addressable location on a generally two-dimensional solid surface; or a bead or other similar individual solid capture object; and multiple copies of a 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, connected at each feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules at other features in the array.

[0306] 59. The spatially encoded capture array according to Example 58, wherein one or more CO nucleic acid molecules are applied to the spatially addressed capture feature in the array by: printing the CO droplets onto the predefined addressable locations on the generally two-dimensional solid surface, or by attaching the CO to beads via the connecting portion.

[0307] 60. A spatially encoded capture array according to Embodiment 58 or Embodiment 59, the array comprising beads, each of which includes a visual barcode operatively coupled to the CO.

[0308] 61. The spatially encoded capture array according to embodiment 60, wherein the visual barcode enables beads to be assigned to positions within the capture array.

[0309] 62. The spatially encoded capture array according to Example 58, wherein the beads or other similar individual capture objects are embedded in a biomolecularly permeable matrix.

[0310] 63. The spatially encoded capture array according to Example 62, wherein: the captured object comprises beads; the biomolecularly permeable matrix comprises a gel; or both.

[0311] 64. The spatially encoded capture array according to Example 63, wherein the biomolecularly permeable matrix of the gel is formatted as a flexible sticker.

[0312] 65. The spatially encoded trapping array according to Example 62, wherein the biomolecules are permeable to a matrix that is structurally stable at a selected temperature between 4-45°C; permeable to proteins such as functional RNase H and polymerases; permeable to ribonucleoside triphosphates (rNTPs) and / or deoxyribonucleotide triphosphates (dNTPs); and permeable to Mg. 2+ Ions are permeable; they are generally inert to biomolecules; and they are flexible enough to allow the relatively thin matrix layer to be applied directly to generally two-dimensional samples or surfaces.

[0313] 66. The spatially encoded capture array according to embodiment 65, which is configured as a three-dimensional thin-layer gel with a width and length significantly greater than its thickness, wherein the spatially identifiable capture features are generally arranged in a single plane on the surface of the gel defined by its length and width.

[0314] 67. The spatially encoded capture array according to embodiment 66, wherein at least the first of the spatially identifiable capture features is directly connected to and / or in contact with the second of the spatially identifiable capture features.

[0315] 68. The spatially encoded capture array according to any one of Examples 62 to 67, wherein the matrix comprises a hydrogel or a polyacrylamide gel.

[0316] 69. The spatially encoded capture array according to Example 68, wherein the thickness of the matrix or gel does not exceed about 2 mm.

[0317] 70. The spatially encoded capture array according to Example 69, wherein the thickness of the matrix or gel is not more than about 1 mm, not more than 500 μm, not more than 250 μm, not more than 200 μm, not more than 150 μm, not more than 125 μm, not more than 100 μm, or less than 100 μm.

[0318] 71. The spatially encoded capture array according to Example 70, wherein the thickness of the matrix or gel is 100-200 μm, 100-150 μm, or about 125 μm.

[0319] 72. The spatially encoded capture array according to any one of embodiments 58 to 71, wherein the array is reinforced by an inert mesh or other support structure.

[0320] 73. The spatially encoded capture array according to any of the embodiments 58 to 72, wherein: the predefined addressable location on a generally two-dimensional solid surface has a surface area of ​​no more than about 1 μm × 1 μm; or the diameter of the bead or other similar individual solid capture object does not exceed about 20 μm.

[0321] 74. The spatially encoded capture array according to Example 73, wherein the diameter of the bead or other similar individual solid capture object is not more than 18 μm, not more than 15 μm, not more than 12 μm, not more than 10 μm, not more than 8 μm, not more than 5 μm, not more than 3 μm, not more than 1 μm or about 100 nm.

[0322] 75. The spatially encoded capture array according to Example 74, wherein the diameter of the bead or other similar individual solid capture object is between 1 and 3 μm.

[0323] 76. A semi-ordered spatially encoded capture array comprising: a grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide being applied to the spatially addressable location by clamping together: an x-coordinate connective oligonucleotide for labeling all spatially addressable locations within a row of the grid; and a y-coordinate connective oligonucleotide for labeling all spatially addressable locations within a column of the grid.

[0324] 77. The semi-ordered spatial coding capture array according to embodiment 76, which at least partially uses Figures 17A-17C The methods provided in the documentation can be used to construct it.

[0325] 78. The semi-ordered spatial coding capture array according to embodiment 76, which has the following characteristics: Figure 10 The grid format within the grid shown.

[0326] 79. A semi-ordered spatially coded capture array comprising: an array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray comprising uniquely identifiable capture features, the location of the uniquely identifiable capture features being at least partially specified by identification of the subarrays within the semi-ordered spatially coded capture array.

[0327] 80. A semi-ordered spatially encoded capture array comprising: a group of two or more subarrays, each subarray including a plurality of capture features, wherein each capture feature within each subarray is connected to a capture oligonucleotide including a unique position tag, the capture features within each subarray being randomly arranged, and the capture features within each subarray further including an oligonucleotide tag identifying the subarray, the oligonucleotide tag identifying the subarray being attached to the capture oligonucleotide of each feature in the subarray via a clamping connection.

[0328] 81. A method for detecting and / or quantifying and / or locating a target in a substantially two-dimensional (2D) sample, the method comprising: contacting a substantially 2D sample with at least one labeled probe to produce a substantially 2D stained sample, the labeled probe comprising: a hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode, or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule including a linker portion and a tag ID barcode; and a probe molecule connected via the linker portion to the hairpin-tagged nucleic acid molecule including the tag ID barcode or the DNA / RNA hybrid hairpin-tagged nucleic acid molecule including the tag ID barcode; contacting the surface of the substantially 2D stained sample with a permeable spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: Multiple spatially identifiable features; and multiple copies of a capture oligonucleotide (CO) nucleic acid molecule or a DNA / RNA chimeric CO nucleic acid molecule linked at each spatially identifiable feature, wherein each CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules at other features in the array; placing a flow cell or other solution-containing cap above the sample-array sandwich to form a shell containing the sample-array sandwich; adding a solution comprising reaction components to the shell to form a reaction mixture, said components comprising: a lyase selected from RNase H or at least one restriction endonuclease (RE); at least one polymerase; a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); Mg 2+ Ions; and buffers; incubating the sample-array sandwich in contact with the reaction mixture at analytical temperature for 30-60 minutes to form a reaction product mixture; removing at least a portion of the reaction product mixture from the shell; and analyzing the reaction product mixture to detect and / or quantify the target in the substantially 2D sample and / or define the location of the target.

[0329] 82. The method for detecting and / or quantifying and / or locating a target in a generally two-dimensional (2D) sample according to Example 81, wherein the housing includes a flow cell.

[0330] 83. The method according to Example 81 or Example 82 is carried out at a single temperature (isothermal) or within a range of about 5°C of a single temperature.

[0331] 84. The method according to any one of Examples 81 to 83, wherein the analysis temperature has a range of 20-55°C or 37-42°C.

[0332] 85. The method according to any one of Examples 81 to 84, wherein the method provides location information of more than one target within the generally 2D sample.

[0333] 86. The method according to any one of Examples 81 to 85, wherein the at least one polymerase provides enzymatic activity of DNA polymerase, reverse transcriptase or RNA polymerase.

[0334] 87. The method according to any one of Examples 81 to 86, wherein analyzing the reaction product mixture comprises sequence analysis of a plurality of nucleic acid molecules containing spatial barcodes and tag ID barcodes.

[0335] 88. The method according to Example 87, wherein analyzing the reaction product mixture comprises next-generation sequencing (NGS) of a plurality of nucleic acid molecules containing spatial barcodes and tag ID barcodes.

[0336] 89. The method according to Example 87 or Example 88, wherein the plurality of nucleic acid molecules containing spatial barcodes and tag ID barcodes are fully extended products released from the spatially identifiable features by cleavage at the cleavage site of the CO.

[0337] 90. The method according to Example 89, wherein the lysis includes RNase H activity or restriction endonuclease activity.

[0338] 91. The method according to any one of Examples 81 to 90, wherein the analysis includes assigning the spatial location of at least one nucleic acid molecule containing a spatial barcode and a tag ID barcode within the substantially 2D sample.

[0339] 92. A method for isothermal spatial encoding of a biological sample, the method comprising the method according to any one of Examples 81 to 91, wherein the generally 2D sample is a biological sample.

[0340] 93. Use of a group of hairpin-tagged nucleic acid molecules according to any one of Examples 16 to 18 or a group of captured oligonucleotide (CO) nucleic acid molecules according to Example 49, or both, for transcriptomic analysis of biological samples.

[0341] 94. A spatially encoded surface, such as a trapping array, for example a bead-based trapping array, generally as described or illustrated herein.

[0342] 95. A spatial coding workflow comprising: contacting a hybrid RNA / DNA tag including a spatial barcode conjugated to an antibody probe with a substantially two-dimensional (2D) tissue sample to produce a stained sample; positioning a capture array including a capture feature in contact with the stained sample to produce a sample / array sandwich; placing a fluid-containing housing on top of the sample / array sandwich; introducing an analytical solution including an active RNase H and an active polymerase into the fluid-containing housing, thereby contacting the analytical solution with the sample / array sandwich; and incubating the sample / array sandwich in the sample solution at a temperature and time sufficient to allow the RNase H activity to at least partially digest the hybrid RNA / DNA tag to produce a lysed tag, thereby generating a stained sample. The cleaved tag is released into the analytical solution near the capture feature; the cleaved tag is allowed to interact with the capture oligonucleotide (CO) on the nearby capture feature to provide a captured cleaved tag; the sample / array sandwich is incubated in the sample solution at a temperature and time sufficient to allow polymerase activity to extend the captured cleaved tag using the CO as a template to produce an extension product; after sufficient extension, the extension product is cleaved with RNase H in the sample solution to produce a complementary RNA / DNA region based on the RNA bases in the CO, thereby releasing a full-length extension product; at least a portion of the released full-length extension product is collected; and at least one of the released full-length extension products is amplified and / or sequenced.

[0343] 96. A computer-readable medium or digital resource or digital database containing spatial location information of features of a spatially encoded array according to any one of embodiments 58 to 80.

[0344] 97. The computer-readable medium or digital resource or digital database according to embodiment 96, which contains spatial location information of substantially all of the features of the spatial coding array.

[0345] 98. Use of a computer-readable medium or digital resource or digital database according to embodiment 96 or 97 for providing a user with location information of one or more targets related to spatial location information of a spatially coded array.

[0346] 99. The use according to embodiment 98, wherein the correlation is generated by using the spatial coding array in a workflow or method as described or illustrated herein.

[0347] 100. A kit comprising one or more of the following: two or more hairpin-tagged nucleic acid molecules according to any one of Examples 1 to 13; two or more DNA / RNA hybridization hairpin-tagged nucleic acid molecules according to Example 14 or Example 15; a group of two or more hairpin tags according to any one of Examples 16 to 18; a group of two or more labeled probes according to any one of Examples 19 to 28; two or more capture oligonucleotide (CO) nucleic acid molecules according to any one of Examples 30 to 46; two or more according to Example 47 or Example 48. DNA / RNA chimeric CO nucleic acid molecules; a group of two or more CO nucleic acid molecules according to any one of Examples 30 to 45; a group of two or more DNA / RNA chimeric CO nucleic acid molecules according to any one of Examples 46 to 48; two or more CO nucleic acid molecules according to any one of Examples 50 to 53, each of which is linked to a capture feature; at least one spatially encoded capture array according to any one of Examples 58 to 75; at least one semi-ordered spatially encoded capture array according to any one of Examples 76 to 80; or a spatially encoded surface according to Example 94.

[0348] 101. The kit according to Example 100, wherein the spatial coding capture array, the semi-ordered spatial coding capture array, or the spatial coding surface is in the format of a flexible sticker.

[0349] 102. The kit according to Example 100 further comprises one or more of the following: a container containing a functional RNase H; a container containing a functional polymerase; a container containing a functional restriction enzyme; a container containing one or a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); a container containing Mg 2+ A container for a solution of ions; or a container containing a buffer solution.

