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WO2024196728A2 - Sondes d'acide nucléique pour séquençage et analyse spatiale combinés - Google Patents

Sondes d'acide nucléique pour séquençage et analyse spatiale combinés Download PDF

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Publication number
WO2024196728A2
WO2024196728A2 PCT/US2024/020073 US2024020073W WO2024196728A2 WO 2024196728 A2 WO2024196728 A2 WO 2024196728A2 US 2024020073 W US2024020073 W US 2024020073W WO 2024196728 A2 WO2024196728 A2 WO 2024196728A2
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nucleic acid
nucleotides
domain
target
acid sequence
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WO2024196728A3 (fr
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Margaret HOANG
Joseph M. Beechem
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NS Wind Down Co Inc
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Nanostring Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • BACKGROUND Although there are currently a variety of methods for detecting nucleic acid sequences in a biological sample, a need remains for improved, accurate, rapid, and sensitive multiplexed detection, identification, and quantification of target nucleic acids within a biological sample that has maintained its original morphology. Specifically, there is a need for the ability to: a) detect the in situ abundance and spatial location of certain target nucleic acid sequences present within tissue samples; and b) subsequently identify the sequence of the nucleic acid molecule that contains said target nucleic acid sequence.
  • the present disclosure provides, for the first time, compositions and methods that allow for combined sequencing analysis and spatial abundance analysis of target nucleic acids located within tissue samples.
  • the present disclosure provides a nucleic acid probe comprising: a target binding domain and a barcode domain, wherein the target binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid, wherein the target binding domain comprises D-DNA, wherein the target binding domain further comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, and wherein the barcode domain is a single-stranded polynucleotide comprising at least about one attachment region, wherein each attachment region comprises about one attachment sequence, and wherein the sequences of each of the attachment sequences are different, and wherein the barcode domain comprises L-DNA.
  • nucleic acid probe comprising: a single-stranded target binding domain and a single-stranded identifier oligonucleotide; wherein the single- stranded target binding domain comprises: a reversible terminator at one terminus and a photocleavable linker at the other terminus; and a sequence that binds to a target nucleic acid sequence, wherein single-stranded target binding domain and the single-stranded identifier oligonucleotide are linked together via the cleavable linker, wherein the reversible terminator comprises a photocleavable moiety such that when the reversible terminator is excited by light of a sufficient wavelength, the photocleavable moiety is cleaved, thereby rendering a terminal 3′-OH moiety accessible to a polymerase, wherein the single-stranded identifier oligonucleotide comprises at least one identifier sequence that is specific
  • the reversible terminator comprises a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase, preferably wherein the cleavable moiety is a photocleavable moiety, a chemically-cleavable moiety or an enzymatically-cleavable moiety.
  • the reversible terminator is selected from:
  • the photocleavable linker is selected from: [0009] In some embodiments, each of the attachment sequences is about 14 nucleotides in length. [0010] In some embodiments, the target binding domain is about 35 to about 40 nucleotides in length. In some embodiments, the target binding domain consists of D-DNA and the barcode domain consists of L-DNA. [0011] In some embodiments, the barcode domain or the identifier oligonucleotide of the nucleic acid probes comprises at least two, or at least three, or at least four attachment positions, and optionally wherein the barcode domain or the identifier oligonucleotide is about 56 nucleotides in length.
  • the probe further comprises a reporter probe comprising at least one detectable label hybridized to at least one attachment region of the barcode domain or the identifier oligonucleotide.
  • the reporter probe comprises: a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, and wherein each of the tertiary nucleic acid molecules comprise at least one detectable
  • At least one of the primary nucleic acid molecule, the secondary nucleic acid molecules, and/or the tertiary nucleic acid molecules comprises L-DNA.
  • a plurality of the nucleic acid probes of any one of the above aspects or embodiments wherein the plurality of nucleic acid probes comprises at least two species of nucleic acid probes, wherein the two species of nucleic acid probes comprise unique target binding domains that bind to different target nucleic acid sequences, thereby allowing for the spatial detection of at least two target nucleic acid sequences in the biological sample.
  • the plurality comprises a plurality of each different species of nucleic acid probe.
  • a method comprising: a1) contacting the biological sample with a plurality of nucleic acid probes such that the nucleic acid probes bind to one or more copies of a target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; wherein the nucleic acid probes comprise: a target binding domain that binds to the target nucleic acid sequence; and a barcode domain specific for the at least one target nucleic acid sequence, wherein the barcode domain comprises at least one attachment position; b1) contacting the biological sample with a plurality of reporter probes, thereby binding a reporter probe to an attachment region of the barcode domain of nucleic acid probes bound to the target nucleic acid sequence, wherein each reporter probe comprises at least one detectable label and at least one photocleavable linker; c1) recording the identity and spatial position of the detectable labels of the bound reporter probes; d1) illuminating a location of the biological sample with light of a
  • the method further comprises determining the abundance and/or spatial position of the one or more copies of the target nucleic acid sequence in the biological sample based on the detectable labels that were recorded in step (c1).
  • the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions, and the method further comprises, after step (d1): (d2) repeating steps (b1) – (d1) until each attachment position in the barcode domains of the nucleic acid probes bound to the target nucleic acid sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein determining the abundance and spatial position of the target nucleic acid sequence in the biological sample comprises determining the abundance and/or spatial position of the nucleic acid sequence based on the sequence in which the detectable labels were recorded.
  • the nucleic acid probes comprise: the target binding domain and the barcode domain, wherein the target binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid, wherein the target binding domain comprises D-DNA, wherein the target binding domain further comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, and wherein the barcode domain is a single-stranded polynucleotide comprising at least about one attachment region, wherein each attachment region comprises about one attachment sequence, wherein the sequences of each of the attachment sequences are different, and wherein the barcode domain comprises L-DNA.
  • a method comprising: (a) contacting a biological sample with a plurality of nucleic acid probes such that the nucleic acid probes bind to one or more copies of the target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; (b) illuminating a location of the biological sample with light of a sufficient wavelength to cleave the reversible terminators and photocleavable linkers of the nucleic acid probes bound to one or more copies of the target nucleic acid sequence in that location, thereby: releasing the identifier oligonucleotides of the nucleic acid probes bound to the one or more copies of the target nucleic acid sequence in that location, and rendering terminal 3′-OH moieties of the target binding domains bound to the one or more copies of the target nucleic acid sequence in that location accessible to a polymerase; (c) collecting the released identifier oligonucleotides; (d) spatially
  • sequencing the amplification products in step (d)(ii) comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • the nucleic acid probes comprise: a single-stranded target binding domain and a single-stranded identifier oligonucleotide; wherein the single-stranded target binding domain comprises: a reversible terminator at one terminus and a photocleavable linker at the other terminus; and a sequence that binds to a target nucleic acid sequence, wherein single-stranded target binding domain and the single-stranded identifier oligonucleotide are linked together via the cleavable linker, wherein the reversible terminator comprises a photocleavable moiety such that when the reversible terminator is excited by light of a sufficient wavelength, the photocleavable moiety is cleaved, thereby rendering a terminal
  • performing a sequencing reaction comprises: i) purifying the nucleic acid molecules that are bound to the target binding domains; ii) performing a template- switching reverse transcription reaction using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; and iii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • performing a sequencing reaction comprises: i) performing a template-switching reverse transcription reaction in situ within the biological sample using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; and ii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • sequencing the cDNA molecules comprises performing next- generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • the target nucleic acid sequence is in proximity to a splice junction.
  • FIG.1 is a schematic diagram of an exemplary nucleic acid probe of the present disclosure comprising a reversible terminator.
  • FIG.2A – FIG.2B are schematic diagrams of an exemplary nucleic acid probe of the present disclosure comprising a reversible terminator, a target binding domain and a barcode domain (FIG.2A) or comprising a reversible terminator and a cleavable linker located between the target binding domain and the identifier oligonucleotide (FIG.2B).
  • FIGs.3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I are exemplary schematics of the steps of a method of detecting the abundance and spatial location target nucleic acid sequences and sequencing the molecules that contain said target nucleic acid sequences.
  • FIG.4 is a schematic diagram of an exemplary reporter probe of the present disclosure.
  • FIGs.5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 5I are exemplary schematics of the steps of a method of detecting the abundance and spatial location target nucleic acid sequences and sequencing the molecules that contain said target nucleic acid sequences.
  • the present disclosure relates to nucleic acid probes, compositions, methods, and kits for simultaneous, multiplexed, spatial detection and quantification of target nucleic acid sequences in a biological sample, in combination with subsequent sequencing of the nucleic acid molecules that contain said target nucleic acid sequences.
  • the compositions and methods of the present disclosure provide a comparison of the identity and abundance of target nucleic acid sequences present in a first region of interest (e.g., tissue type, a cell (including normal and abnormal cells), and a subcellular structure within a cell) and the identity and abundance of target nucleic acid sequences present in a second region of interest.
  • a first region of interest e.g., tissue type, a cell (including normal and abnormal cells), and a subcellular structure within a cell
  • the upper limit relates to the size of the region of interest relative to the size of the sample.
  • the upper limit relates to the size of the region of interest relative to the size of the sample.
  • a section may have hundreds to thousands of regions of interest; however, if a tissue section includes only two cell types, then the section may have only two regions of interest (each including only one cell type).
  • the present disclosure provides a higher degree of multiplexing than is possible with standard immunohistochemical or in situ hybridization methods. In situ hybridization methods are generally limited to simultaneous detection of fewer than ten nucleic acid targets. The present disclosure provides spatial detection of large combinations of nucleic acid targets.
  • compositions and methods of the present disclosure provide an increase in objective measurements by digital quantification and increased reliability and consistency.
  • the compositions and methods of the present disclosure also allow for subsequent sequencing analysis of the target nucleic acids. That is, a specific target nucleic acid sequence can first be detected within the tissue sample through binding of a probe of the present disclosure in order to determine the spatial position and abundance of said target nucleic acid, and then said probe can be subsequently used to sequence the nucleic acid molecule that contains said target nucleic acid sequence, thereby providing sequence-level information about said nucleic acid molecule.
  • compositions and methods described herein can be multiplexed to interrogate the spatial abundance and sequence of large combinations of target nucleic acids.
  • the ability to determine the spatial abundance as well as sequence information is particularly advantageous to study specific populations of target nucleic acids within a cell.
  • TCR T-cell Receptors
  • SNVs single nucleotide variant
  • CNVs Copy Number Variants
  • the nucleic acid probes of the present disclosure comprise a reversible terminator at one terminus that allows for their dual use in detecting the spatial abundance of a target nucleic acid sequence and the subsequent sequencing of the nucleic acid molecule containing said target nucleic acid sequence.
  • a reversible terminator is used herein to refer to a chemical modification located at a terminus of a nucleic acid probe that comprises a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase.
  • the reversable terminator is cleaved to result in a natural base at the site of the cleavage. In some embodiments, the reversable terminator is cleaved by a chemical reaction. In some embodiments, the reversable terminator is cleaved by an enzymatic reaction. In some embodiments, the reversable terminator comprises a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase. Prior to cleavage of the reversible terminator, the reversible terminator renders the terminus of the nucleic acid probe to which it is attached inaccessible to enzymes.
  • FIG.1 shows a schematic of an exemplary nucleic acid probe comprising a reversible terminator.
  • a sufficient stimulus e.g., light, chemical, enzyme
  • the 3′ terminus of the probe is inaccessible to enzymes such as polymerases.
  • a reversible terminator can be selected from any of the reversible terminators described in Chen, F., Dong, M., Ge, M., Zhu, L., Ren, L., Liu, G., & Mu, R. (2013) Genomics, proteomics & bioinformatics, 11(1), 34–40, which is incorporated herein by reference in its entirety for all purposes.
  • the reversible terminator is a blocking group that is covalently bound to the 3′-OH of a terminal nucleotide.
  • reversible terminators include, but are not limited to a 3′-ONH2 reversible terminator, a 3′-O-allyl reversible terminator, and 3′-o-azidomethyl reversible terminator (see Chen et al.2013, which is incorporated herein by reference in its entirety for all purposes).
  • a reversible terminator is a virtual terminator nucleotide as put forth in Bowers J, Mitchell J, Beer E, Buzby PR, Causey M, Efcavitch JW, Jarosz M, Krzymanska- Olejnik E, Kung L, Lipson D, Lowman GM, Marappan S, McInerney P, Platt A, Roy A, Siddiqi SM, Steinmann K, Thompson JF. Virtual terminator nucleotides for next-generation DNA sequencing. Nat Methods.2009 Aug;6(8):593-5, which is incorporated herein by reference in its entirety for all purposes.
  • a virtual terminator nucleotide can have the formula: wherein R and R1 are selected from the combinations put forth in Table 1. Table 1
  • a reversible terminator can be any of the reversible terminators put forth in Wu W, Stupi BP, Litosh VA, Mansouri D, Farley D, Morris S, Metzker S, Metzker ML. Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′- deoxyadenosine triphosphates. Nucleic Acids Res.2007;35(19):6339-49, which is incorporated herein by reference in its entirety for all purposes. [0049] In some aspects, a reversible terminator can be selected from:
  • a reversible terminator can be any of the reversible terminators put forth in Gardner, A. F., Wang, J., Wu, W., Karouby, J., Li, H., Stupi, B. P., Jack, W. E., Hersh, M. N., & Metzker, M. L. (2012). Rapid incorporation kinetics and improved fidelity of a novel class of 3′-OH unblocked reversible terminators. Nucleic acids research, 40(15), 7404–7415, which is incorporated herein by reference in its entirety for all purposes. [0051] In some aspects, a reversible terminator can be selected from:
  • a reversible terminator can be any of the reversible terminators put forth in Stupi, B. P., Li, H., Wang, J., Wu, W., Morris, S. E., Litosh, V. A., Muniz, J., Hersh, M. N., & Metzker, M. L. (2012). Stereochemistry of benzylic carbon substitution coupled with ring modification of 2-nitrobenzyl groups as key determinants for fast-cleaving reversible terminators. Angewandte Chemie (International ed. in English), 51(7), 1724–1727, which is incorporated herein by reference in its entirety for all purposes. [0053] In some aspects, a reversible terminator can be selected from:
  • a reversible terminator can be selected from any of the reversible terminators put forth in Litosh, V. A., Wu, W., Stupi, B. P., Wang, J., Morris, S. E., Hersh, M. N., & Metzker, M. L. (2011). Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates. Nucleic acids research, 39(6), e39, which is incorporated herein by reference in its entirety. [0055] In some aspects, a reversible terminator can be selected from:
  • any of the above reversible terminators that are shown covalently linked to a specific nucleobase can be adapted, using standard methods known in the art, to be covalently linked to any other nucleobase.
  • nucleic Acid Probes I. Probe Design A the nucleic acid probes of the present disclosure comprise: i) a target binding domain; and ii) a barcode domain. In some aspects, the target binding domain is operably linked to the barcode domain.
  • a target binding domain can be a single-stranded, a double-stranded, or a partially double-stranded nucleic acid molecule. In some aspects, a target binding domain is a single-stranded nucleic acid molecule.
  • the target binding domain can comprise a nucleic acid sequence that is complementary to the target nucleic acid sequence that is specific to that probe (i.e., the target nucleic acid sequence that is to be bound by and measured and/or sequenced using said probe). [0061] In some aspects, the entire target binding domain can be complementary to the target nucleic acid.
  • a target binding domain can comprise at least one portion that is complementary to a target nucleic acid sequence and at least one portion that is not complementary to a target nucleic acid sequence.
  • a portion that is not complementary to a target nucleic acid sequence can be located 5′ to a portion that is complementary to a target nucleic acid sequence.
  • a portion that is not complementary to a target nucleic acid sequence can be located 3′ to a portion that is complementary to a target nucleic acid sequence.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one amplification primer binding site.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one modified nucleotide, wherein the modified nucleotide comprises at least one biotin moiety. Without wishing to be bound by theory, the at least one biotin moiety can be used in subsequent affinity purification steps.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one chemical moiety that prevents primer extension using the target binding domain.
  • the at least one chemical moiety that prevents primer extension using the target binding domain can be a cleavable chemical moiety such that when the chemical moiety is cleaved (e.g., enzymatically, chemically or via photocleavage), primer extension using the target binding domain is possible.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one capture sequence that is suitable for use as a capture sequence in a hybridization-capture purification protocol. That is, the capture sequence can be complementary to one or more specific capture probes that are to be used in the hybrid capture purification protocol.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one spacer sequence.
  • a target binding domain can be at least about 5 nucleotides, or at least about 10 nucleotides, or at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 25 nucleotides, or at least about 30 nucleotides, or at least about 35 nucleotides, or at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 50 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 65 nucleotides, or at least about 70 nucleotides, or at least about 75 nucleotides, or at least about 80 nucleotides, or at least about 85 nucleotides, or at least about 90 nucleotides, or at least about 95
  • a target binding domain can be at least 5 nucleotides, or at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 35 nucleotides, or at least 40 nucleotides, or at least 45 nucleotides, or at least 50 nucleotides, or at least 55 nucleotides, or at least 60 nucleotides, or at least 65 nucleotides, or at least 70 nucleotides, or at least 75 nucleotides, or at least 80 nucleotides, or at least 85 nucleotides, or at least 90 nucleotides, or at least 95 nucleotides, or at least 100 nucleotides in length.
  • a target binding domain can be about 5 nucleotides, or about 10 nucleotides, or about 15 nucleotides, or about 20 nucleotides, or about 25 nucleotides, or about 30 nucleotides, or about 35 nucleotides, or about 40 nucleotides, or about 45 nucleotides, or about 50 nucleotides, or about 55 nucleotides, or about 60 nucleotides, or about 65 nucleotides, or about 70 nucleotides, or about 75 nucleotides, or about 80 nucleotides, or about 85 nucleotides, or about 90 nucleotides, or about 95 nucleotides, or about 100 nucleotides.
  • a target binding domain can be about 35 nucleotides in length.
  • a target binding domain can be 5 nucleotides, or 10 nucleotides, or 15 nucleotides, or 20 nucleotides, or 25 nucleotides, or 30 nucleotides, or 35 nucleotides, or 40 nucleotides, or 45 nucleotides, or 50 nucleotides, or 55 nucleotides, or 60 nucleotides, or 65 nucleotides, or 70 nucleotides, or 75 nucleotides, or 80 nucleotides, or 85 nucleotides, or 90 nucleotides, or 95 nucleotides, or 100 nucleotides.
  • a target binding domain can be 35 nucleotides in length. [0072] In some aspects, a target binding domain in be at least about 35 nucleotides in length to at least about 40 nucleotides in length. In some aspects, a target binding domain can be about 35 nucleotides to about 40 nucleotides in length.
  • a target binding domain can comprise about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides, or about 26 nucleotides, or about 27 nucleotides, or about 28 nucleotides, or about 29 nucleotides, or about 30 nucleotides, or about 31 nucleotides, or about 32 nucleotides, or about 33 nucleotides, or about 34 nucleotides, or about 35 nucleotides, or about 36 nucleotides, or about 37 nucleotides, or about 38 nucleotides, or about 39 nucleotides, or about 40 nucleotides, or about 41 nucleotides, or about 42 nucleotides, or about 43 nucleotides, or about 45 nucleotides in length.
  • a target binding domain comprises D-DNA. In some aspects, a target binding domain consists of D-DNA. [0074] In some aspects, a target binding domain can be about 35 nucleotides to about 40 nucleotides in length and comprises D-DNA. In some aspects, a target binding domain can be about 35 nucleotides to about 40 nucleotides in length and consists of D-DNA. [0075] In some aspects, a target binding domain can comprise a reversible terminator at one terminus and is connected to the barcode domain at the other terminus. Barcode Domain [0076] In some aspects, a barcode domain can be a single stranded polynucleotide.