[0350] 103. The kit according to Example 101, wherein at least one of the containers contains at least two of the following: the RNase H, the polymerase, the restriction endonuclease, the rNTP and / or dNTP, and the Mg... 2+ Ions or the buffer solution.

[0351] 104. The kit according to any of Examples 100 to 103 further comprises one or more of the following: a component that can be used to prepare a sample for analysis using the methods provided herein; a cover containing a solution adapted to be placed over a sample-array sandwich on a glass slide to form a fluid-containing shell for the sample-array sandwich; a flow cell cover, or a glass slide or other surface adapted to accommodate a generally two-dimensional sample.

[0352] Example 1: Constructing DNA / RNA hybridization hairpin tags

[0353] A demonstrative DNA / RNA hybrid hairpin tag is constructed by conjugating two separate nucleic acids; the first nucleus (tag stem oligonucleotide) contains RNA bases in its 5' region; and the second nucleic acid (tag loop oligonucleotide) is entirely composed of DNA, containing bases at the 3' end designed to complement the RNA bases of the first nucleic acid, followed by the hairpin loop region, and a stem region at the 5' end. In all cases, the first nucleic acid (tag stem oligonucleotide) contains a 5' flexible linker that links the nucleic acid 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 oligonucleotide) contains an internal amino-modified base for covalently labeling the amine reactive chemical (IDT: / iAmMC6T / ). Prior to labeling the nucleic acid, all remaining free amino groups resulting from the synthesis reaction are removed by sodium acetate / EtOH precipitation using the published protocol (when standard desalted oligonucleotides are ordered from the supplier). The precipitated amino-modified oligonucleotides (and any internally amino-modified oligonucleotides, or ordered as pre-purified oligonucleotides obtained by lyophilization from the manufacturer) were resuspended in H2O to 400 μM and subsequently diluted 1:1 with 200 mM sodium phosphate buffer (pH 8.5). The resulting sample contained 200 μM of each amino-modified oligonucleotide in a final N-hydroxysuccinimide (NHS) conjugation buffer composition in 100 mM sodium phosphate (pH 8.5) for downstream labeling with NHS-modified reagents.

[0354] To each 100 μl oligonucleotide sample, an approximately 10-fold molar excess of 10 μl of an appropriate NHS-carrying labeling reagent (20 mM in anhydrous DMF) was added for overnight conjugation at room temperature. The oligonucleotide labeling reaction was then quenched by adding 25 μl of 1 M Tris pH 7.5 to each sample. All oligonucleotide labeling reactions were analyzed using a 15% TBU gel (Thermo Fisher EC6885 BOX), where unmodified oligonucleotides of the same type were run side-by-side with modified oligonucleotides in adjacent lanes to observe a clear upward shift of the band within the lane, corresponding to successful and complete modification with the appropriate NHS reagent (no band corresponding to the unmodified oligonucleotide was observed in any modified oligonucleotide lane); 2 pmol of each band was loaded and analyzed using SYBR Green. TM Gold (Thermo Fisher Scientific S11494) staining. To remove excess labeling reagent from each reaction, oligonucleotides were precipitated with sodium acetate / EtOH, resuspended in 100 μl H2O, and then stained with 0.5 mL of 7.5 K MWCO Zeba solution equilibrated with H2O. TM The column (Thermo Fisher Scientific 89883) is used for desalting to further remove excess labeling reagent. A second desalting step is performed by repeating the above desalting procedure using oligonucleotides labeled with a surface- or probe-ligated chemical (biotin or trans-cyclooctene (TCO) is used here) to more completely remove any remaining free label. A single desalting step is typically performed using fluorophore-labeled oligonucleotides. Any T4 DNA ligase (MO202S or...) is used. The ligase (NEB M0375S) enzymatically covalently ligated the two oligonucleotide species (tag stem oligonucleotide and tag ring oligonucleotide) together with relatively equivalent efficiency, as determined by denaturing nucleic acid gel analysis using 10% TBU gel (Thermo Fisher Scientific EC68752BOX).

[0355] Different types of DNA / RNA hybridization hairpin tags (referred to herein as v1-v6) were tested to compare their relative performance when conjugated to surface and antibody probes, where they were used in analyte binding assays and tissue staining workflows. These experiments concluded that smaller, more compact tag types containing fewer unpaired bases in their stem and loop regions exhibited less nonspecific interaction with tissue samples under optimized staining procedures. Tag type v6 was selected for downstream experiments due to its compact size (and relatively fewer single-stranded bases exposed to solvent, which are responsible for generating nonspecific interactions with the sample) and its overall performance in tissue staining procedures. Tag type v6 (its full sequence is provided herein) is available as a full-length tag from the vendor (purchased herein from IDT) containing a 5' flexible linker carrying a primary amino group (IDT: / 5AmMC12 / ) for conjugation with a labeled agent carrying NHS without requiring conjugation of two separate species. However, for all dual-labeled v6 tags used herein, a two-step labeling and conjugation procedure as described was employed prior to gel purification of the conjugation product.

[0356] Example 2: Optimize the tag capture area

[0357] A series of oligonucleotides (Ab_DRD-tag 1 cleavage v1-v6, shown below) were designed and ordered from IDT to determine the optimal capture of the cleaved hairpin tags by the capturing oligonucleotides. These capture experiments were performed at an isothermal analysis temperature (42°C) in isothermal analysis buffer (1×RNase H buffer from NEB). The oligonucleotides within the hairpins in this series differed only in the length of their capture regions, allowing for a series of titrations around the capture Tm. The underlined sequences correspond to the regions of each oligonucleotide in the series that were captured by the capturing oligonucleotides used in the experiments. The salt-regulated Tm for these sequences was calculated using the Northwestern Oligo Calc website and analyzed at 50 nM oligonucleotide concentration and 75 mM salt (monovalent cation: Na+) concentration.

[0358] Ab_DRD-tag 1 cleavage v1; salt-regulated Tm = 60.4 (SEQ ID NO:8)

[0359] Ab_DRD-tag 1 cleavage v2; salt-regulated Tm = 54.3 (SEQ ID NO:9)

[0360] Ab_DRD-tag 1 cleavage v3; salt-regulated Tm = 52.9 (SEQ ID NO: 10)

[0361] Ab_DRD-tag 1 cleavage v4; salt-regulated Tm = 46.4 (SEQ ID NO: 11)

[0362] Ab_DRD-tag 1 cleavage v5; salt-regulated Tm = 43.8 (SEQ ID NO: 12)

[0363] Ab_DRD-tag 1 cleavage v6; salt-regulated Tm = 40.9 (SEQ ID NO: 13)

[0364] For the capture assay, MyOne T1 streptavidin dinobeads (Dynabeads) were coated with a single biotin-modified capture oligonucleotide type (DRD1_CO_6sUMI-DNA bases; SEQ ID NO:14) consisting of whole DNA bases, and simultaneously mixed at room temperature in SA binding buffer (10 mM Tris pH 7.5, 500 mM NaCl) at a bead concentration of 1 mg / mL and an oligonucleotide concentration of 200 nM. The capture beads were washed as described above, and 1 mg / mL beads were mixed with various hairpin tags (lysed v1-v6; SEQ ID NO:8-13) at a hairpin tag concentration of 50 nM for incubation at 42°C for 30 min in 1×RNase H buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT (pH 8.3)). After incubation, the sample was rapidly transferred from the temperature block to a magnetic rack and allowed 3–4 minutes to ensure complete magnet-driven agitation of the beads. The supernatant was collected, and the beads were resuspended in an equal volume of 1×RNase H buffer. Equal volumes of both fractions were run on a 10% TBU denaturing gel (Thermo Fisher Scientific EC68752BOX), and the amount of hairpin oligonucleotides contained in the bead fraction was compared to the amount in the supernatant fraction to determine the ratio of each hairpin oligonucleotide tested. Gel analysis revealed that hairpin oligonucleotide v4 was the most effectively captured of the six hairpins tested under these conditions, and therefore, the length and composition of this capture region became the basis for the final capture length of the optimized v6 hairpin tag design.

[0365] Example 3: Capturing Oligonucleotides

[0366] The capture oligonucleotides (COs) used here are composed entirely of DNA (DRD1_CO_6sUMI-DNA bases; SEQ ID NO: 14) or designed to contain RNA bases at their 5' end (DRD1_CO_6sUMI-RNA bases; SEQ ID NO: 38). The capture oligonucleotides contain a flexible linker carrying a primary amino group at their 5' end for conjugation downstream of the NHS-modified label. A 3'-reverse T base modification is included to cap the 3' end of the CO in the experiment shown here (to prevent elongation). As described in the labeling procedure outlined above, CO in NHS conjugation buffer (200 μM oligonucleotide in 100 mM sodium phosphate buffer (pH 8.5)) was modified with NHS-LC-Biotin (Thermo Fisher Scientific 21336) with a 10-fold molar excess of the labeling reagent and allowed to react overnight at room temperature. Subsequently, as described above, the conjugation reaction was quenched with 1M Tris at pH 7.5, precipitated with sodium acetate / EtOH using a standard procedure, and the aggregated precipitate was resuspended in 100 μl H2O. Then, each sample of resuspended oligonucleotides underwent two reverse desalting steps using a 0.5 mL 7.5 M W CO Zeba desalting column (Thermo Fisher Scientific 89883) to remove as much free biotin as possible. To construct CO containing 5'-RNA bases, two oligonucleotide clips were joined together using T4 DNA ligase, with the clip oligonucleotides designed to be complementary to both the 3' end of the biotinylated oligonucleotide containing RNA bases and the 5' end of the downstream oligonucleotide fragment encoding the remainder of the capture oligonucleotide sequence. After conjugation, the full-length biotinylated CO product containing 5'-RNA bases was purified by gel electrophoresis for downstream immobilization experiments.

[0367] Example 4: Capturing Beads

[0368] Capture beads were generated by coating MyOne T1 streptavidin dinopra beads (Thermo Fisher Scientific 65601) with CO. In short, the beads were washed according to the manufacturer's instructions, sonicated to reduce clumping, and resuspended to 1 mg / mL in SA binding buffer (500 mM NaCl, 10 mM Tris pH 7.5) with 200 nM biotin-labeled CO, and the tube was rotated for continuous mixing at room temperature for 30 minutes. Once coated with CO, the beads were washed six times in SA binding buffer, sonicated between washes to remove any free oligonucleotides. Subsequently, the capture beads were resuspended to a concentration of 1–2 mg / mL in an appropriate buffer (different buffers are used for different intended applications), sonicated to reduce clumping, and used in various downstream tag capture experiments.

[0369] Example 5: Antibody probe conjugation with DNA / RNA hybridization hairpin tag

[0370] Gel analysis of tagged antibodies typically consists of a 3-8% Tris acetate gel (Thermo Fisher Scientific WG1602BOX) with three lanes for each conjugation reaction: Lane 1 contains a protein-size marker; Lane 2 contains 1 μg of the initial unconjugated antibody; and Lane 3 contains approximately 2-3 μg of the tagged conjugation reaction mixture. In typical results, two bands are present in Lane 2; one migrates to approximately 65 kD relative to the protein-size marker, corresponding to the BSA carrier protein in the antibody solution; and the second migrates to approximately 150 kD, corresponding to the unconjugated antibody. In Lane 3, two major bands are always present, migrating to higher positions in the gel (relative to the initial unconjugated antibody), corresponding to the antibody modified with one or two tag molecules; and a very small band is present, corresponding to the initial unconjugated antibody (typically <5%) and the approximately 65 kD band of the BSA carrier. This typical result indicates successful conjugation of the DNA / RNA hybridization hairpin tag to the antibody probe, resulting in a reaction solution containing primarily one or two tag / antibody molecules (as expected). For analysis of antibodies conjugated to fluorescently labeled DNA / RNA hybridization hairpin tags, the gel is first imaged using a fluorescent gel scanner (Thermo Fisher iBright Imaging System) with an appropriate excitation wavelength and emission filters for the corresponding fluorophores, followed by Coomassie blue staining. Two fluorescent bands are typically observed in lanes containing the conjugated reactants, and after Coomassie blue staining, a similar band pattern to that seen in the non-fluorescent analytical gels of the conjugated compounds (described above) is observed.

[0371] Example 6: In vitro binding analysis

[0372] To determine whether conjugation to a DNA / RNA hybridization hairpin tag affects antibody-target analyte binding, in vitro binding assays were performed using an Octet instrument (OctetRED 384, forteBIO, a division of Pall Life Sciences). Here, anti-human IgG (Jackson Immuno Research 109-005-003) was conjugated to a DNA / RNA hybridization tag v1 (SEQ ID NO:3) and used in the experiment along with unconjugated and conjugated antibody samples, which had been pre-incubated with RNase H (NEB M0297S). Binding of unmodified antibody and both conjugated samples to biotin-labeled human IgG, immobilized on a high-precision streptavidin-coated Octet tip (SATORIUS-) in a 3-fold dilution series from 18 μM to 74 nM, was tested. The high-precision streptavidin (SAX) biosensor (18-5117) was used. This procedure was performed at 37°C and 500 RPM and included a 15-minute tip 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 (1×PBS, supplemented with 100 nM biotinylated human IgG), a 1-minute wash step in blocking buffer (50% Superblock / 50% PBS, supplemented with 1 mg / ml BSA), a 10-minute binding step (exposed to antibody samples at different concentrations in the blocking buffer), and a 10-minute dissociation step. These results showed similar binding kinetics across all three samples at all tested concentrations, but a slightly reduced dissociation rate of the conjugates was observed. It was hypothesized that using 10% DMSO in the dissociation step could increase the dissociation rate of the conjugates, and this was also observed in the experiments. It is assumed that including a small amount of detergent (0.01-0.2% Tween-20) or increasing the salt concentration (to 300 mM NaCl) in the dissociation step could further increase the dissociation rate of the conjugate. The extent to which these components increase the dissociation rate of the conjugate is less than that observed with the inclusion of 10% DMSO. This slower dissociation was observed using only the DNA / RNA hybridization hairpin tag v1 (SEQ ID NO:3), and may not be the case for tag v6 (SEQ ID NO:7).

[0373] Example 7: Antibody stability of DNA / RNA hybridization tag conjugation

[0374] To test the stability of the conjugates stored at 4°C for several weeks, tagged v6 conjugated antibodies (α-ER [EPR4007] (Abcam: ab108398) and α-HER2 / ErbB2 (R&D Systems: MAB1129) were stored at 4°C for 7 weeks in PBS with 0.25% BSA. Unconjugated antibodies (1 μg per lane) and HPv6 conjugated antibodies (2–3 μg per lane) were run on 3–8% Tris-acetate gels in separate lanes and visualized by Coomassie blue staining. Lanes containing the stored conjugated antibodies showed that, after 7 weeks, the majority (>95%) of the antibodies remained stably conjugated to one or both tagged v6 molecules.

[0375] Example 8: Exemplary Antibody Staining Procedure

[0376] The following protocol was used to perform antibody staining on cells in 2D culture:

[0377] 1) Fix the cells with 4% PFA for 10 minutes.

[0378] 2) Infiltration was performed by incubating at room temperature with 1×PBS containing 0.25% Triton-X for 15 minutes.

[0379] 3) Features Image-iT TM FX signal enhancer (Life Technologies, I36933) was sealed at room temperature for 30 minutes.

[0380] 4) SuperBlock containing BSA (50 mg / ml) and cleaved salmon DNA (Thermo, AM9680) TM (Life Technologies, 37580) After being sealed at room temperature for 30 minutes, the ratio was 93%:2%:5%.

[0381] 5) Incubate the antibody-tag conjugate overnight at 4°C or incubate at room temperature for 2 hours.

[0382] 6) Unbound antibodies were washed away by three 5-minute washes in PBST (0.02% Tween-20 in 1×PBS).

[0383] For FFPE tissue sections, perform the following steps:

[0384] 1) Bake the FFPE slices at 65℃ for 30 minutes.

[0385] 2) Washing: Wash 3× in xylene (5 minutes), wash 2× in 100% EtOH (5 minutes), wash 2× in 95% EtOH (3 minutes), wash 2× in 70% EtOH (3 minutes), and wash 2× in H2O (3 minutes).

[0386] 3) Under high pressure, antigen retrieval was performed at 110°C in sodium citrate buffer (pH 6.0) for 15 minutes.

[0387] 4) After antigen retrieval, this procedure continues from step 3 of the antibody staining procedure above.

[0388] Use labeled antibodies at the following concentrations:

[0389] α-ER [EPR4007] (Abogen: ab108398): Used at a ratio of 1:150

[0390] α-HER2 / ErbB2 (Andy Biotech: MAB1129): Use at a 1:100 ratio.

[0391] Example 9: Imaging

[0392] All imaging was performed on a LeicaTHUNDER wide-field deconvolution microscope equipped with one of four objectives (HC PL APO 20x.8NA, HCX PL FLUOTAR L40x.6NA CORR PH2, HC APO 40x 1.25NA GLYC CORR CS2, or HC PL APO 63x 1.4NA OIL). Excitation was provided by a Lumencor SPECTRAX light engine housing the following LEDs (395, 440, 470, 510, 550, 640, and 750). The emission filters consist of quad cubes (Ex: 375-407, 462-496, 542-566, 622-654; Dc: 415, 500, 572, 660; Em: 420-450, 506-532, 578-610, 666-724) and Y7 cubes (Ex: 672-748; Dc: 760; Em: 765-855). Before collection on a Leica DFC9000s CMOS camera, a final fast filter wheel with the following LP filters (440, 510, 590, 700, 100%) is used to remove the left side of the emission signal from the quad cubes.

[0393] Example 10: Tag recovery and qPCR analysis of released tags

[0394] In some experiments, DNA / RNA hybridization hairpin tags were lysed from MyOne T1 streptavidin dinobeads (Thermo Fisher Scientific), and in others, these tag types were lysed from conjugated probes (antibodies) that were attached to analytes immobilized on a glass slide (e.g., fixed cells or tissue sections). After labeling the analytes (probing Her2 or ER here) within the sample with appropriate antibodies conjugated to two different barcoded HPv6 tags (Her2-v6 tag 1 and ER-v6 tag 2, respectively), the sample was simply washed in 1×RNase H buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT (pH 8.3)). Following this wash, 1×RNase H buffer containing RNase H (NEB M0297S) (75 units of enzyme in 300 μl buffer) was placed directly onto the sample for tag lysis (release). The reaction was carried out on a heating block set to 37°C for 30 minutes, and the solution was recovered by aspiration from the sample (cells or tissue sections). Subsequently, 300 μl of the aspirate was divided in half. One half received capture beads coated with a capture oligonucleotide (DRD1_CO_6sUMI-DNA bases; SEQ ID NO:14) (final 0.1 mg / mL), which contained full-DNA bases. The other half (150 μl) received capture beads coated with a capture oligonucleotide (DRD1_CO_6sUMI-RNA bases; SEQ ID NO:38) (final 0.1 mg / mL), which contained RNA at its 5' end.

[0395] DRD1_CO_6sUMI-DNA bases (SEQ ID NO:14)

[0396] DRD1_CO_6sUMI-RNA bases (SEQ ID NO:38)

[0397] DRD1_CO_6sUMI-RNA base oligonucleotides are generated by splicing the following two smaller oligonucleotides:

[0398] CO5 RNA (SEQ ID NO:15)

[0399] CO_3_invT(SEQ ID NO:16)

[0400] CO-clamp (for binding CO5 RNA and CO3 invT) (SEQ ID NO:17)

[0401] dNTPs (finally 200 μM each) and 1 μl of Maxima reverse transcriptase (NEB) [200 U / μl] containing RNase H activity were added to both samples for template extension of the 3' end of the captured oligonucleotide tag. The extension reaction was incubated at 37 °C for 45 min. After this incubation, the beads were pulled on a magnet, and the supernatant (approximately 125 μl) was recovered from each of the two reactions by aspiration. The remaining beads were resuspended in 125 μl of 1×RNase H buffer and incubated at 85 °C for 3 min to allow unwinding from the extension of the captured oligonucleotide. The sample was rapidly moved from the heat source to the magnet, and the supernatant was collected once the beads were fully pulled by the magnet. For each sample, these two portions were labeled as supernatant and beads, respectively. The portions were then subjected to qPCR for quantification. In short, using Thermo Fisher Scientific PowerUp SYBR master mix, 10 μl of the sample was analyzed by qPCR with the following primers:

[0402] 5'DRD LNA v4 (SEQ ID NO:18; where + indicates locked nucleic acid; LNA)

[0403] 3'DRD common primer v4 (SEQ ID NO:19)

[0404] The first assay uses an Hpv6 antibody conjugated to α-ER [EPR4007] (Abogen, ab108398) or α-HER2 / ErbB2 (MAB1129) for tag recovery, capture, extension and amplification, the Hpv6 antibody being used to stain a mixture of SKBR3 (Her2+, ER-) and MCF-7 (Her2-, ER+) cells cultured on a glass slide.

[0405] Example 11: Staining of α-HER2-v6 tag 1 antibody conjugates in cultured SKBR3 and MCF7 cells

[0406] For SKBR3 and MCF7 cell experiments, approximately 50,000 cells were grown on coverslips to approximately 70% cell density, fixed and infiltrated as described above, and used in downstream staining experiments. As described above, capture beads were coated with one of two different types of capture oligonucleotides to capture the tag released from the probe-tag conjugate bound to the biological sample: one containing full-length DNA bases (DRD1_CO_6sUMI-DNA bases; SEQ ID NO:14), and the other containing RNA bases at the 5' end (DRD1_CO_6sUMI-RNA bases; SEQ ID NO:38). The inclusion of full-length DNA bases in the capture oligonucleotides enables capture and extension of the 3' end of the capture tag; while the inclusion of RNA bases at the 5' end of the capture oligonucleotides enables capture, extension, and release of the extended product via a completely "one-pot isothermal" reaction due to the generation of a new DNA / RNA hybridization region following tag extension by polymerase. The RNase H activity of *E. coli* RNase H (NEBM0297S) and the RNase H activity of Maxima polymerase (Thermo Fisher Scientific Maxima EP0741) can release tags from probes bound to the surface, as well as release tags bound to capture oligonucleotides that extend through newly formed DNA / RNA hybridization regions. Following α-HER2-v6 tag 1 staining, samples were washed with 1×RNase H buffer and exposed to a solution containing *E. coli* RNase H for tag cleavage from bound conjugates as previously described. This solution was incubated at an isothermal reaction temperature, then removed from the samples, divided into two equal volumes, and two different types of capture beads were added to capture released α-HER2-v6 tag 1 hairpins in the presence of Maxima RT and dNTPs. In experiments using capture oligonucleotides composed of whole DNA bases, qPCR results demonstrated that the level of α-HER2-v6 tag 1 hairpins released in the beaded portion of SKBR3 cells increased approximately 13.7-fold compared to the beaded portion after probing MCF7 cells with this conjugate, consistent with the corresponding expression level of the known HER2 protein. These results indicate that the tag cleavage (hairpin release), capture, and extension steps of the procedure function as expected.