  • a barcode domain can comprise at least one attachment region.
  • a barcode domain can comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions.
  • a barcode domain can comprise about 4 attachment regions.
  • a barcode domain can comprise about 3 attachment regions.
  • An attachment region can comprise at least one nucleic acid sequence that is capable of being reversibly bound by a reporter probe of the present disclosure.
  • a nucleic acid sequence that is capable of being reversibly bound by a reporter probe of the present disclosure is herein referred to as an attachment sequence.
  • an attachment region of a barcode domain can comprise at least one attachment sequence.
  • the attachment sequences within a single attachment region can be identical; thus, the reporter probes that bind within that single attachment region will be identical.
  • the attachment sequences within a single attachment can be different; thus, the reporter probes that bind within that single attachment will be different.
  • the attachment sequences in each of the different attachment regions can be different; thus, different reporter probes will bind to each attachment region in the barcode domain.
  • an attachment sequence can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length. In some aspects, an attachment sequence can be about 14 nucleotides in length.
  • a barcode domain comprises L-DNA. In some aspects, a barcode domain consists of L-DNA. [0083] In some aspects, a barcode domain can comprise about 4 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 56 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain comprises L-DNA.
  • a barcode domain can comprise about 4 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 56 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain consists of L-DNA.
  • a barcode domain can comprise about 3 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 42 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain comprises L-DNA.
  • a barcode domain can comprise about 3 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 42 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain consists of L-DNA.
  • a barcode domain can be used to identify the target analyte bound to the target binding domain.
  • the barcode domain comprises a unique nucleic acid sequence (apportioned into attachment regions, as described above) that identifies the target analyte bound to the target binding domain of the probe.
  • a probe with a target binding domain that binds to the protein p53 comprises a barcode domain with specific combination of attachment regions such that the sequence of the barcode domain corresponds to p53, while a probe with a target binding domain that binds to the protein p97 comprises a barcode domain with a specific combination of attachment regions such that the sequence of the barcode domain corresponds to p97.
  • a barcode domain can comprise at least one amplification primer binding site.
  • a barcode domain can comprise at least two amplification primer binding sites.
  • An amplification primer binding site is a nucleic acid sequence capable of binding to an amplification primer.
  • An amplification primer can be used to amplify the nucleic molecule to which it is bound using methods known in the art, including, but not limited to, polymerase chain reaction (PCR).
  • An amplification primer can also be used to amplify the nucleic acid molecule to which it is bound as part of a method of producing a sequencing library.
  • a barcode domain can comprise a sequence that is unique to a specific cell within a biological sample, allowing identification of said cell.
  • a barcode domain can comprise a sequence that is unique to a specific region of interest within a biological sample, allowing identification of said region of interest.
  • a barcode domain comprises a specific nucleic acid sequence that is a priori assigned to the specific target analyte bound to the target binding to which the barcode domain is attached.
  • a probe designated as “probe X” designed to spatially detect “target analyte X” comprises a target binding domain designated “target binding domain X” linked to a barcode domain designated “barcode domain X”.
  • Target binding domain X binds to target analyte X
  • barcode domain X comprises a nucleic acid sequence, designated as “nucleic acid sequence X”, which corresponds to target analyte X.
  • a barcode domain can comprise at least one unique molecular identifier.
  • a unique molecular identifier is a sequence that can be used to perform error correction during sequencing analysis.
  • a barcode domain can be a single-stranded, a double-stranded, or a partially double-stranded nucleic acid molecule. In some aspects, a barcode domain is a single-stranded nucleic acid molecule.
  • a barcode domain can be at least about 5 nucleotides, or at least about 10 nucleotides, or at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 25 nucleotides, or at least about 30 nucleotides, or at least about 35 nucleotides, or at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 50 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 65 nucleotides, or at least about 70 nucleotides, or at least about 75 nucleotides, or at least about 80 nucleotides, or at least about 85 nucleotides, or at least about 90 nucleotides, or at least about 95 nucleotides, or at least about 100 nucleotides in length.
  • a barcode domain can be at least 5 nucleotides, or at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 35 nucleotides, or at least 40 nucleotides, or at least 45 nucleotides, or at least 50 nucleotides, or at least 55 nucleotides, or at least 60 nucleotides, or at least 65 nucleotides, or at least 70 nucleotides, or at least 75 nucleotides, or at least 80 nucleotides, or at least 85 nucleotides, or at least 90 nucleotides, or at least 95 nucleotides, or at least 100 nucleotides in length.
  • a barcode domain can be about 5 nucleotides, or about 10 nucleotides, or about 15 nucleotides, or about 20 nucleotides, or about 25 nucleotides, or about 30 nucleotides, or about 35 nucleotides, or about 40 nucleotides, or about 45 nucleotides, or about 50 nucleotides, or about 55 nucleotides, or about 56 nucleotides, or about 60 nucleotides, or about 65 nucleotides, or about 66 nucleotides, or about 70 nucleotides, or about 75 nucleotides, or about 80 nucleotides, or about 85 nucleotides, or about 90 nucleotides, or about 95 nucleotides, or about 100 nucleotides.
  • a barcode domain can be about 56 nucleotides in length. In some aspects, a barcode domain can be about 42 nucleotides in length. [0095] In some aspects, a barcode domain can be 5 nucleotides, or 10 nucleotides, or 15 nucleotides, or 20 nucleotides, or 25 nucleotides, or 30 nucleotides, or 35 nucleotides, or 40 nucleotides, or 45 nucleotides, or 50 nucleotides, or 55 nucleotides, or 56 nucleotides, or 60 nucleotides, or 65 nucleotides, or 70 nucleotides, or 75 nucleotides, or 80 nucleotides, or 85 nucleotides, or 90 nucleotides, or 95 nucleotides, or 100 nucleotides.
  • a barcode domain can be 56 nucleotides in length.
  • the present disclosure provides nucleic acid probes comprising a target binding domain and a barcode domain, wherein the target binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid, wherein the target binding domain is about 35 to about 40 nucleotides in length, wherein the target binding domain comprises D-DNA, wherein the target binding domain further comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, and wherein the barcode domain is a single-stranded polynucleotide comprising about four attachment regions, wherein each attachment region comprises about one attachment sequence, wherein each of the attachment sequences is about 14 nucleotides in length, and wherein the sequences of each of the attachment sequences are different, and wherein the barcode domain comprises L-DNA.
  • the present disclosure provides nucleic acid probes comprising a target binding domain and a barcode domain, wherein the target binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid, wherein the target binding domain is about 35 to about 40 nucleotides in length, wherein the target binding domain consists of D-DNA, wherein the target binding domain further comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, and wherein the barcode domain is a single-stranded polynucleotide comprising about four attachment regions, wherein each attachment region comprises about one attachment sequence, wherein each of the attachment sequences is about 14 nucleotides in length, and wherein the sequences of each of the attachment sequences are different, and wherein the barcode domain consists of L-DNA.
  • the nucleic acid probes of the present disclosure comprise: i) a target binding domain; and ii) an identifier oligonucleotide.
  • the target binding domain and the identifier oligonucleotide are linked together via a cleavable linker.
  • the cleavable linker is cleavable via an enzymatic reaction.
  • the cleavable linker is cleaved via a chemical reaction.
  • the cleavable linker is a photocleavable linker and is cleaved via exposure to light of a specific wavelength.
  • the photocleavable linker comprises a phosphoramidite.
  • the photocleavable linker e.g., phosphoramidite
  • the cleavage reaction results in a 5′-phosphorylated oligonucleotide.
  • the cleavage reaction results in both 3′ and 5′ phosphorylated segments, e.g., a 5′ and a 3′ monophosphate.
  • a 3′ monophosphate can be removed using methods as known in the art, e.g., using T4 polynucleotide kinase (New England Biolabs®).
  • the photocleavable linker can be selected from: ,
  • a target binding domain can be a single-stranded, a double-stranded, or a partially double-stranded nucleic acid molecule. In some aspects, a target binding domain is a single-stranded nucleic acid molecule. [00104] In some aspects, the target binding domain can comprise a nucleic acid sequence that is complementary to the target nucleic acid sequence that is specific to that probe (i.e., the target nucleic acid sequence that is to be bound by and measured and/or sequenced using said probe). [00105] In some aspects, the entire target binding domain can be complementary to the target nucleic acid.
  • a target binding domain can comprise at least one portion that is complementary to a target nucleic acid sequence and at least one portion that is not complementary to a target nucleic acid sequence.
  • a portion that is not complementary to a target nucleic acid sequence can be located 5′ to a portion that is complementary to a target nucleic acid sequence.
  • a portion that is not complementary to a target nucleic acid sequence can be located 3′ to a portion that is complementary to a target nucleic acid sequence.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one amplification primer binding site.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one modified nucleotide, wherein the modified nucleotide comprises at least one biotin moiety. Without wishing to be bound by theory, the at least one biotin moiety can be used in subsequent affinity purification steps.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one chemical moiety that prevents primer extension using the target binding domain.
  • the at least one chemical moiety that prevents primer extension using the target binding domain can be a cleavable chemical moiety such that when the chemical moiety is cleaved (e.g., enzymatically, chemically or via photocleavage), primer extension using the target binding domain is possible.
  • portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one capture sequence that is suitable for use as a capture sequence in a hybridization-capture purification protocol. That is, the capture sequence can be complementary to one or more specific capture probes that are to be used in the hybrid capture purification protocol.
  • a portion of the target binding domain that is not complementary to a target nucleic acid sequence can comprise at least one spacer sequence.