[0407] Within the probed SKBR3 cells, qPCR revealed a 15-fold increase in α-HER2-v6 tag 1 hairpin signal released in the bead fraction compared to the supernatant fraction, and a 40-fold increase compared to the background signal. Following experiments using capture oligonucleotides containing RNA bases at their 5' ends, qPCR results demonstrated an approximately 11.7-fold increase in α-HER2-v6 tag 1 hairpin signal released in the supernatant fraction after probed MCF7 cells, compared to the signal in the supernatant fraction. This recovered signal is similar to the signal obtained from the bead fraction when using whole-DNA capture oligonucleotides. Furthermore, an approximately 1.5-fold increase in α-HER2-v6 tag 1 hairpin signal released in the supernatant fraction compared to the bead fraction was observed, indicating that approximately 60% of the extended product was released into the supernatant after hairpin extension on the capture oligonucleotides. This result indicates that during a one-pot isothermal information transfer reaction, the cleaved α-HER2-v6 tag 1 hairpin is released from the bound probe, captured by a capture oligonucleotide containing 5'-RNA bases, followed by tag extension, and subsequently, as predicted, the extension product is released from the capture bead. Future experiments will optimize the extension product release steps to achieve a conversion rate of over 90% from the bead fraction to the supernatant fraction. In these final experiments, an approximately 120-fold increase in the signal of the released extension product compared to the background signal was observed in the supernatant fraction following the detection of SKBR3 cells.

[0408] Example 12: Staining of α-ER-v6 tag 2 antibody conjugates in cultured SKBR3 and MCF7 cells

[0409] To test different antibody-tag conjugates, for staining with the α-ER-v6 tag 2 antibody conjugate, the same cell culture samples and capture beads prepared and used in staining with the α-HER2-v6 tag 1 antibody conjugate in cultured SKBR3 and MCF7 cells were used. Following experiments using capture oligonucleotides composed of whole DNA bases, qPCR results demonstrated an approximately 0.82-fold increase in detectable released α-ER-v6 tag 2 hairpin signal in the bead fraction obtained after staining SKBR3 cells compared to the signal obtained after staining SKBR3 cells. Within MCF7 cells, an approximately 3.5-fold increase in released α-ER-v6 tag 2 hairpin signal was observed on the capture beads compared to the supernatant fraction and an approximately 10-fold increase relative to the background signal. Following experiments using capture oligonucleotides containing RNA bases at their 5' ends, qPCR results demonstrated an approximately 0.97-fold increase in α-ER-v6 tag 2 hairpin signal released in the supernatant fraction after staining MCF7 cells compared to the signal observed in the supernatant fraction after staining SKBR3 cells. Within MCF7 cells, an approximately 5.9-fold increase in α-ER-v6 tag 2 hairpin signal was observed in the supernatant fraction compared to the bead fraction. These results suggest that estrogen receptor expression levels are roughly equivalent between MCF7 and SKBR3 cells, which is incorrect based on previously published studies and inconsistent with internal experiments where these identical cells were stained with unconjugated ER antibodies and visualized using a fluorescently labeled secondary antibody (Thermo Fisher Scientific A32732). These initial studies indicate that additional blocking steps may be required to accurately stain and count tags released from antibody conjugates used to detect low expression levels of proteins, in addition to using SuperBlock supplemented with 1 mg / mL BSA and salmon sperm DNA. TM The blocking solution, and the experiment using the α-HER2-v6 tag 1 conjugate to detect HER2 protein levels (which is known to be expressed at much higher levels in SKBR3 cells than in the nuclei of MCF7 cells), does not require an additional blocking step.

[0410] Example 13: Improved blocking and washing conditions for antibody conjugate staining

[0411] Adding nucleic acid tags to antibodies can lead to nonspecific interactions between the conjugated antibody and other nucleic acids or positively charged molecules within the sample. These nonspecific interactions can increase the “background” signal in staining experiments. Therefore, a series of experiments were designed to determine the optimal blocking conditions for staining with the antibody-tag conjugates used in this paper. In short, nonspecific staining under different blocking conditions was tested by exposing MCF7 cells (low HER2 protein expression levels) to the α-HER2-v6 tag 1 conjugate or SKBR3 cells (low to no ER protein expression) to the α-ER-v6 tag 2 conjugate. Initial characterization of the background signal was performed using quantitative immunofluorescence (IF) staining, and the results of interest were further confirmed by qPCR analysis.

[0412] After staining MCF7 cells with α-HER2-v6 tag 1, test the following blocking or washing steps: Use SuperBlock to seal the control staining group. TM Blocked with (PBS) blocking buffer (Thermo Fisher Scientific 37515) and subsequently washed with PBST. A considerable amount of background staining was observed in these experiments during IF staining and qPCR analysis. Figure 15A , 15B ).

[0413] Next, we tested the following conditions and measured the changes in background signal obtained by IF staining:

[0414] Wash with 10% DMSO (background staining reduced by 1.2 times).

[0415] Additional blocking with Denhardt's seal (background staining reduced by 1.3 times)

[0416] Additional blocking with salmon sperm DNA (background staining reduced by 67-fold)

[0417] The use of salmon sperm DNA as a blocking agent was further tested in the following experiments, in which SKBR3 cells (high HER2 protein expression) were stained with α-HER2-v6 tag 1, and this blocking agent showed minimal effect on the overall staining signal. To confirm the benefits observed from using salmon sperm DNA as a blocking agent, MCF7 and 1954 cells were blocked and stained with α-HER2-v6 tag 1, washed, and RNase H was added to capture the released hairpins, extend them onto the capturing oligonucleotides bound to the capture beads, and quantify the samples by qPCR as described above. From these experiments, the following observations were made:

[0418] Quantitative analysis by qPCR showed that when MCF7 cells (HER2-negative) were stained with α-HER2-v6 tag 1, the addition of salmon sperm DNA as a blocking agent reduced background staining by approximately 62-fold, which was very similar to the level observed in IF staining experiments.

[0419] Quantitative analysis by qPCR showed that when 1954 cells (HER2 positive) were stained with α-HER2-v6 tag 1, the addition of salmon sperm DNA as a blocking agent reduced the positive staining signal by about 1.2-fold, further confirming the IF results, which showed that this blocking agent did not interfere with the specific staining of the antibody.

[0420] Although the addition of salmon sperm DNA effectively reduced background staining when using the α-HER2-v6 tag 1 conjugate, this blocking agent was insufficient to effectively restore accurate signals in experiments using the α-ER-v6 tag 2 conjugate. To further improve the blocking conditions, an infusion assay was performed after staining SKBR3 cells (ER protein negative) to determine whether background staining from the α-ER-v6 tag 2 conjugate could be further eliminated. Here, the control conditions included salmon sperm DNA in the blocking solution, and the following washing and blocking conditions were tested:

[0421] Washing with 10% DMSO (background staining reduced by 1.13 times)

[0422] Washing with 20% DMSO (background staining reduced by 1.03 times)

[0423] Image-iT TM FX signal enhancer (background staining reduced by 4.9 times)

[0424] RNase A treatment (background staining reduced by 1.9-fold)

[0425] DNase A treatment (background staining reduced by 1.27-fold)

[0426] In these experiments, background signals observed in the cell nucleus and cytoplasm of these cells were calculated independently, and it was clear that the sealing step was equally effective in both compartments of the cell. This was achieved using Image-iT... TM The above experiments were repeated using FX signal enhancer as a blocking agent (SKBR3 and MCF7 cells stained with α-ER-v6 tag 2 antibody conjugate) to test whether the signals in these experiments could be restored. Image-iT was added. TMThe use of FX signal enhancer as a blocking agent (in an end-to-end workflow with qPCR readout) enabled the observation of an approximately 1.8-fold increase in ER expression in MCF7(ER+) cells relative to SKBR3(ER-) cells. While significant, this recovery of ER signaling in these experiments may not fully reflect ER levels within the cells. Therefore, further optimization of blocking and washing conditions could be beneficial.

[0427] Example 14: Library Construction:

[0428] To test sequence preference during hairpin recovery and PCR amplification, a hairpin library was designed to mimic the structure of released hairpin species. This library consists of four random nucleotide positions (N) within the loop of the hairpin, representing the positions of various tag barcodes. The library was prepared by IDT using machine mixing of the four distinct bases at each position. The ordered oligonucleotide library was subsequently purified internally by PAGE using a 10% TBU gel. The oligonucleotide sequence for the library amplification preference test is shown in SEQ ID NO:37.

[0429] The PAGE-purified oligonucleotides were then diluted to 0.5 pM in 1×RNase H buffer. 22.5 μl of this sample and approximately 2.5 μl each of primers were added to 25 μl of... In the high-fidelity 2× master mixture at thermal initiation:

[0430] DRD forward protrusion P5-tag (SEQ ID NO:20)

[0431] DRD reverse protruding end P7-tag (SEQ ID NO:21)

[0432] The primer stock solution was 1 μM, bringing the final concentration in the reaction to 50 nM. The following PCR cycling conditions were used: 98 °C (30 sec) (1×), 98 °C (10 sec) -> 72 °C (15 sec) (25×), 72 °C (4 min) (1×). This reaction produced a single band of approximately 156 bp, which was visualized on a 6% TBE gel stained with SYBR gold. The PCR reaction mixture (50 μl) was purified using AMPure XP beads at a 1.5× ratio to remove excess (unextended) primers. The clean product from this “first step” of library preparation was eluted from the beads in 30 μl of IBI Scientific PCR-grade water, yielding approximately 5 ng / μl of product.

[0433] For the "second step" of library preparation, 1 μl (5 ng) of the purified AMPure XP product was added to the [missing information - likely a specific ingredient or product]. In 50 μl of the heat-started high-fidelity 2× master mixture, perform 18 PCR cycles under the same cycling conditions as in the "first step" of library construction, except that the following primers are used:

[0434] P7-G7 index primer (SEQ ID NO:22)

[0435] P5-G7 index primer (SEQ ID NO:23)

[0436] When developed on a 6% TBE gel stained with SYBR gold, this reaction produced a major band shifting (ran) of approximately 225 bp. This 225 bp band was further cleaned (0.6× ratio -> 1.5×) using two-step size selection with AMPure XP beads. The cleaned 225 bp band was then submitted for Sanger sequencing reactions in both directions using the following primers:

[0437] KAPA primer 1 (SEQ ID NO:24)

[0438] KAPA primer 2 (SEQ ID NO:25)

[0439] Example 15: Sanger sequencing of extended products

[0440] Sanger sequencing was performed using Azenta / GeneWiz for analysis of PCR-amplified circular library test oligonucleotides. The trace file revealed a random, homogeneous mix of A, T, C, and G bases at barcode location “NNNN.” While it was difficult to determine whether any specific barcode loss occurred via Sanger sequencing, a preference for G or C appears to exist at the first position after the initiation site in the tag. To address this, one or more G bases can be included immediately downstream of the initiation site, as in the optimized form of tag v6. NGS analysis of hairpin circular library test oligonucleotides achieved higher resolution to reveal this phenomenon.

[0441] Example 16: NGS Analysis of Extended Products

[0442] The same bands used for Sanger sequencing were also submitted for NGS on an Illumina NovaSeq 6000S4 flow cell (150 end pairs). This analysis yielded a total of 52,000,000 viable reads for Geneious Prime. To construct a reference genome library for analysis, sequences corresponding to all possible 256 (4-base) barcodes were generated using the R programming language, with 10 nucleotides side-glued on either side of the barcode to match the expected side-glued v6 tag sequence of the template. The paired-end (PE) reads and the “256-barcode” reference genome were input into Geneious Prime. In version 2023.0.4, for trimming and alignment, paired-end reads were trimmed to 10 nucleotides on either side of the barcode location and aligned with the “Geneious” mapper after 5 iterations of refinement. This alignment demonstrated that 98.6% of the reads mapped to the reference genome library. The percentage of representation for 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. Annex B, submitted together with this, provides a table containing read counts.

[0443] Figure 16A The graph shown is generated from 1 million (randomly selected) reads from 26,000,000 PE reads aligned to a reference genome library; then, the percentage representation for each barcode is plotted. This is performed 5 times, each time randomly selecting 1 million reads to allow for error calculation. Subsequently, GraphPad Prism is used to plot the percentage of reads obtained for each of the 256 barcodes.

[0444] Figure 16B The graphic “sequence identifier” (graphic representation of patterns within multiple sequence alignments) generated by uploading 10,000 reads (which have been successfully aligned with the reference genome) to WebLogo (online at weblogo.berkeley.edu / logo.cgi) is shown. See Crooks et al., Genome Research 14(6):1188-1190, 2004.