  • a target binding domain can be at least about 5 nucleotides, or at least about 10 nucleotides, or at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 25 nucleotides, or at least about 30 nucleotides, or at least about 35 nucleotides, or at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 50 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 65 nucleotides, or at least about 70 nucleotides, or at least about 75 nucleotides, or at least about 80 nucleotides, or at least about 85 nucleotides, or at least about 90 nucleotides, or at least about 95
  • a target binding domain can be at least 5 nucleotides, or at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 35 nucleotides, or at least 40 nucleotides, or at least 45 nucleotides, or at least 50 nucleotides, or at least 55 nucleotides, or at least 60 nucleotides, or at least 65 nucleotides, or at least 70 nucleotides, or at least 75 nucleotides, or at least 80 nucleotides, or at least 85 nucleotides, or at least 90 nucleotides, or at least 95 nucleotides, or at least 100 nucleotides in length.
  • a target binding domain can be about 5 nucleotides, or about 10 nucleotides, or about 15 nucleotides, or about 20 nucleotides, or about 25 nucleotides, or about 30 nucleotides, or about 35 nucleotides, or about 40 nucleotides, or about 45 nucleotides, or about 50 nucleotides, or about 55 nucleotides, or about 60 nucleotides, or about 65 nucleotides, or about 70 nucleotides, or about 75 nucleotides, or about 80 nucleotides, or about 85 nucleotides, or about 90 nucleotides, or about 95 nucleotides, or about 100 nucleotides.
  • a target binding domain can be about 35 nucleotides in length.
  • a target binding domain can be 5 nucleotides, or 10 nucleotides, or 15 nucleotides, or 20 nucleotides, or 25 nucleotides, or 30 nucleotides, or 35 nucleotides, or 40 nucleotides, or 45 nucleotides, or 50 nucleotides, or 55 nucleotides, or 60 nucleotides, or 65 nucleotides, or 70 nucleotides, or 75 nucleotides, or 80 nucleotides, or 85 nucleotides, or 90 nucleotides, or 95 nucleotides, or 100 nucleotides.
  • a target binding domain can be 35 nucleotides in length.
  • a target binding domain can comprise a reversible terminator at one terminus and a photocleavable linker at the other terminus, wherein the target binding domain and identifier oligonucleotide are linked together via the cleavable linker.
  • Identifier Oligonucleotides [00117] An identifier oligonucleotide is a nucleic acid molecule that identifies the target analyte bound to the target binding domain.
  • the identifier oligonucleotide comprises a unique nucleic acid sequence that identifies the target analyte bound to the target binding domain of the probe, herein referred to as the “identifier sequence”.
  • a probe with a target binding domain that binds to the protein p53 comprises an identifier oligonucleotide with an identifier sequence that corresponds to p53
  • a probe with a target binding domain that binds to the protein p97 comprises an identifier oligonucleotide with an identifier sequence that corresponds to p97.
  • An identifier oligonucleotide can be DNA, RNA, or a combination of DNA and RNA.
  • an identifier oligonucleotide comprises DNA. In some aspects, an identifier oligonucleotide consists of DNA. [00120] In some aspects, an identifier oligonucleotide can comprise at least one amplification primer binding site. In some aspects, an identifier oligonucleotide can comprise at least two amplification primer binding sites.
  • An amplification primer binding site is a nucleic acid sequence capable of binding to an amplification primer.
  • An amplification primer can be used to amplify the nucleic molecule to which it is bound using methods known in the art, including, but not limited to, polymerase chain reaction (PCR).
  • an amplification primer can also be used to amplify the nucleic acid molecule to which it is bound as part of a method of producing a sequencing library.
  • an identifier oligonucleotide can comprise a sequence that is unique to a specific cell within a biological sample, allowing identification of said cell.
  • an identifier oligonucleotide can comprise a sequence that is unique to a specific region of interest within a biological sample, allowing identification of said region of interest.
  • the identifier oligonucleotide comprises a specific nucleic acid sequence that is a priori assigned to the specific target analyte bound to the target binding to which the identifier oligonucleotide is attached.
  • a probe designated as “probe X” designed to spatially detect “target analyte X” comprises a target binding domain designated “target binding domain X” linked to an identifier oligonucleotide designated “identifier oligonucleotide X”.
  • Target binding domain X binds to target analyte X
  • identifier oligonucleotide X comprises a nucleic acid sequence, designated as “nucleic acid sequence X”, which corresponds to target analyte X.
  • the amount, or number of sequencing reads, of nucleic acid sequence X can be used to determine the quantity, in absolute or relative terms, the amount of target analyte X within the region of interest.
  • a specific nucleic acid sequence that is specific to the target analyte bound to the target binding domain can be about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 nucleotides in length.
  • a specific nucleic acid sequence that is specific to the target analyte bound to the target binding domain can be about 12 nucleotides in length.
  • an identifier oligonucleotide can comprise at least one unique molecular identifier.
  • a unique molecular identifier is a sequence that can be used to perform error correction during sequencing analysis.
  • a unique molecule identifier can be about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 nucleotides in length.
  • a unique molecular identifier can be about 14 nucleotides in length.
  • an identifier oligonucleotide can be a single-stranded, a double- stranded, or a partially double-stranded nucleic acid molecule. In some aspects, an identifier oligonucleotide is a single-stranded nucleic acid molecule.
  • an identifier oligonucleotide can be at least about 5 nucleotides, or at least about 10 nucleotides, or at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 25 nucleotides, or at least about 30 nucleotides, or at least about 35 nucleotides, or at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 50 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 65 nucleotides, or at least about 70 nucleotides, or at least about 75 nucleotides, or at least about 80 nucleotides, or at least about 85 nucleotides, or at least about 90 nucleotides, or at least about 95 nucleotides, or at least about 100 nucleotides in length.
  • an identifier oligonucleotide can be at least 5 nucleotides, or at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 35 nucleotides, or at least 40 nucleotides, or at least 45 nucleotides, or at least 50 nucleotides, or at least 55 nucleotides, or at least 60 nucleotides, or at least 65 nucleotides, or at least 70 nucleotides, or at least 75 nucleotides, or at least 80 nucleotides, or at least 85 nucleotides, or at least 90 nucleotides, or at least 95 nucleotides, or at least 100 nucleotides in length.
  • an identifier oligonucleotide can be about 5 nucleotides, or about 10 nucleotides, or about 15 nucleotides, or about 20 nucleotides, or about 25 nucleotides, or about 30 nucleotides, or about 35 nucleotides, or about 40 nucleotides, or about 45 nucleotides, or about 50 nucleotides, or about 55 nucleotides, or about 60 nucleotides, or about 65 nucleotides, or about 66 nucleotides, or about 70 nucleotides, or about 75 nucleotides, or about 80 nucleotides, or about 85 nucleotides, or about 90 nucleotides, or about 95 nucleotides, or about 100 nucleotides.
  • an identifier oligonucleotide can be about 66 nucleotides in length.
  • an identifier oligonucleotide can be 5 nucleotides, or 10 nucleotides, or 15 nucleotides, or 20 nucleotides, or 25 nucleotides, or 30 nucleotides, or 35 nucleotides, or 40 nucleotides, or 45 nucleotides, or 50 nucleotides, or 55 nucleotides, or 60 nucleotides, or 65 nucleotides, or 66 nucleotides, or 70 nucleotides, or 75 nucleotides, or 80 nucleotides, or 85 nucleotides, or 90 nucleotides, or 95 nucleotides, or 100 nucleotides.
  • an identifier oligonucleotide can be 66 nucleotides in length.
  • Exemplary Nucleic Acid Probes [00130]
  • the present disclosure provides nucleic acid probes comprising: a target binding domain and an identifier oligonucleotide; wherein the target binding domain comprises: a reversible terminator at one terminus and a cleavable linker at the other terminus; and a sequence that binds to a target nucleic acid sequence, wherein the target binding domain and the identifier oligonucleotide are linked together via the cleavable linker, wherein the identifier oligonucleotide comprises at least one identifier sequence that is specific to the target binding domain of the probe.
  • Some embodiments include wherein the reversible terminator comprises a photocleavable moiety such that when the reversible terminator is excited by light of a sufficient wavelength, the photocleavable moiety is cleaved, thereby rendering a terminal 3′- OH moiety accessible to a polymerase.
  • nucleic acid probes comprising: a single-stranded target binding domain and a single-stranded identifier oligonucleotide; wherein the single-stranded target binding domain comprises: a reversible terminator at one terminus and a cleavable linker at the other terminus; and a sequence that binds to a target nucleic acid sequence, wherein the single-stranded target binding domain and the single stranded identifier oligonucleotide are linked together via the cleavable linker, wherein the identifier oligonucleotide comprises at least one identifier sequence that is specific to the target binding domain of the probe.
  • Some embodiments include wherein the reversible terminator comprises a photocleavable moiety such that when the reversible terminator is excited by light of a sufficient wavelength, the photocleavable moiety is cleaved, thereby rendering a terminal 3′-OH moiety accessible to a polymerase.
  • FIG.2B An exemplary schematic of the preceding probes is shown in FIG.2B.
  • the present disclosure provides pluralities the nucleic acid probes of the present disclosure, wherein the plurality of nucleic acid probes comprises at least two different species of nucleic acid probes, wherein the sequence of the target binding domain of each species is distinct, such that the single-stranded target binding domain of each species binds to a distinct target nucleic acid sequence, wherein the barcode domain of a single species comprises a nucleic acid sequence that is specific to the target binding domain of that single species, and wherein the barcode domains of different species are distinct.
  • the present disclosure provides pluralities of the nucleic acid probes of the present disclosure, wherein the plurality of nucleic acid probes comprises at least two different species of nucleic acid probes, wherein the sequence of the target binding domain of each species is distinct, such that the single-stranded target binding domain of each species binds to a distinct target nucleic acid sequence, wherein the identifier oligonucleotide of a single species comprises at least one identifier sequence that is specific to the target binding domain of that single species, and wherein the at least one identifier sequences of different species are distinct.
  • a plurality of nucleic acid probes can comprise at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000, or at least about 100,000, or at least about 1,000,000 different species of nucleic acid probes.
  • a plurality of nucleic acid probes can comprise at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or at least 160, or at least 170, or at least 180, or at least 190, or at least 200, or at least 210, or at least 220, or at least 240, or at least 250, or at least 260, or at least 270, or at least 280, or at least 290, or at least 300, or at least 500, or at least 1,000, or at least 10,000, or at least 100,000, or at least 1,000,000 different species of nucleic acid probes.
  • the pluralities of nucleic acid probes can comprise a plurality of each different species of nucleic acid probe.
  • Reporter probes of the present disclosure [00137] The present disclosure provides reporter probes for use in the methods of the present disclosure. The reporter probes of the present disclosure bind to the attachment sequences within the attachment regions of the barcode domains of the nucleic acid probes of the present disclosure. The reporter probes comprise at least one detectable label, e.g., a fluorescent moiety, that allows them to be detected in the methods of the present disclosure. [00138] A reporter probe can comprise at least two domains, wherein the first domain hybridizes to an attachment sequence and the second domain comprises at least one detectable label.
  • a reporter probe can comprise at least about 10, or at least about 15, or at least about 20, or at least about 25, or at least about 30, or at least about 35, or at least about 40, or at least about 45, or at least about 50 detectable labels. In some aspects, a reporter probe can comprise about 10, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40, or about 45, or about 50 detectable labels. [00140] In some aspects, a reporter probe can be pre-assembled prior to being contacted with a biological sample. [00141] In some aspects, a reporter probe can comprise a primary nucleic acid molecule. A primary nucleic acid molecule can be a single-stranded polynucleotide.
  • a primary nucleic acid molecule can comprise L-DNA. In some aspects, a primary nucleic acid molecule can consist of L-DNA. [00142] A primary nucleic acid molecule can comprise at least two domains. In some aspects, the first domain of a primary nucleic acid molecule can hybridize to an attachment sequence in an attachment region of a barcode domain of a nucleic acid probe of the present disclosure. In some aspects, the second domain of a primary nucleic acid molecule comprises at least one detectable label. [00143] In some aspects, the second domain of a primary nucleic acid molecule can hybridize to at least one secondary nucleic acid molecule.
  • a primary nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten secondary nucleic acid molecules. In some aspects, a primary nucleic acid molecule can hybridize to about 6 secondary nucleic acid molecules. [00144] In some aspects, a primary nucleic acid molecule can further comprise a cleavable linker moiety. In some aspects, the cleavable linker moiety can be located between the first domain and the second domain, such that when the cleavable linker moiety is cleaved, the first domain and the second domain are separated.
  • the cleavable linker is a photocleavable linker.
  • the first domain of a primary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length.
  • the first domain of a primary nucleic acid molecule can be about 14 nucleotides in length.
  • the second domain of a primary nucleic acid molecule can be about 75 nucleotides, or about 76 nucleotides, or about 77 nucleotides, or about 78 nucleotides, or about 79 nucleotides, or about 80 nucleotides, or about 81 nucleotides, or about 82 nucleotides, or about 83 nucleotides, or about 84 nucleotides, or about 85 nucleotides, or about 86 nucleotides, or about 87 nucleotides, or about 88 nucleotides, or about 89 nucleotides, or about 90 nucleotides in length.
  • the second domain of a primary nucleic acid molecule can be about 84 nucleotides in length.
  • a primary nucleic acid molecule can be about 90 nucleotides, or about 91 nucleotides, or about 92 nucleotides, or about 93 nucleotides, or about 94 nucleotides, or about 95 nucleotides, or about 96 nucleotides, or about 97 nucleotides, or about 98 nucleotides, or about 99 nucleotides, or about 100 nucleotides, or about 101 nucleotides, or about 102 nucleotides, or about 103 nucleotides, or about 104 nucleotides, or about 105 nucleotides, or about 106 nucleotides, or about 107 nucleotides, or about 108 nucleotides, or about 109 nucleotides, or about 110 nucleotides in length.
  • a primary nucleic acid can be about 98 nucleotides in length.
  • a reporter probe can comprise at least one secondary nucleic acid molecule.
  • a reporter probe can comprise at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten secondary nucleic acid molecules.
  • a reporter probe can comprise about six secondary nucleic acid molecules.
  • a secondary nucleic acid molecule can be a single-stranded polynucleotide.
  • a secondary nucleic acid molecule can comprise L-DNA.
  • a secondary nucleic acid molecule can consist of L-DNA.
  • a secondary nucleic acid molecule can comprise at least two domains.
  • the first domain of a secondary nucleic acid molecule can hybridize to a primary nucleic acid molecule.
  • the second domain of a secondary nucleic acid molecule can comprise at least one detectable label.
  • a secondary nucleic acid molecule can further comprise a cleavable linker.
  • the cleavable linker can be located between the first domain and the second domain, such that when the cleavable linker is cleaved, the first domain and the second domain of the secondary nucleic acid molecule are separated.
  • the cleavable linker is a photocleavable linker.
  • the second domain of a secondary nucleic acid molecule can hybridize to at least one tertiary nucleic acid molecule. In some aspects, the second domain of a secondary nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten tertiary nucleic acid molecules. In some aspects, the second domain of a secondary nucleic acid molecule can hybridize to about five tertiary nucleic acid molecules.
  • the first domain of a secondary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length.
  • the first domain of a secondary nucleic acid molecule can be about 14 nucleotides in length.
  • the second domain of a secondary nucleic acid molecule can be about 65 nucleotides, or about 66 nucleotides, or about 67 nucleotides, or about 68 nucleotides, or about 69 nucleotides, or about 70 nucleotides, or about 71 nucleotides, or about 72 nucleotides, or about 73 nucleotides, or about 74 nucleotides, or about 75 nucleotides, or about 76 nucleotides, or about 77 nucleotides, or about 78 nucleotides, or about 79 nucleotides, or about 80 nucleotides, or about 81 nucleotides, or about 82 nucleotides, or about 83 nucleotides, or about 84 nucleotides, or about 85 nucleotides in
  • a reporter probe can comprise at least one tertiary nucleic acid molecule.
  • a reporter probe can comprise at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, or at least about 31, or at least about 32, or at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40 tertiary nucleic acid molecules.
  • a tertiary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides in length.
  • a tertiary nucleic acid molecule can be about 15 nucleotides in length.
  • all of the detectable labels of the reporter probe can have the same emission spectrum.
  • the detectable labels are fluorescent labels
  • reporter probes wherein all of the detectable labels have the same emission spectrum can be referred to as “single-color” reporter probes.
  • the reporter probe can have two or more detectable labels that each have a different emission spectra.
  • reporter probes that have two or more detectable labels that each have a different emission spectrum can be referred to as “multi-color” reporter probes.
  • the present disclosure provides a reporter probe comprising a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at least one detect
  • the first domain of the primary nucleic acid molecule is about 14 nucleotides in length
  • the second domain of the primary nucleic acid molecule is about 84 nucleotides in length
  • the first domain of the secondary nucleic acid molecules is about 14 nucleotides in length
  • the second domain of the secondary nucleic acid molecules is about 75 nucleotides in length
  • each of the tertiary nucleic acid molecules is about 15 nucleotides in length.
  • the first domain of the primary nucleic acid molecule is about 14 nucleotides in length
  • the second domain of the primary nucleic acid molecule is about 84 nucleotides in length
  • the first domain of the secondary nucleic acid molecules is about 14 nucleotides in length
  • the second domain of the secondary nucleic acid molecules is about 75 nucleotides in length
  • each of the tertiary nucleic acid molecules is about 15 nucleotides in length.
  • a photocleavable linker can be cleaved upon exposure to UV light.
  • the light can be provided by a light source selected from the group consisting of an arc-lamp, a laser, a focused UV light source, and light emitting diode.
  • the photocleavable linker comprises a phosphoramidite.
  • the photocleavable linker e.g., phosphoramidite
  • the cleavage reaction results in a 5′-phosphorylated oligonucleotide.
  • the cleavage reaction results in both 3′ and 5′ phosphorylated segments, e.g., a 5′ and a 3′ monophosphate.
  • a 3′ monophosphate can be removed using methods as known in the art, e.g., using T4 polynucleotide kinase (New England Biolabs®).
  • the photocleavable linker can be selected from:
  • the nucleic acid probes of the present disclosure comprising a target binding domain and a barcode domain or identifier oligonucleotide, wherein the target binding domain and the identifier oligonucleotide are connected via a cleavable linker, in combination with the reporter probes of the present disclosure, can be used to spatially detect one or more species of target nucleic acid sequences within a biological sample (e.g., a tissue sample). Spatial detection can comprise identifying the presence of one or more species of target nucleic acid sequences within a particular region of interest of a biological sample (e.g., a tissue sample).
  • a target nucleic acid sequence can be spatially detected within a biological sample based on the following method: a 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to target nucleic acid sequence, wherein the nucleic acid probes comprise: i) a target binding domain that binds to the target nucleic acid sequence; and ii) a barcode domain specific for the at least one target nucleic acid sequence, wherein the barcode domain comprises at least one attachment position; b 1 ) contacting the biological sample with a plurality of reporter probes, thereby binding a reporter probe to an attachment region of the barcode domain of nucleic acid probes bound to the target nucleic acid sequence wherein each reporter probe comprises at least one detectable label; and
  • the preceding method can further comprise: d 1 ) determining the abundance and/or spatial position of the at least one target nucleic acid sequence in the biological sample based on the detectable labels that were recorded in step (c1).
  • the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
  • the method can further comprise, after step (c1) (and optionally prior to step (d1)): (c2) removing the detectable labels of the bound reporter probes; and (c 3 ) repeating steps (b 1 ) – (c 2 ) until each attachment position in the barcode domains of the nucleic acid probes bound to the target nucleic acid sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (d 1 ) comprises determining the abundance and spatial position of the target nucleic acid sequence in the biological sample based on the sequence in which the detectable labels were recorded.
  • removing the detectable labels of the bound reporter probes can comprise illuminating a location of the biological sample with light of a sufficient wavelength to cleave the photocleavable linkers of the reporter probes bound to said nucleic acid probes in that location, thereby removing the detectable labels of the reporter probes bound to the nucleic acid probes in that location.