[0445] Example 17: Capturing Surfaces

[0446] In some experiments, amino-modified glass slides (AutoMate Scientific Po-104 000 406) or coverslips (AutoMate Scientific 104 000 406) are first modified with NHS conjugation buffer containing 1 mM NHS-LC-biotin to create a biotin-labeled surface. In these experiments, MyOne T1 streptavidin beads, resuspended in SA binding buffer at 2 mg / ml, are attached to the biotin-labeled surface by allowing beads to settle onto the surface. The bead-coated surface is then washed to remove any unbound beads. Generally, this procedure produces a dense layer of beads immobilized on the biotin-labeled surface, as determined by bright-field imaging at various magnifications. The beads are then coated with a single biotin-labeled trapping oligonucleotide (500 nM in SA binding buffer) to create a dense monolayer of trapping beads attached to the surface for various proof-of-concept experiments. These initial experiments aimed to obtain visual confirmation of the successful transfer of information (tags) from the sample surface (slide) to the capture array via isothermal information transfer reactions. To test this, fluorescently labeled DNA / RNA hybridization hairpin tags, modified to contain a 5' TCO linker and an internal fluorophore (AF-647 or AF-550) within their loop region, were fixed onto methyltetraazine-modified glass slides. While holding the slide on ice, add dropwise a solution containing all the components required for the isothermal information transfer reaction (200 μM dNTP, 5000 U / mL Maxima [RNase] H minus RT (Thermo Fisher Scientific EP0751), including (slide 1; Experiment 1, see pages 39-41 of Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023) or excluding (slide 2; Experiment 2, see pages 39-41 of Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023) 500 U / mL RNase H) to the sample surface. Subsequently, a glass capture array (thin coverslip) consisting of immobilized capture beads coated with a single type of capture oligonucleotide (containing full DNA bases to make the capture step visible) is placed face down to create a "sandwich" that brings the capture beads very close to the sample surface in the presence of the two solutions being tested. These sandwiched slide-capture arrays are then moved to a pre-set 42°C and 0 RPM heating block (with a block-mounted heat mixer, Thermo Fisher Scientific 13687711) for incubation at an isothermal transfer reaction temperature.After a 30-minute incubation, the capture array was removed from the slide by dropwise addition of 1×PBS buffer (Thermo Fisher Scientific J61196.AP) to allow the buffer to increase the gap between the capture array and the sample slide, enabling gentle separation. The slide and capture array were then washed with additional 1×PBS buffer and imaged using the described imaging parameters. Tag transfer from the sample surface to the capture array (slide 1; Experiment 1, see pages 39-41 of Appendix A included in U.S. Provisional Application No. 63 / 487,575, filed February 28, 2023) was observed only when performed in the presence of a solution containing RNase H. In earlier attempts to transfer information from the sample surface to this type of capture array (using fluorescently labeled tag v6 (SEQ ID NO:7) DNA / RNA hybridization hairpin tags attached to a glass slide in a described isothermal information transfer reaction; data not shown), it was determined that capture beads attached to rigid surfaces (such as glass) are not optimal for high-resolution information transfer. In practice, larger features (several millimeters in diameter) of fluorescently labeled tags can be efficiently transferred from glass slides to capture beads attached to rigid surfaces, while preserving the 2D information contained within the sample surface. However, in early attempts to use such glasses to transfer cellular-level information at subcellular resolution (from a sample surface composed of immobilized Her2+ cells stained with an anti-Her2 antibody conjugated to a fluorescently labeled tag v6 (SEQ ID NO:7)), capture arrays were only successful when the two surfaces were placed extremely close; this required pressing the two surfaces together during the transfer step. This pressure pushed the capture beads into the cells of the sample, and these capture beads exhibited high fluorescence due to the captured tag signal.

[0447] It is then assumed that trap beads immobilized within a flexible material (such as a gel) will increase the proximity of the trap beads to the sample surface without requiring as much pressure. Furthermore, if the flexible material used for the trap array can be made permeable to macromolecules, it is assumed that after the trap array has been positioned in close proximity to the sample slide to initiate the transfer reaction, the enzyme and cofactor components required for the isothermal information transfer reaction (at least to initiate it) can be added to the permeable gel. Moreover, if RNA bases are included within the 5' end of the trap oligonucleotide, the extended product of the isothermal information transfer reaction (the complete reaction, in which the tag is released from the probe bound to the sample surface, traps onto the trap oligonucleotide, extends to include the trap oligonucleotide information, and is released as a product into the surrounding solution) can proceed entirely through the gel matrix. These subsequent experiments for testing the flexible trap array are outlined below.

[0448] In other experiments, flexible, macromolecularly permeable gels were used instead of glass as the matrix material for the capture array. First, as described above, MyOne T1 streptavidin beads were coated with a single type of whole-DNA capture oligonucleotide, followed by two washes 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 clumping and applied directly to an amino-modified glass surface (Automation Technologies, PO-104 000 406). The capture beads were allowed to settle to the surface over several hours, where they likely bind primarily through 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. Generally, this procedure results in a dense monolayer of capture beads immobilized on the amino-modified surface, as determined by bright-field imaging at various magnifications. A bead layer is generated within a specific shape defined by a 125 μm thick sticker (GRACE BIO-LABS 6544008), the sticker containing eight individual pores, for example, 9 mm in diameter. The captured bead surface is then placed on a heating block set to 45°C (with the bead-coated surface facing upwards). Melted agarose (high melting temperature, catalog number and manufacturer) prepared at 10 mM Tris pH 7.5 with an agarose percentage in the range of 0.5–2% is cooled to approximately 40–45°C and slowly applied dropwise directly to the bead surface while allowing any remaining buffer solution (10 mM Tris pH 7.5) to evaporate from the bead-coated surface.

[0449] Subsequently, a standard glass slide or coverslip is gently pressed down onto the molten gel to flatten the agarose layer to approximately the thickness of a sticker. After cooling to room temperature, the slide or coverslip used to flatten the gel is gently removed by sliding it in one direction, revealing the agarose gel "pad" cast into a defined shape containing a dense layer of embedded beads. In this illustrated gel casting procedure, if the molten agarose is prepared with a buffer containing little or no salt (10 mM Tris pH 7.5 was successfully used here), and cooled sufficiently to prevent the beads from thermally dissociating from the surface upon exposure to the molten agarose (here, below 50°C yielded successful results), then electrostatic interactions will maintain the adhesion of the trapped beads to the amino-modified glass surface. Mechanical damage to bead adhesion can also be caused by adding molten agarose to the bead monolayer at excessively low temperatures, causing the slide or coverslip used to flatten the semi-hardened agarose into a thin pad to physically destroy the bead monolayer. Therefore, these experiments are not optimal (in many cases, the beads are unstablely embedded within the gel), but nevertheless, this type of array serves as a proof-of-concept for subcellular capture (using capture oligonucleotides containing full DNA bases) to obtain information from cells. When properly prepared, the flexible bead capture array can be peeled off the glass surface (almost like a thin "skin" for the capture beads) and used in capture experiments.

[0450] As outlined above, during sandwich construction, an early transfer experiment is performed on ice using a flexible trap array to reduce enzyme activity, and these are then moved to a pre-set 42°C heating block to initiate the transfer reaction. To construct the sandwich on ice, sample slides are placed on an ice bed in an ice bucket, and a drop of reaction mixture is placed on the sample slide, away from the cells bound to the slide. This keeps the reaction solution at a low temperature before dragging the trap array “skin” through the solution for immediate direct placement (beads down) onto the sample slide. In some cases, a coverslip or glass slide is placed on top of the trap array to apply a small amount of pressure. Generally, this type of flexible trap array, in the presence of isothermal information transfer reaction components (RNase H, Maxima RT (RNase H minus), and dNTP cofactor in 1×RNase H buffer), achieves highly successful subcellular capture of fluorescently labeled tags released from probes bound to cells. This was determined using a sample surface composed of immobilized Her2+ cells stained with an anti-Her2 antibody conjugated to a fluorescently labeled tag v6 (SEQ ID NO:7). Images of sample slides encasing the flexible trapping array showed that, after 10 minutes of incubation at isothermal reaction temperatures (37–42 °C), almost all signals derived from the fluorescent tag had been transferred from the probe (bound to cells on the sample slide) to the trapping beads of the trapping array.

[0451] In later experiments, RNase H was removed from the isothermal reaction mixture used during sandwich construction and included only in the solution applied to the back side of the capture array. In these experiments, the transfer reaction was observed to be initiated by diffusion across the gel upon the addition of a trigger enzyme (RNase H in this case). In subsequent experiments, capture oligonucleotides containing RNA bases at their 5' ends were used under the same conditions as previously described, and a significant enrichment 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 back side of the capture array gel. Figure 12 These results demonstrate that not only can one or more of the components required for the initial isothermal information transfer reaction be applied to the assembled sandwich via diffusion, but the full-length products of the reaction can also be recovered via diffusion through the gel. This enables an extremely simple workflow for isothermally transferring information from probed biological samples to a flexible trap array, coupled with the recovery of information from the reactants via aspiration, without additional user handling.

[0452] Example 18: Transformation of DNA-encoded molecules for isothermal spatial coding

[0453] Here, it is demonstrated that a T7 (RNA polymerase) promoter containing an adaptor is directly bound to the free end of dsDNA, which is ligated to a probe molecule (biotin) (a mimic probe or small molecule library) at its opposite 5' end. Following an optional gel purification step, this bound species or library can be used in binding experiments, for example, upstream of the isothermal space-coding workflow described herein.

[0454] To demonstrate this, an adaptor-modified DNA-encoded probe (biotin) was bound to streptavidin beads, unbound probes were washed away, and these beads were placed in the same reaction tube with mixed beads containing capture oligonucleotides (CO). In these experiments, the CO did not contain a 3'-reverse T base, but it did contain an RNA base at its 5' end. A solution containing T7 RNA polymerase and Maxima reverse transcriptase was introduced to initiate a one-pot reaction by generating RNA transcripts in vitro, which were then captured onto the capture oligonucleotides bound to the capture beads, and the 3' end of the CO was extended by the Maxima enzyme to combine information from both species (the transcribed tag and the capture oligonucleotide) within a single, continuous DNA sequence.

[0455] It was also demonstrated that by adding DNA polymerase (Bst 2.0 or 3.0; an enzyme capable of extending the 3' end of the captured RNA strand), full-length double-stranded extension products can be produced, and these products can be released upon introduction of RNase H. This alternative analytical chemical can be incorporated into the spatially encoded platform described herein, thereby placing spatially encoded capture beads over the sample bound to the probe. As with other workflows described herein, the resulting sequential sequences produced by this method also contain information relating to which probe molecule is present at a given location in the sample, as the information contained within the CO provides spatial coordinates.

[0456] Experimental methods:

[0457] T7p adaptor conjugation: Using 1 μM dsDNA T7p translocators and 400 nM dsDNA short tags, pre-annealed dsDNA T7p translocators (T7p conjugation adaptor annealed to T7p conjugation adaptor_rc; SEQ ID NO: 31 and 32, respectively) were conjugated to pre-annealed short tag 1 dsDNA oligonucleotides (short tag 1 with an A tail annealed to short tag 1_rc; SEQ ID NO: 29 and 3-, respectively) 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) in a 200 μl reaction volume. Conjugation was carried out overnight at room temperature. The next day, the conjugation reactants were precipitated with EtOH, resuspended in ultrapure H2O, and then pre-equilibrated with Zeba O2 using ultrapure H2O. TM Cleaned on a 7K MWCO rotary desalting column (Thermo Fisher Scientific 89882). The same procedure was performed for conjugating the dsDNA T7p adaptor to a short tag 1 dsDNA oligonucleotide without an A-base overhang (short tag 1 requiring an A tail annealed to short tag 1_rc; SEQ ID NO: 28 and 30, respectively); however, prior to the conjugation step, a [preparation procedure was performed using...] Ultra TMII-terminal repair / dA-tailing (NEB#E7546S) was used to perform an A-tailing reaction on this oligonucleotide to add an A-base overhang to this oligonucleotide (SEQ ID NO:29). The A-tailing reaction was cleaned using a QIAquick PCR purification kit column (Qiagen 28104), and the oligonucleotide was eluted in 30 μl of ultrapure H2O before conjugation with the dsDNA T7p adaptor. The conjugation product was analyzed on a 15% TBE-urea gel (Thermo Fisher Scientific EC6885BOX), where the formation of a 64-base conjugation product was observed after two reactions. The conjugated construct was cleaned by EtOH precipitation, resuspended in ultrapure H2O, and further processed using Zeba gel pre-equilibrated with ultrapure H2O. TM The constructs were cleaned using a 7K MWCO rotary desalting column (Thermo Fisher Scientific 89882) and then used as templates in downstream in vitro transcription reactions.