  • determining the presence, abundance, and/or spatial position of the target nucleic acid sequence in the biological sample based on the detectable labels that were recorded in step (c 1 ) comprises determining the presence, abundance, and/or spatial position of the target nucleic acid sequence based on the identity of the detectable labels recorded in step (c 1 ) (e.g., in aspects wherein the detectable labels are fluorescent labels, the identity can correspond the color of the detectable label) and/or the order in which the specific detectable labels appeared.
  • determining the presence, abundance, and/or spatial position of the target nucleic sequence in the biological sample based on the detectable labels that were recorded in step (c1) can comprise the use of a computer and an accompanying program.
  • Said program can contain a list of patterns of detectable labels that correspond to specific nucleic acid sequences that are to be detected. For example, a detectable label pattern of “Green-Blue-Red” can correspond to a first target nucleic acid sequence and a detectable label pattern of “Red-Yellow-Red” can correspond to a second target nucleic acid sequence.
  • the use of nucleic acid probes comprising barcode domains and reporter probes comprising detectable labels can allow the methods of the present disclosure to be multiplexed such that more than one target nucleic acid sequence can be probed.
  • the plurality of nucleic acid probes comprises at least two species of nucleic acid probes, wherein the two species of nucleic acid probes comprise unique target binding domains that bind to different target nucleic acid sequences, thereby allowing for the determination of the presence, abundance, and/or spatial position of at least two target nucleic acid sequences in a biological sample.
  • nucleic acid probes comprising barcode domains, in combination with labeled-reporter probes, for detecting one or more specific nucleic acid sequences are further detailed in PCT Application Publication No. WO 2022/06097, US Patent No.11,549,139, US Patent No.10,415,080, US Patent Publication No. US 2017-0327876 A1 and US Patent Publication No. US 2016-0194701 A1.
  • the contents of each of the aforementioned patents and patent application publications are incorporated in their entireties for all purposes.
  • each species of target nucleic acid sequence that is to be detected in a biological sample is assigned a predetermined and unique nucleic acid probe that comprises: a) a target binding domain that is complementary to that specific species of target nucleic acid (i.e., that is designed such that it only hybridizes to that specific species of target nucleic acid); and b) a unique barcode domain comprising a unique nucleic acid sequence that is specific to that species of target nucleic sequence.
  • the unique nucleic acid sequence of the barcode domain is designed such that a specific sequence of reporter probes of the present disclosure will bind sequentially to the different attachment regions in the barcode domain, thereby creating a “linear order of detectable labels” which is specific to that species of target nucleic acid.
  • FIGs.5A-5I A schematic of a non-limiting example of these methods is shown in FIGs.5A-5I, which shows the detection of two different species of target nucleic acid sequence in a biological sample using the nucleic acid probes of the present disclosure and reporter probes of the present disclosure.
  • the method begins in FIG.5A with a biological sample that comprises two copies of target nucleic acid sequence #1 (one in the upper left part of the sample and one in the lower right part of the sample) and one copy of target nucleic acid sequence #2 (in the upper right part of the biological sample).
  • the biological sample is contacted with a plurality of ISH probes of the present disclosure.
  • the nucleic acid (NA) probes with target binding domains that are complementary to target nucleic acid sequence #1 hybridize to target nucleic acid #1
  • NA probes with target binding domains that are complementary to target nucleic acid #2 hybridize to target nucleic acid sequence #2.
  • a third type of probe which has a target binding domain complementary to a third type of target nucleic acid sequence does not hybridize within the biological sample, because the biological sample does not contain the third type of target nucleic acid sequence.
  • the nucleic acid probes further comprise a reversible terminator (denoted by a star) at the terminus of the target binding domain that is not attached to the barcode domain.
  • the reversible terminator comprises a photocleavable moiety such that when the photocleavable moiety is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase.
  • the non-hybridized NA probes are washed off of the biological sample.
  • the non-hybridized NA probes can be washed off with a buffer solution or with water.
  • the biological sample is contacted with a plurality of reporter probes comprising detectable labels.
  • the detectable labels are fluorescent labels.
  • the barcode domain of NA probe type #1 is designed such that the first attachment region hybridizes to a reporter probe with a red fluorescent label and NA probe type #2 is designed such that the first attachment region hybridizes to a reporter probe with a green fluorescent label.
  • a fourth step shown in FIG.5C, the identity and spatial location of the detectable labels of the hybridized reporter probes are recorded. Accordingly, during the first round of imaging, a red label was detected in “Location 1”, a green label was detected in “Location 2” and a red label was detected in “Location 3”.
  • the detectable labels are removed from the hybridized reporter probes.
  • the reporter probes comprise photocleavable moieties that can be cleaved by illumination with UV light, which releases the detectable labels, which are subsequently washed away.
  • the UV light also results in the cleavage of the reversible terminators located on the target binding domains of the nucleic acid probes.
  • the biological sample is contacted with a second plurality of reporter probes comprising detectable labels.
  • the barcode domain of NA probe type #1 is designed such that the second attachment region hybridizes to a reporter probe with a yellow fluorescent label and MA probe type #2 is designed such that the second attachment region hybridizes to a reporter probe with a red fluorescent label.
  • a seventh step as shown in FIG.5F, the identity and spatial location of the detectable labels of the hybridized reporter probes are recorded.
  • the detectable labels are removed from the hybridized reporter probes by UV- induced cleavage of photocleavable moieties within the reporter probes. [00184] These steps are repeated until each of the attachment regions in each NA probe has been bound by a reporter probe, and the identity of the detectable label of the reporter probe has been recorded. Thus, at the end of the method, a “linear order of detectable labels” will have been recorded at each location of interest.
  • the linear order of detectable labels at Location 1 and Location 3 was red-yellow- green-red and the linear order of detectable labels at Location 2 was green-red-yellow- yellow.
  • red-yellow-green-red is specific to target nucleic acid sequence #1
  • green-red-yellow-yellow is specific to target nucleic acid sequence #2
  • the method has allowed for the identification of two copies of target nucleic acid sequence #1 in the biological sample, with one of the copies being present at Location 1 and one of the copies being present at Location 3, and the identification of one copy of target nucleic acid sequence #2 at Location 2.
  • the above method can be multiplexed to detect any number of target nucleic acids and/or target proteins at any number of locations with a biological sample.
  • the methods of the present disclosure can be used to determine the spatial abundance of at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000,
  • the above method can be multiplexed to detect any number of target nucleic acids and/or target proteins at any number of locations with a biological sample.
  • the methods of the present disclosure can be used to determine the spatial abundance of about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 240, or about 250, or about 260, or about 270, or about 280, or about 290, or about 300, or about 500, or about 1,000, or about 10,000, or about 100,000, or about 1,000,000 different species of target nucleic acids and/or target proteins within a biological sample.
  • the methods of the present disclosure can further comprise after step (b 1 ) and prior to step (c 1 ), removing unbound reporter probes. [00188] In some aspects, the methods of the present disclosure can further comprise, prior to the addition of reporter probes, removing unbound nucleic acid probes from the sample. [00189] These methods can be used to spatially detect any number of target nucleic acids within any number of locations within a biological sample. A specific location of a biological sample is also referred to herein as a “region of interest” or an “ROI”. A region of interest may be a tissue type present in a sample, a cell type, a cell, or a subcellular structure within a cell.
  • region of interest and “ROI” are used in their broadest sense to refer to a specific location within a sample that is to be analyzed using the methods of the present disclosure.
  • spatialally detecting is used in its broadest sense to refer to the identification of the presence of a specific target analyte within a specific region of interest in a sample. Spatially detecting can comprise quantifying the amount of a specific target analyte present within a specific region of interest in a sample.
  • Spatially detecting can further comprise quantifying the relative amount of a first target analyte within a specific region of interest in a sample as compared to the amount of at least a second target analyte within a specific region of interest in a sample.
  • Spatially detecting can also comprise quantifying the relative amount of a specific target analyte within a first region of interest in a sample compared to the amount of the same target analyte in at least a second region of interest in the same sample or different sample.
  • a target nucleic acid sequence can be spatially detected within a biological sample based on the following method: (a) contacting a biological sample with a plurality of nucleic acid probes of the present disclosure such that the nucleic acid probes bind to one or more copies of the target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; (b) illuminating a location of the biological sample with light of a sufficient wavelength to cleave the reversible terminators and photocleavable linkers of the nucleic acid probes bound to one or more copies of the target nucleic acid sequence in that location, thereby releasing the identifier oligonucleotides of the nucleic acid probes bound to the one or more copies of the target nucleic acid sequence in that location, and (c) collecting the released identifier oligonucleotides from the location that was illuminated; (d) spatially detecting the one or more copies of the target nucleic acid sequence
  • FIGs.3A- 3I A schematic of a non-limiting example of the preceding method is shown in FIGs.3A- 3I.
  • the method begins in FIG. 3A with a sample.
  • the sample comprises four cells, each cell comprising at least one copy of the specific mRNA comprising a target nucleic acid sequence to be measured.
  • Cell #1, Cell #2 and Cell #3 comprise one copy of the said mRNA and Cell #3 comprises two copies of the said mRNA.
  • the sample can be subdivided into regions of interest (ROI) as shown in FIG.3B.
  • each ROI comprises one of the cells.
  • FIG.3B regions of interest
  • each ROI comprises one of the cells.
  • the sample is contacted with a plurality of probes, wherein the probes comprise a unique target binding domain that binds to the target mRNA, a unique identifier oligonucleotide specific for the target analyte that comprises a unique identifier sequence specific for the target analyte, a photocleavable linker between the target binding domain and the identifier oligonucleotide, and a reversible terminator, as shown in the top left hand corner of FIG. 3C.
  • the target binding domain is specific for the target mRNA and hybridizes to each target mRNA within each cell.
  • the sample may be contacted with a plurality of probes comprising any number of different species of probes wherein each species of probe comprises a unique target binding domain that binds to one of the at least two target analytes and a unique identifier oligonucleotide specific for the target analyte.
  • FIGs.3A-3I illustrate a single species of probe for exemplary purposes only.