[0458] In vitro transcription: In vitro transcription reactions were established to test various tag constructs containing the T7 promoter (encoding FLT7p short tag 1), which were generated by three different methods described above: (1) purchased fully synthetic and biotinylated constructs (FL T7p short tag 1 annealed to FL T7p short tag 1_rc; SEQ ID NO: 26 and 27, respectively); (2) annealed T7p adaptors (T7p conjugating adaptors annealed to T7p conjugating adaptors_rc; SEQ ID NO: 31 and 32, respectively) with short tag 1 oligonucleotides containing an artificial A-base overhang (short tag 1 with an A tail annealed to short tag 1_rc; SEQ ID NO: 29 and 30, respectively); and (3) annealed T7p adaptors with short tag 1 oligonucleotides without an A tail (short tag 1 requires an A tail annealed to short tag 1_rc; SEQ ID NO: 28 and 30, respectively). For the tag constructs described in (1) above, MyOne T1 streptavidin dino beads (Thermo Fisher Scientific 65601) were used to mimic the "target" for biotin "probes" (linked to this oligonucleotide construct) binding, followed by standard washing (described elsewhere in this application after coating the streptavidin beads with biotin-labeled oligonucleotides) to remove unbound probes. These beads were then equilibrated in 1× transcription buffer prior to in vitro transcription reactions. Transcription production of the remaining two tag species (2 and 3) in solution was tested. All in vitro transcription reactions were performed in... The reaction mixture was prepared using the T7 High-Yield RNA Synthesis Kit (NEB#E2040S) and incubated at 37°C for 1 hour. After this incubation, 1 / 3 volume of each reaction mixture was removed and RNase was cocked. TMThe enzyme mixture (Thermo Fisher Scientific AM2286) was treated at 37°C for one hour, or with TURBO. TM Treatment with DNase (Thermo Fisher Scientific AM2239) was performed. All nuclease reactions were carried out in 1×DNase I buffer (Thermo Fisher Scientific AM2239). Gel analysis of the transcription reactions using 15% TBE-urea gel (Thermo Fisher Scientific EC6885BOX) showed approximately 10-20 fold RNA amplification during the reaction, based on a comparison of the template band and the RNA band (41 bases) intensity. In all three reactions (1-3), a band of RNA product (41 bases) of the expected size was observed, and this band was completely eliminated after treatment with RNase, and this band was unaffected by treatment with DNase I.

[0459] RNA Capture and Elongation: To demonstrate the capture of RNA transcribed from a dsDNA tag modified with a dsDNA T7p adaptor, and to demonstrate the templated elongation of the RNA 3' end of the captured oligonucleotide (CO) and the elongation of the CO 3' end, we utilized the same CO as previously described in this application, but without the inverse T (invT) base at the 3' end of the CO (containing 5'-RNA bases), so that this CO could elongate after capturing the transcribed RNA. In a similar manner to the previous CO, 3'-invT-free RNA COs were constructed by sandwich conjugation between RNA CO without invT and CO_5_RNA (SEQ ID NO: 35 and 15, respectively), the sandwich conjugation utilizing a CO_sandwich (SEQ ID NO: 17) as a template for conjugation. After conjugation, this oligonucleotide was PAGE purified and bound to MyOne T1 streptavidin dinopills (Thermo Fisher Scientific 65601) for downstream applications.

[0460] To test whether the transcribed RNA could be captured on CO and extended, the transcribed RNA was added to a solution containing CO bound to beads at a concentration of 0.25 mg / ml in 1× transcription buffer supplemented with 5% DTT, 50 mM KCl, NTPs, and dNTPs. The T7 High-Yield RNA Synthesis Kit (NEB#E2040S) was used as the buffer for testing this alternative one-pot isothermal reaction. 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, like the Klenow fragment, this polymerase is known to extend RNA primers. After this reaction, the beads were pulled off and transferred to 10 mM Tris, heated to 85°C to dehybridize the extended RNA, which was then recovered from the supernatant for gel analysis. The supernatant of this reaction was observed on a 15% TBE-urea gel (Thermo Fisher Scientific EC6885BOX), where a prominent band moved to approximately 80 bp, corresponding to the (RNA chain) extension product that was efficiently completed by the DNA templated region of CO rather than by the RNA bases that templated the 5' region of CO. Here, if Bst 3.0 can effectively extend RNA bases, then we would expect a 96-base product, indicating that Bst 3.0 contains weak reverse transcriptase activity under these conditions. An 80bp product represents a single-stranded product containing RNA at the 5' end and newly extended DNA bases at the 3' end; this product would contain primer landing sites for CO primers DS1 and DS2. To confirm that this observed approximately 80bp product indeed corresponds to captured and extended RNA, PCR was performed to produce a 79bp product. No product was produced in the negative controls (ultrapure water, unextended RNA, or CO alone).

[0461] To test whether CO could be extended via captured transcribed RNA, 1 μl of reverse transcriptase Maxima (Thermo Fisher Scientific EP0741) with RNase H activity (RNase H+) was added to a solution containing CO immobilized on beads and RNA transcribed from a T7p FL short tag. This step was performed in the same buffer (1× transcription buffer) described above and incubated at 37°C for 1 hour. Subsequently, the beads were pulled off by a magnet and resuspended in ultrapure H2O. The beads were then heated to 85°C to disrupt the biotin / streptavidin interaction between the biotin-bound CO and the streptavidin beads. After heating, the beads were pulled off by a magnet, and the supernatant was analyzed on a 15% TBE-urea gel (Thermo Fisher Scientific EC6885BOX), where a prominent band was observed at approximately 100 bases, corresponding to a fully extended CO of the expected size (templated by the captured RNA strand). As described above, to demonstrate that the observed approximately 100-base-long product indeed corresponds to the extended CO, PCR was performed using CO primers DS1 and DS2 to produce a 100 bp product, as determined by gel analysis. While it was possible to amplify the expected product, amplification of the negative control (CO-coated beads + transcribed RNA, but excluding Maxima enzyme) subjected to the same heating procedure to disrupt the oligonucleotide ligation (from the biotinylate portion of the streptavidin beads) could not be achieved using PCR with the same primers.

[0462] Alternative one-pot isothermal reactions:

[0463] Streptavidin beads coated with biotin-labeled FL T7p short tags (as described above) were subjected to an in vitro transcription reaction at a final concentration of 0.05 mg / ml, supplemented with 1 μl of T7 RNA polymerase (MO255AVial), Bst3.0 (8 units, NEB M0374S), and Maxima reverse transcriptase (200 units, Thermo Fisher Scientific EP0741), as well as CO-coated streptavidin beads (as described above). This experiment was performed to simulate the transfer of information from a T7p transducer-modified tag bound to one surface to a CO-containing surface. Here, the reaction was carried out at 37°C for 1 hour. Optionally, RNase H (5 units, NEB M0297S) was introduced into the reaction following the described in vitro transcription-mediated information transfer reaction. In both cases, magnetic beads were pulled down onto a magnet at room temperature, and the product in the supernatant was collected. Subsequently, both supernatants were run on a 15% TBU gel. In both cases, a band was detected at the expected size of the extended CO (approximately 100 bases); however, this band was more prevalent in further reactions treated with RNase H. In both cases, amplification of the 79 bp product using CO primers DS1 and DS2 indicated that this approximately 100-base band was indeed the extended CO.

[0464] The above results provide support. Figure 7B and Figure 8 The data.

[0465] Restriction enzyme-mediated elongation products are released from the captured beads:

[0466] To demonstrate the release of full-length elongation products mediated by restriction enzyme (RE) via the alternative one-pot isothermal signal transfer reaction described above, a novel capture oligonucleotide containing the XhoI restriction site (Biotin TEG-DNACO without InvT; SEQ ID NO:36) was synthesized. This oligonucleotide was conjugated to MyOne T1 streptavidin dino beads (Thermo Fisher Scientific 65601) as described above for CO containing an RNA base at its 5' end. Following the in vitro transcription-mediated signal transfer reaction, half of the reaction mixture was aspirated using a pipette and placed in the same tube. Subsequently, both reactions were subjected to magnetization to aggregate the beads, the supernatant was removed, and the beads were resuspended in 1×rCutSmart. TMIn a buffer (NEB B6004S), 60 units of XhoI (NEB R0146S) were added to a tube (+XhoI) and mixed. Both tubes were incubated at 37°C for 15 minutes. After this incubation, both reactions (with and without XhoI) were pulled down onto a magnet, and the supernatant was collected from each tube. These supernatants were used in PCR reactions with CO primers DS1 and DS2 (SEQ ID NO: 33 and 34, respectively), followed by gel analysis. A band of the expected size of 59 bp was detected in the reaction containing supernatant from the sample treated with XhoI, but this band was not present in the reaction containing supernatant from the sample not treated with XhoI. This result demonstrates that transfer information (transcribed RNA) is captured by CO, subsequently extended to include information from both the probe tag and the captured oligonucleotides (full-length extension product), and this information is subsequently released into solution upon introduction of the XhoI enzyme. These results support this finding. Figure 9E and 9B The data.

[0467] List of oligonucleotides used in Example 18

[0468] FL T7p short label 1: PAGE purified, T7p, Capture region CO DS primer 1 landing site (SEQ ID NO:26)

[0469] FL T7p short label 1_biotin_rc: PAGE purified (SEQ ID NO:27)

[0470] Short tag 1 requires an A tail: PAGE purified (SEQ ID NO:28)

[0471] Short tag 1 with an A tail: PAGE purified (SEQ ID NO:29)

[0472] Short tag 1_rc: PAGE purified (SEQ ID NO:30)

[0473] T7p binding adaptor: purified by HPLC (SEQ ID NO:31)

[0474] T7p binding adaptor rc: purified by HPLC (SEQ ID NO:32)

[0475] CO DS primer 1: Standard desalting Tm = 47-51 (SEQ ID NO: 33)

[0476] CO DS primer 2: Standard desalting Tm = 47-51 (SEQ ID NO: 34)

[0477] RNA CO without invT: Page purified (SEQ ID NO:35)

[0478] CO5 RNA (SEQ ID NO:15)

[0479] CO_3_invT(SEQ ID NO:16)

[0480] CO-clamp (SEQ ID NO:17)

[0481] Biotin-TEG-DNA CO InvT-free: HPLC purified, XhoI restriction site (SEQ ID NO:36)

[0482] Selecting references

[0483] Fu et al., Cell 185:4621-4633, 2022, doi.org / 10.1061 / j.cell.2022.10.021

[0484] Rodrigues et al., Science 363:1463-1467, 2019

[0485] Troll et al., BMC Genomics 20:1023, 2019, doi.org / 10.1186 / s12864-019-6355-0

[0486] Yang et al., ChemBiochem 16(9):1365-1370, 2015

[0487] U.S. Patent No. 10,002,316, "Spatially Addressable Molecular Barcoding"

[0488] US2022 / 0033802 "Method and apparatus for encoding cellular spatial position information"

[0489] US2020 / 0190583 "Methods and Compositions for Sequentially Detecting Targets"

[0490] WO 2018 / 087539 "Tagless encoding chemical library"

[0491] Ending paragraph

[0492] As will be understood by those skilled in the art, each embodiment disclosed herein may include, or substantially consist of, the elements, steps, ingredients, or components specifically stated herein. Therefore, the terms “include” or “including” should be interpreted as stating: “comprising, consisting of, or substantially consisting of.” The transitional terms “comprise” or “comprises” mean having, but not limited to, and allowing the inclusion of even a major amount of unspecified elements, steps, ingredients, or components. The transitional phrase “consisting of” excludes any unspecified elements, steps, ingredients, or components. The transitional phrase “substantially consisting of” limits the scope of the embodiments to the specified elements, steps, ingredients, or components and those that do not significantly affect the embodiments.