  • the released identifier oligonucleotides from ROI #1 are collected from the sample.
  • the released identifier oligonucleotides can be collected from a solution immediately adjacent to the sample surface.
  • a holding vessel e.g., a sample tube, a well of a microtiter plate, etc.
  • FIG.3F This collection is shown in FIG.3F.
  • the steps shown in FIGs. 3D-3F are then repeated for each ROI, resulting in the collection of released identifier oligonucleotides from each ROI, as shown in FIG. 3G. Note that two identifier oligonucleotides were collected from ROI #3 as two target nucleic acid sequences were present in said ROI.
  • FIG.3H The method continues in FIG.3H, where the collected identifier oligonucleotides are then amplified such that the amplified products in each of the separate holding vessels comprise a nucleic acid sequence that identifies the specific location from which the identifier oligonucleotide was released.
  • amplification products can then be sequenced, thereby allowing for the spatial detection of the target nucleic acid sequence based on the sequence of the identifier sequence and the sequence specific to the ROI, which will be revealed in each of the sequencing reads. Based on the number of sequencing reads, the abundance of the target nucleic acid sequence in that region can also be determined based on methods known in the art. The method can further continue as shown in FIG.3I, which is described in further detail herein.
  • a comparison of the identity and abundance of the target nucleic acids present in a first region of interest e.g., tissue type, a cell type (including normal and abnormal cells), and a subcellular structure within a cell
  • a first region of interest e.g., tissue type, a cell type (including normal and abnormal cells), and a subcellular structure within a cell
  • the nucleic acid probes of the present disclosure comprise: a target binding domain and a barcode domain, wherein the target binding domain comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, or a target binding domain and an identifier oligonucleotide, wherein the target binding domain and the identifier oligonucleotide are connected via a cleavable linker, and further comprising a reversible terminator, which can be used to both: i) spatially detect one or more species of target nucleic acid sequence (using the methods described supra); and ii) sequence the nucleic acid molecules that contain said target nucleic acid sequences within specific regions of a biological sample.
  • the present disclosure provides methods comprising: a 1 ) contacting the biological sample with a plurality of nucleic acid probes of the present disclosure, such that the nucleic acid probes bind to one or more copies of a target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; wherein the nucleic acid probes comprise: i) a target binding domain that binds to the target nucleic acid sequence; and ii) a barcode domain specific for the at least one target nucleic acid sequence, wherein the barcode domain comprises at least one attachment position; b1) contacting the biological sample with a plurality of reporter probes, thereby binding a reporter probe to an attachment region of the barcode domain of nucleic acid probes bound to the target nucleic acid sequence wherein each reporter probe comprises at least one detectable label and at least one photocleavable linker; c 1 ) recording the identity and spatial position of the detectable labels of the bound reporter probes; d1)
  • the preceding method can further comprise determining the abundance and/or spatial position of the one or more copies of the target nucleic acid sequence in the biological sample based on the detectable labels that were recorded in step (c 1 ).
  • the barcode domains of the nucleic acid probes can comprise at least two, or at least three, or at least four attachment positions, and the method can further comprises, after step (d 1 ): (d 2 ) repeating steps (b 1 ) – (d 1 ) until each attachment position in the barcode domains of the nucleic acid probes bound to the target nucleic acid sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein determining the abundance and spatial position of the target nucleic acid sequence in the biological sample comprises determining the abundance and/or spatial position of the nucleic acid sequence based on the sequence in which the detectable labels were recorded.
  • steps (d 1 ) – (e 1 ) are performed in two or more locations within the biological sample, thereby spatially detecting the one or more copies of the target nucleic acid sequence and sequencing the one or more nucleic acid molecules present in the two or more locations within the biological sample.
  • performing a sequencing reaction in step (e1) can comprise: i) purifying the nucleic acid molecules that are bound to the target binding domains; ii) performing a template-switching reverse transcription reaction using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; iii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • sequencing the cDNA molecules comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • performing a sequencing reaction in step (e 1 ) can comprise: i) performing a template-switching reverse transcription reaction in situ within the biological sample using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; and ii) sequencing the cDNA molecules, thereby sequencing the nucleic acid molecules.
  • the sequencing can be performed in situ within the biological sample.
  • sequencing the cDNA molecules comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof. Accordingly, an example of the preceding method is shown in FIGs.5A-5I (see description of FIGs.5A-5H above).
  • the present disclosure provides methods comprising: (a) contacting a biological sample with a plurality of nucleic acid probes of the present disclosure such that the nucleic acid probes bind to one or more copies of the target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; (b) illuminating a location of the biological sample with light of a sufficient wavelength to cleave the reversible terminators and photocleavable linkers of the nucleic acid probes bound to one or more copies of the target nucleic acid sequence in that location, thereby: releasing the identifier oligonucleotides of the nucleic acid probes bound to the one or more copies of the target nucleic acid sequence in that location, and rendering terminal 3′-OH moieties of the target binding domains bound
  • steps (d) and (e) can be performed sequentially, in any order. In some aspects of the preceding methods, steps (d) and (e) are performed concurrently. [00210] As described herein, steps (b) – (e) of the preceding method can be performed in two or more locations within the biological sample, thereby spatially detecting the one or more copies of the target nucleic acid sequence and sequencing the nucleic acid molecules containing said target nucleic acid sequences in the two or more locations within the biological sample. [00211] In some aspects of the preceding methods, sequencing the amplification products in step (d)(ii) can comprise performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • performing a sequencing reaction in step (e) can comprise: i) purifying the nucleic acid molecules that are bound to the target binding domains; ii) performing a template-switching reverse transcription reaction using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; iii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • sequencing the cDNA molecules in step (e)(iii) comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • performing a sequencing reaction in step (e) can comprise: i) performing a template-switching reverse transcription reaction in situ within the biological sample using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; and ii) sequencing the cDNA molecules, thereby obtaining the sequence of the nucleic acid molecules.
  • the sequencing can be performed in situ within the biological sample.
  • sequencing the cDNA molecules in step (e)(ii) comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • FIGs.3A-3I an example of the preceding method is shown in FIGs.3A-3I (see description of FIGs.3A-3H above).
  • reverse transcriptase is added to the sample and the bound probes are used to prime the reverse transcriptase reaction in order to create cDNA that is subsequently sequenced.
  • the cDNA molecules can be sequenced in situ.
  • the cDNA molecules can be removed from the tissue sample and sequenced.
  • removing the cDNA molecules from the tissue sample can comprise removing cDNA from specific ROIs and sequencing them individually and/or tagging them prior to sequencing with nucleic acid tags specific to that specific ROI.
  • cDNA can first be removed from ROI #1 and tagged with a nucleic acid sequence specific to ROI #1. cDNA can then be removed from ROI#2 and tagged with a nucleic acid sequence specific to ROI #2. This process can continue to cDNA from each ROI is collected. In some aspects, the cDNA can be removed from the entire tissue sample at once.
  • Target Nucleic Acid Sequences can be any target nucleic acid sequence that is present or that is suspected of being present in a biological sample.
  • a target nucleic acid may be a DNA sequence.
  • a DNA sequence can be a gDNA sequence.
  • a DNA sequence can be an mtDNA sequence. In some aspects, a DNA sequence can be an exogenous DNA sequence that has been introduced into the biological sample (e.g., a vector that has been introduced into a modified cell).
  • a target nucleic acid may be an RNA sequence. In some aspects, an RNA sequence can be an mRNA sequence. In some aspects, an RNA sequence can be a miRNA sequence. In some aspects, an RNA sequence can be a tRNA sequence. In some aspects, an RNA molecule can be an rRNA sequence.
  • a target nucleic acid sequence can comprise at least one nucleic acid sequence encoding a T-cell receptor, or a portion thereof.
  • a target nucleic acid sequence can comprise at least one nucleic acid sequence encoding a CDR3 region of a T-cell receptor, or a portion thereof. [00219] In some aspects, a target nucleic acid sequence can comprise a single nucleotide variant of interest. [00220] In some aspects, a target nucleic acid sequence can be a nucleic acid sequence that is not endogenous to the tissue sample within which it is located. In a non-limiting example, the target nucleic acid sequence can be a microbe-derived (e.g., bacterial or fungal) nucleic acid sequence that is present within a mammalian tissue sample.
  • a microbe-derived e.g., bacterial or fungal
  • the target nucleic acid sequence can be a viral-derived nucleic acid sequence that is present within a mammalian tissue sample.
  • the target nucleic acid sequence is located in proximity to a splice junction.
  • the target nucleic acid sequence is located in proximity to a single nucleotide variant.
  • the target nucleic acid sequence is located in proximity to a single nucleotide polymorphism.
  • the target nucleic acid sequence is located in proximity to or within a copy number variant.
  • the target nucleic acid sequence is located proximity to or within a chromosomal rearrangement.
  • a biological sample can be a tissue sample.
  • a tissue sample can be a fresh frozen tissue sample.
  • a tissue can be a fixed tissue sample.
  • a fixed tissue sample can be a formalin-fixed, paraffin-embedded (FFPE) tissue sample.
  • a biological sample can be an FFPE microtome section that is at least about 1 ⁇ m, or at least about 2 ⁇ m, or at least about 3 ⁇ m, or at least about 4 ⁇ m, or at least about 5 ⁇ m, or at least about 6 ⁇ m, or at least about 7 ⁇ m, or at least about 8 ⁇ m, or at least about 9 ⁇ m, or at least about 10 ⁇ m thick.
  • the biological sample is an FFPE microtome section that is at least about 5 ⁇ m thick.
  • a biological sample can be an FFPE microtome section that is about 1 ⁇ m, or about 2 ⁇ m, or about 3 ⁇ m, or about 4 ⁇ m, or about 5 ⁇ m, or about 6 ⁇ m, or about 7 ⁇ m, or about 8 ⁇ m, or about 9 ⁇ m, or about 10 ⁇ m thick.
  • the biological sample is an FFPE microtome section that is about 5 ⁇ m thick.