[0493] Unless otherwise indicated, all figures used in this specification and claims to represent the amount of components, properties such as molecular weight, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations, which may vary depending on the desired properties sought to be obtained by the invention. To a minimum, and without attempting to limit the application of the equivalence principle to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying general rounding techniques. When further clarification is required, the term “about” when used in conjunction with the stated values ​​or ranges has the meaning reasonably attributable to it by one skilled in the art, meaning slightly larger or smaller than the stated value or range, within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0494] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​described in specific examples are reported as precisely as possible. However, any value inherently contains some error, which is necessarily caused by the standard deviation found in its corresponding test measurement.

[0495] Unless otherwise indicated herein or clearly contradicted by the context, the terms “a,” “an,” and “the,” and similar designations used in the context of describing the invention (particularly in the context of the appended claims) should be interpreted as encompassing both the singular and plural. The description of ranges of values ​​herein is intended only as a simplified way of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were described separately herein. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all example or exemplary language provided herein (e.g., “such as”) is intended only to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0496] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may be mentioned and claimed individually or in any combination with other members in the group or other elements seen herein. It is contemplated that one or more members in a group may be included in or removed from the group for convenience and / or patentability reasons. When any such inclusion or removal occurs, this specification is deemed to contain the modified group and thus satisfy the written description of all Markush groups as used in the appended claims.

[0497] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art after reading the above description. The inventors expect those skilled in the art to adopt these variations where appropriate, and the inventors intend to practice the invention in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter set forth in the appended claims, where permitted by applicable law. Furthermore, unless otherwise indicated herein or otherwise expressly contradicted by the context, the invention covers any combination of the elements described above in all possible variations.

[0498] Furthermore, numerous references have been made throughout the specification to patents, print publications, journal articles, other written texts, and website content (materials cited herein). From the filing date of the first application in the priority chain, including the specific references, each of the cited materials is incorporated herein by reference in its entirety. For example, from the filing date of an application in the priority chain, information from database entries relating to compounds, nucleic acids, and amino acid sequences cited herein that are available in public databases is incorporated herein by reference, wherein the database identifier of the compound or sequence is included herein for the first time.

[0499] It should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications may be made within the scope of the invention. Therefore, alternative configurations of the invention can be utilized based on the teachings herein, rather than as examples. Thus, the invention is not limited to what is precisely shown and described.

[0500] The details shown herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the invention, and are intended to provide the most useful and readily understood description of the principles and concepts of various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a basic understanding of the invention, and this specification, along with the drawings and / or examples, makes it clear to those skilled in the art how to implement the various forms of the invention in practice.

[0501] Unless clearly and explicitly modified in one or more instances or when the application of meaning renders any construct meaningless or substantially meaningless, the definitions and interpretations used in this disclosure are intended to control any future constructs. Where the construction of a term would render it meaningless or substantially meaningless, the definition shall be taken from Webster's Dictionary, 11th edition, or a dictionary known to a person of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2nd edition (edited by Anthony Smith, Oxford University Press, Oxford, 2006) and / or the Dictionary of Chemistry, 8th edition (edited by J. Law and R. Rennie, Oxford University Press, 2020).

Claims

1. A hairpin-tagged nucleic acid molecule comprising functionally linked portions ABCDE in a 5' to 3' sequence, wherein: Part A contains a cleavable site, which includes one of the following: (1) RNA base string, or (2) A DNA base string containing restriction enzyme (RE) recognition sites; Part B contains a DNA base string with a tagged PCR handle; Part C contains a DNA base string containing a tag ID barcode, and the DNA base string forms part of the loop of the hair clip; Part D contains a DNA base string having the reverse complementary sequence of the tag PCR handle, thereby forming part of the stem of the hairpin; and The E portion comprises a DNA base string having (1) the RNA base string having the A portion or (2) the DNA base string having at least a portion of the reverse complementary sequence of the DNA base string having the RE recognition site, thereby forming a portion of the stem of the hairpin.

2. The hairpin-tagged nucleic acid molecule according to claim 1, further comprising a linker portion attached to the 5' end of the A portion.

3. The hairpin-tagged nucleic acid molecule of claim 2, wherein the linker portion provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

4. The hairpin-tagged nucleic acid molecule according to claim 2, further comprising a linker between the linker portion and portion A.

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

6. The hairpin-tagged nucleic acid molecule according to claim 1, wherein the tag ID barcode is at least 4 bases long.

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

8. The hairpin-tagged nucleic acid molecule according to claim 1, wherein the tag PCR handle in part B and the reverse complementary sequence in part D are each at least 10 bases long.

9. The hairpin-tagged nucleic acid molecule according to claim 8, wherein the tag PCR handle in part B and the reverse complementary sequence in part D are each 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length.

10. The hairpin-tagged nucleic acid molecule according to any one of claims 1 to 9, which is constructed at least in part using a templated conjugation reaction.

11. The hairpin-tagged nucleic acid molecule according to any one of claims 1 to 10, wherein part A comprises the DNA base string containing the RE recognition site, and the RE recognition site is at least 4 bases long.

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

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

14. The hairpin-tagged nucleic acid molecule according to any one of claims 1 to 10, wherein it is a DNA / RNA hybrid hairpin-tagged nucleic acid molecule, and wherein part A contains the RNA base string, and the RNA base string in part A is at least 5 bases long.

15. The DNA / RNA hybrid hairpin-tagged nucleic acid molecule according to claim 14, wherein the RNA base string in part A is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length.

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

17. The group of two or more hairpin-tagged nucleic acid molecules and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules according to claim 16, further comprising at least one attenuated tag-like nucleic acid molecule, said attenuated tag-like nucleic acid molecule differing from the hairpin-tagged nucleic acid molecules or DNA / RNA hybrid hairpin-tagged nucleic acid molecules of the group in that it lacks a functional cleavable site.

18. The group of two or more hairpin-tagged nucleic acid molecules and / or DNA / RNA hybrid hairpin-tagged nucleic acid molecules according to claim 17, wherein the weakened tag-like nucleic acid molecule differs from the hairpin-tagged nucleic acid molecule or DNA / RNA hybrid hairpin-tagged nucleic acid molecule of the group in that (1) the RNA base of part A is replaced with a DNA base, or (2) the RE recognition site of part A is lacking.

19. A labeled probe comprising a probe molecule connected via the connecting portion to a hairpin-tagged nucleic acid molecule according to any one of claims 2 to 13 or a DNA / RNA hybrid hairpin-tagged nucleic acid molecule according to claim 14 or 15.

20. The labeled probe of claim 19, wherein the probe molecule comprises an affinity molecule having binding affinity to the target molecule.

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

22. The labeled probe of claim 19, wherein the probe molecule comprises one or more of the following: an antibody or a binding fragment thereof, a nucleic acid, a small molecule, an organic or inorganic chemical substance, a presumed drug target, an identified drug or biomacromolecule complex.

23. The labeled probe of claim 19, wherein the probe molecule is one of a group of probe molecules, each of which comprises one of a plurality of members of: a small molecule library, a drug target library, a bioaffinity molecule library, a natural product library, a bioactive compound library, a genomic library, a transcriptome library, a metabolome library, or a drug screening library.

24. The labeled probe of claim 20, wherein the target molecule comprises addressable features of a biomolecule, an inorganic object, or an array.

25. The labeled probe of claim 24, wherein the target molecule comprises a biomolecule, and the biomolecule comprises one or more of the following: protein, lipid, carbohydrate, nucleic acid molecule, or a combination of protein, lipid, carbohydrate and / or nucleic acid molecule.

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

27. The labeled probe according to any one of claims 19 to 26, further comprising an amplified sequence containing a polymerase promoter sequence.

28. The labeled probe of claim 27, wherein the amplification sequence comprises a T7 promoter sequence, such as a T7 promoter adaptor.

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

30. A capture oligonucleotide (CO) nucleic acid molecule comprising functionally linked portions I-II-III-IV in a 5' to 3' sequence, wherein: Part I contains cleavable sites that contain one of the following: (1) A single RNA base or a continuous string of RNA bases, or (2) A DNA base string containing restriction enzyme (RE) recognition sites; Part II contains a DNA base string containing a CO PCR handle; Part III contains a DNA base string containing spatial barcodes; and The IV portion contains a DNA base string containing the tag-capture region.

31. The CO nucleic acid molecule of claim 30, further comprising a linker portion conjugated to the 5' end of the I portion.

32. The CO nucleic acid molecule of claim 31, wherein the linker portion provides amine-reactive crosslinking agent activity or thiol-reactive crosslinking agent activity.

33. The CO nucleic acid molecule according to claim 31, further comprising a linker between the linker portion and the I portion.

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

35. The CO nucleic acid molecule according to claim 30, wherein the spatial barcode is at least 4 bases long.

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

37. The CO nucleic acid molecule according to claim 30, wherein the CO PCR stem in part II is at least 5 bases long.

38. The CO nucleic acid molecule according to 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 bases in length.

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

40. The CO nucleic acid molecule according to any one of claims 30 to 38, wherein its sequence has no more than two consecutive self-complementary internal sequences.

41. The CO nucleic acid molecule according to claim 40, wherein its sequence 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 self-complementary consecutive bases.

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

43. The CO nucleic acid molecule according to any one of claims 30 to 42, wherein part I comprises the DNA base string containing the RE recognition site, and the RE recognition site is at least 4 bases long.

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

45. The CO nucleic acid molecule according to claim 43, wherein the RE recognition site is an IIS type restriction enzyme recognition site, a site where a RE with a 3' overhang is left, or both.

46. ​​The CO nucleic acid molecule according to any one of claims 30 to 42, wherein it is a DNA / RNA chimeric CO nucleic acid molecule, and wherein part I comprises the single RNA base.

47. The CO nucleic acid molecule according to any one of claims 30 to 42, wherein it is a DNA / RNA chimeric CO nucleic acid molecule, and wherein part I comprises the continuous RNA base string.

48. The DNA / RNA chimeric CO nucleic acid molecule according to claim 47, wherein the continuous RNA base string in part I is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases long or longer than 20 bases.

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

50. The CO nucleic acid molecule according to any one of claims 31 to 45 or the DNA / RNA chimeric CO nucleic acid molecule according to any one of claims 46 to 48, wherein the ligation portion is connected to the capture feature.

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

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

53. The CO nucleic acid molecule according to 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 connected via a linker portion to a 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.

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

56. The spatially encoded capture feature according to claim 54 or claim 55, wherein it is one spatially encoded capture feature within an array of at least 100 different spatially encoded capture features, and wherein the CO nucleic acid molecule on each of the at least 100 different spatially encoded capture features in the array each contains a different spatial barcode.

57. A capture pair comprising: The hairpin-tagged nucleic acid molecule according to any one of claims 2 to 13, or the DNA / RNA hybridization hairpin-tagged nucleic acid molecule according to claim 14 or claim 15, or the released hairpin-tagged nucleic acid molecule according to claim 29; and The capture oligonucleotide (CO) nucleic acid molecule according to any one of claims 31 to 42, or the DNA / RNA chimeric CO nucleic acid molecule according to any one of claims 43 to 48, or the linked CO according to any one of claims 50 to 73, The sequences of the hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule are at least partially complementary, such that when the hairpin-tagged nucleic acid molecule is released from its linker near the CO nucleic acid molecule, the released hairpin-tagged nucleic acid molecule is captured by the complementary sequence bound to the 3' end of the CO nucleic acid molecule, such that the resulting complex of the released hairpin-tagged nucleic acid molecule and the CO nucleic acid molecule is capable of undergoing downstream extension reactions by polymerase.

58. A spatially coded capture array comprising a defined array of spatially addressed capture features, wherein each capture feature includes: Spatial identifiable features include: Predefined addressable locations on a generally two-dimensional solid surface; or Beads or other similar individual solid objects are captured. as well as Multiple copies of the CO nucleic acid molecule according to any one of claims 31 to 42 or the DNA / RNA chimeric CO nucleic acid molecule according to any one of claims 43 to 48 are linked at each feature, wherein the CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules at other features in the array.

59. The spatially encoded capture array of claim 58, wherein one or more CO nucleic acid molecules are applied to the spatially addressed capture features of the array in the following manner: The CO droplets are printed onto the predefined addressable locations on the generally two-dimensional solid surface, or The CO is attached to the beads via the connecting portion.

60. The spatially encoded capture array of claim 58 or claim 59, the array comprising beads, each of which contains a visual barcode operatively coupled to the CO.

61. The spatially encoded capture array of claim 60, wherein the visual barcode enables beads to be assigned to positions within the capture array.

62. The spatially encoded capture array of claim 58, wherein the beads or other similar individual capture objects are embedded in a biomolecularly permeable matrix.

63. The spatially encoded capture array according to claim 62, wherein: The captured object contains beads; The biomolecularly permeable matrix comprises a gel; or Both.

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

65. The spatially encoded trapping array of claim 62, wherein the biomolecules are permeable to the matrix: The structure is stable at the selected temperature range of 4-45℃; It is permeable to proteins such as functional RNase H and polymerase; It is permeable to ribonucleoside triphosphate (rNTP) and / or deoxynucleotide triphosphate (dNTP); For Mg 2+ Ions are permeable; It is generally inert to biomolecules; and It is flexible enough to allow the relatively thin matrix layer to be applied directly to a generally two-dimensional sample or surface.

66. The spatially encoded capture array of claim 65, configured as a three-dimensional thin-layer gel, the width and length of which are significantly greater than the thickness of which the spatially identifiable capture features are generally arranged in a single plane on the surface of the gel defined by its length and width.

67. The spatially encoded capture array of claim 66, wherein at least the first of the spatially identifiable capture features is directly connected to and / or in contact with the second of the spatially identifiable capture features.

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

69. The spatially encoded capture array of claim 68, wherein the thickness of the matrix or gel does not exceed about 2 mm.

70. The spatially encoded capture array of claim 69, wherein the thickness of the matrix or gel is not more than about 1 mm, not more than 500 μm, not more than 250 μm, not more than 200 μm, not more than 150 μm, not more than 125 μm, not more than 100 μm, or less than 100 μm.

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

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

73. The spatially encoded capture array according to any one of claims 58 to 72, wherein: The predefined addressable locations on a generally two-dimensional solid surface have a surface area not exceeding approximately 1 μm × 1 μm; or The diameter of the beads or other similar individual solid traps does not exceed about 20 μm.

74. The spatially encoded capture array of claim 73, wherein the diameter of the bead or other similar individual solid capture object is not more than 18 μm, not more than 15 μm, not more than 12 μm, not more than 10 μm, not more than 8 μm, not more than 5 μm, not more than 3 μm, not more than 1 μm or about 100 nm.

75. The spatially encoded capture array of claim 74, wherein the diameter of the bead or other similar individual solid capture object is between 1 and 3 μm.

76. A semi-ordered spatially encoded capture array, comprising: A grid of spatially addressable locations, each labeled with an oligonucleotide having a unique XY coordinate sequence, the oligonucleotide being applied to the spatially addressable location via a clamping mechanism: x-coordinate connector oligonucleotides, wherein the x-coordinate connector oligonucleotides are used to mark all spatially addressable locations within a row of the grid; and The y-coordinate linker oligonucleotide is used to mark all spatially addressable locations within a column of the grid.

77. The semi-ordered spatially encoded capture array of claim 76, which is constructed at least in part using the methods provided in Figures 17A to 17C.

78. The semi-ordered spatial coding capture array according to claim 76, having a grid-within-a-grid format as shown in FIG10.

79. A semi-ordered spatially encoded capture array, comprising: An array of uniquely identifiable capture features, the array comprising two or more subarrays, each subarray containing uniquely identifiable capture features, the location of which is specified at least in part by the identification of the subarrays within the semi-ordered spatially encoded capture array.

80. A semi-ordered spatially encoded capture array, comprising: A group of two or more subarrays, each subarray containing multiple capture features, wherein each capture feature within each subarray is connected to a capture oligonucleotide containing a unique position tag, the capture features within each subarray being randomly arranged, and each capture feature within each subarray further containing an oligonucleotide tag that identifies the subarray, the oligonucleotide tag that identifies the subarray being attached to the capture oligonucleotide of each feature in the subarray via a clamping connection.

81. A method for detecting and / or quantifying and / or locating a target in a substantially two-dimensional (2D) sample, the method comprising: A substantially 2D sample is contacted with at least one labeled probe to produce a substantially 2D stained sample, said labeled probe comprising: Hairpin-tagged nucleic acid molecules containing a linker portion and a tag ID barcode, or DNA / RNA hybrid hairpin-tagged nucleic acid molecules containing a linker portion and a tag ID barcode; and The probe molecule is connected to the hairpin-tagged nucleic acid molecule containing the tag ID barcode or the DNA / RNA hybrid hairpin-tagged nucleic acid molecule containing the tag ID barcode via the connection portion. The surface of the substantially 2D stained sample is brought into contact with a permeable spatially encoded capture array to form a sample-array sandwich, the spatially encoded capture array comprising: Multiple spatially identifiable features; and Multiple copies of a capture oligonucleotide (CO) nucleic acid molecule or a DNA / RNA chimeric CO nucleic acid molecule are linked at each spatially identifiable feature, wherein each CO nucleic acid molecule at each feature has a unique spatial barcode sequence compared to the CO nucleic acid molecules at other features in the array; A flow cell or other cover containing a solution is placed above the sample-array sandwich to form a shell containing the sample-array sandwich; A solution containing reactive components is added to the shell to form a reaction mixture, said components comprising: Lyase selected from RNase H or at least one restriction endonuclease (RE); At least one polymerase; A mixture of ribonucleotide triphosphates (rNTPs) and / or deoxyribonucleotide triphosphates (dNTPs); Mg 2+ Ions; and Buffer; The sample-array sandwich in contact with the reaction mixture is incubated at the analytical temperature for 30-60 minutes to form a mixture of reaction products; At least a portion of the reaction product mixture is removed from the shell; as well as The reaction product mixture is analyzed to detect and / or quantify the target in the substantially 2D sample and / or define the location of the target.

82. The method for detecting and / or quantifying and / or locating a target in a substantially two-dimensional (2D) sample according to claim 81, wherein the housing comprises a flow cell.

83. The method according to claim 81 or claim 82, wherein it is carried out at a single temperature (isothermal) or within a range of about 5°C of a single temperature.

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

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

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

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

88. The method of claim 87, wherein analyzing the reaction product mixture comprises performing next-generation sequencing on a plurality of nucleic acid molecules containing spatial barcodes and tag ID barcodes; NGS).

89. The method of claim 87 or claim 88, wherein the plurality of nucleic acid molecules containing spatial barcodes and tag ID barcodes are fully extended products released from the spatially identifiable features by cleavage at the cleavage site of the CO.

90. The method of claim 89, wherein the lysis comprises RNase H activity or restriction endonuclease activity.

91. The method according to any one of claims 81 to 90, wherein the analysis comprises assigning the spatial location of at least one nucleic acid molecule containing a spatial barcode and a tag ID barcode within the substantially 2D sample.

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

93. Use of a group of hairpin-tagged nucleic acid molecules according to any one of claims 16 to 18 or a group of captured oligonucleotide (CO) nucleic acid molecules according to claim 49, or both, for transcriptomic analysis of biological samples.

94. A spatially encoded surface, such as a trapping array, for example a bead-based trapping array, generally as described or illustrated herein.

95. A spatial coding workflow, comprising: The hybrid RNA / DNA tag containing a spatial barcode conjugated to an antibody probe is brought into contact with a substantially two-dimensional (2D) tissue sample to produce a stained sample; A capture array containing capture features is placed in contact with the stained sample to create a sample / array sandwich; Place the fluid-containing housing on top of the sample / array sandwich; An analytical solution containing active RNase H and active polymerase is introduced into the fluid-containing housing, thereby bringing the analytical solution into contact with the sample / array sandwich. The sample / array sandwich is incubated in the sample solution at a temperature and time sufficient to allow RNase H activity to at least partially digest the hybrid RNA / DNA tag, in order to produce a lysed tag and thereby release the lysed tag into the analytical solution near the capture feature; The cleaved tag is allowed to interact with the capture oligonucleotide (CO) on the nearby capture feature to provide a tag for the capture cleavage; The sample / array sandwich is cultured in the sample solution at a temperature and time sufficient to allow polymerase activity to extend the captured lysed tag using the CO as a template, in order to produce an extension product. After sufficient extension, the extension product is cleaved with RNase H in the sample solution to generate complementary RNA / DNA regions based on the RNA bases in the CO, thereby releasing the full-length extension product. Collect at least a portion of the released full-length extended product; as well as Amplify and / or sequence at least one of the released full-length extension products.

96. A computer-readable medium or digital resource or digital database containing spatial location information of the features of a spatially encoded array according to any one of claims 58 to 80.

97. The computer-readable medium or digital resource or digital database of claim 96, which contains spatial location information of substantially all of the features of the spatial coding array.

98. Use of a computer-readable medium or digital resource or digital database according to claim 96 or 97 for providing a user with location information of one or more targets related to spatial location information of a spatially coded array.

99. The use according to claim 98, wherein the correlation is generated by using the spatial coding array in a workflow or method as described or illustrated herein.

100. A kit comprising one or more of the following: Two or more hairpin-tagged nucleic acid molecules according to any one of claims 1 to 13; Two or more DNA / RNA hybridization hairpin-tagged nucleic acid molecules as described in claim 14 or claim 15; A group of two or more hair clip tags as described in any one of claims 16 to 18; A group of two or more marked probes according to any one of claims 19 to 28; Two or more capture oligonucleotide (CO) nucleic acid molecules according to any one of claims 30 to 46; Two or more DNA / RNA chimeric CO nucleic acid molecules as described in claim 47 or claim 48; The group consisting of two or more CO nucleic acid molecules according to any one of claims 30 to 45; The group consisting of two or more chimeric CO nucleic acid molecules according to any one of claims 46 to 48; Two or more linked 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-ordered spatial coding capture array according to any one of claims 76 to 80; or The spatially encoded surface according to claim 94.

101. The kit of claim 100, wherein the spatial coding capture array, the semi-ordered spatial coding capture array, or the spatial coding surface is in the format of a flexible sticker.

102. The kit according to claim 100, further comprising one or more of the following: It contains a container with functional RNase H; It contains containers for functional polymerases; It contains containers that contain functional restriction enzymes; A container containing one or a mixture of ribonucleoside triphosphate (rNTP) and / or deoxyribonucleotide triphosphate (dNTP); It contains Mg 2+ A container for a solution of ions; or Containers containing buffer solutions.

103. The kit of claim 101, wherein at least one of the containers contains at least two of the following: the RNase H, the polymerase, the restriction endonuclease, the rNTP and / or dNTP, and the Mg... 2+ Ions or the buffer solution.

104. The kit according to any one of claims 100 to 103, further comprising one or more of the following: It can be used to prepare components of samples for analysis using the methods provided herein; A cover containing a solution, suitable for placement above a sample-array sandwich on a glass slide, to form a fluid-containing shell for the sample-array sandwich; or Flow cell cover, glass slide, or other surface suitable for containing substantially two-dimensional samples.

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