  • the biological sample can be a tissue sample from any organ.
  • the biological sample is a tissue sample from the Intestine, Embryo, Brain, Spleen, Eye, Retina, Liver, Kidney, Breast, Throat, Colon, Lung, Prostate, Lymph node, Tonsil, Pancreas, Cervix, Head, Neck, Liver, Skin, Nevus, Placenta or any other organ.
  • the biological sample can comprise non-cancerous cells.
  • the biological sample can comprise cancerous cells.
  • the biological sample can comprise a combination of both non-cancerous cells and cancerous cells.
  • the cancerous cells can be from a carcinoma, lymphoma, blastoma, sarcoma, leukemia and germ cell tumors.
  • the cancerous cells can be from a adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic
  • cancers include breast cancer, lung cancer, lymphoma, melanoma, liver cancer, colorectal cancer, ovarian cancer, bladder cancer, renal cancer or gastric cancer.
  • Further examples of cancer include neuroendocrine cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, thyroid cancer, endometrial cancer, biliary cancer, esophageal cancer, anal cancer, salivary, cancer, vulvar cancer, cervical cancer, Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Adrenal gland tumors, Anal cancer, Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain tumors, Breast cancer, Cancer of unknown primary (CUP), Cancer spread to bone, Cancer spread to brain, Cancer spread to liver, Cancer spread to lung, Carcinoid, Cervical cancer, Children's cancers, Chronic lymphocytic leukemia (CLL), Chrome myeloid leukemia (CML), Colorectal cancer, Ear cancer, Endo
  • Retinoblastoma Salivary gland cancer, Secondary' cancer, Signet cell cancer, Skin cancer, Small bowel cancer, Soft tissue sarcoma, Stomach cancer, T cell childhood non Hodgkin lymphoma (NHL), Testicular cancer, Thymus gland cancer, Thyroid cancer, Tongue cancer, Tonsil cancer, Tumors of the adrenal gland, Uterine cancer. Vaginal cancer, Vulval cancer, Wilms' tumor, Womb cancer and Gynaecological cancer.
  • cancer also include, but are not limited to, Hematologic malignancies, Lymphoma, Cutaneous T-cell lymphoma, Peripheral T-cell lymphoma, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, Multiple myeloma, Chrome lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Myelodysplastic syndromes, Myelofibrosis, Biliary tract cancer, Hepatocellular cancer, Colorectal cancer, Breast cancer, Lung cancer, Non-small cell lung cancer, Ovarian cancer, Thyroid Carcinoma, Renal Cell Carcinoma, Pancreatic cancer, Bladder cancer, skin cancer, malignant melanoma, merkel cell carcinoma, Uveal Melanoma or Glioblastoma multiforme.
  • the biological sample can be derived from any species, including, but not limited to; humans, mice, rats, dogs, cats, sheep, rabbits, cows, goats or any other species.
  • a biological sample can be derived from a fungi.
  • a biological sample can be derived from a plant.
  • the biological sample can be a mounted biological sample.
  • the mounted biological sample is in a flow cell.
  • any of the methods of the present disclosure can further comprise morphology scanning of the biological sample. In some aspects, morphology scanning can be used to determine one or more regions of interest to be imaged.
  • morphology scanning can be used to identify specific features of the biological sample (e.g., tumorous cells, healthy cells, tumor margins, cellular membranes, cellular nuclei, one or more cellular organelles, vasculature, or any other features known in the art by the skilled artisan).
  • the specific features of the biological sample can be correlated with the abundance and spatial position of target analytes measured using the methods of the present disclosure.
  • morphology scanning can be used to determine the boundaries of individual cells within the biological sample. The determination of the boundaries of individual cells is referred to herein as "cell segmentation".
  • the methods of the present disclosure can further comprise staining the biological sample with a membrane specific-fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular membranes within the sample. This staining can be performed at any step in the protocol.
  • the methods of the present disclosure can further comprise staining the biological sample with a nuclear-specific fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular nuclei in the sample. This staining can be performed at any step in the methods of the present disclosure.
  • the methods of the present disclosure can further comprise, staining the biological sample with a membrane specific-fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular membranes within the sample.
  • the method can further comprise, staining the biological sample with a nuclear-specific fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular nuclei in the sample.
  • membrane and/or nuclear stains are used to perform morphology scanning on the biological sample.
  • the membrane and/or nuclear stains can be used to determine one or more regions of interest to be imaged during determination of the abundance and spatial position of target analytes (e.g., target nucleic acid sequence).
  • target analytes e.g., target nucleic acid sequence.
  • fiducial markers can be added to the biological sample can be used to focus the biological sample using methods standard in the art, as would be appreciated by the skilled artisan. Specifically, the fiducial markers can be used to determine the best z-position for imaging a particular location within the biological sample.
  • a biological sample can be pre-treated using standard methods known in the art to allow for the recombinase proteins, single-stranded DNA binding proteins, primers, nucleic acid probes and reporter probes to permeate throughout the sample, including, but not limited to, within individual cells in the sample. That is, a biological sample can be permeabilized using standard methods known in the art prior to performing the methods of the present disclosure.
  • Multiplexing [00237] The methods of the present disclosure can be multiplexed to detect a plurality of different target nucleic acid sequences (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different target nucleic acid sequences).
  • the methods of the present disclosure can be multiplexed to detect and sequence any number of target nucleic acid sequences at any number of locations within a biological sample.
  • the methods of the present disclosure can be used to determine the spatial abundance of at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least
  • the methods of the present disclosure can be multiplexed to detect and sequence any number of target nucleic acid sequences at any number of locations with a biological sample.
  • the methods of the present disclosure can be used to determine the spatial abundance of about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 240, or about 250, or about 260, or about 270, or about 280, or about 290, or about 300, or about 500, or about 1,000, or about 10,000, or about 20,000, or about 100,000, or about 1,000,000 different species of target nucleic acid sequences within a biological sample.
  • the methods of the present disclosure can be used to detect and sequence target nucleic acid sequences in at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000, or at least about 100,000, or at least about 1,000,000 different locations (ROIs) within a biological sample.
  • ROIs different locations
  • the methods of the present disclosure can be used to detect and sequence target nucleic acid sequence in about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 240, or about 250, or about 260, or about 270, or about 280, or about 290, or about 300, or about 500, or about 1,000, or about 10,000, or about 100,000, or about 1,000,000 different locations (ROIs) within a biological sample.
  • ROIs different locations
  • kits for use in the methods of the present disclosure.
  • a kit of the present disclosure can comprise one or more pluralities of nucleic acid probes, as described herein.
  • a kit of the present disclosure can comprise one or more pluralities of reporter probes, as described herein.
  • a kit of the present disclosure can comprise a system suitable for use in the methods of the present disclosure.
  • a kit of the present disclosure can comprise an apparatus suitable for use in the methods of the present disclosure. Enumerated Embodiments 1.
  • a nucleic acid probe comprising: a target binding domain and a barcode domain, wherein the target binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid, wherein the target binding domain comprises D-DNA, wherein the target binding domain further comprises a reversible terminator at one terminus and is connected to the barcode domain at the other terminus, and wherein the barcode domain is a single-stranded polynucleotide comprising at least about one attachment region, wherein each attachment region comprises about one attachment sequence, and wherein the sequences of each of the attachment sequences are different, and wherein the barcode domain comprises L-DNA.
  • nucleic acid probe of embodiment 1, wherein the reversible terminator comprises a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase, preferably wherein the cleavable moiety is a photocleavable moiety, a chemically-cleavable moiety or an enzymatically- cleavable moiety.
  • the reversible terminator is a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase, preferably wherein the cleavable moiety is a photocleavable moiety, a chemically-cleavable moiety or an enzymatically- cleavable moiety.
  • each of the attachment sequences is about 14 nucleotides in length.
  • the target binding domain is about 35 to about 40 nucleotides in length.
  • the nucleic acid probe of any one of the preceding embodiments, wherein the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions. 8.
  • the reporter probe comprises: a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at least one detectable label
  • nucleic acid probe of embodiment 9 wherein at least one of the primary nucleic acid molecule, the secondary nucleic acid molecules, and the tertiary nucleic acid molecules comprise L-DNA.
  • a method comprising: a 1 ) contacting the biological sample with a plurality of nucleic acid probes of any one of embodiments 1-7, such that the nucleic acid probes bind to one or more copies of a target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; wherein the nucleic acid probes comprise: i) a target binding domain that binds to the target nucleic acid sequence; and ii) a barcode domain specific for the at least one target nucleic acid sequence, wherein the barcode domain comprises at least one attachment position; b1) contacting the biological sample with a plurality of reporter probes, thereby binding a reporter probe to an attachment region of the barcode domain of nucleic acid probes bound to the target nucleic acid sequence, wherein each reporter probe comprises at least one detectable label and at least one photocleavable linker; c 1 ) recording the identity and spatial position of the detectable labels of the bound reporter probes; d1) illuminating a location of
  • step (c1) The method of embodiment 13, further comprising determining the abundance and/or spatial position of the one or more copies of the target nucleic acid sequence in the biological sample based on the detectable labels that were recorded in step (c1).
  • the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions, and the method further comprises, after step (d 1 ): (d2) repeating steps (b1) – (d1) until each attachment position in the barcode domains of the nucleic acid probes bound to the target nucleic acid sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein determining the abundance and spatial position of the target nucleic acid sequence in the biological sample comprises determining the abundance and/or spatial position of the nucleic acid sequence based on the sequence in which the detectable labels were recorded.
  • steps (d 1 ) – (e 1 ) are performed in two or more locations within the biological sample, thereby spatially detecting the one or more copies of the target nucleic acid sequence and sequencing the one or more nucleic acid molecules present in the two or more locations within the biological sample.
  • the plurality of probes is the plurality of probes of embodiment 10 or embodiment 11, and wherein the method comprises spatially detecting two or more target nucleic acid sequences and sequencing the one or more nucleic acid molecules within which at least one of the target nucleic acid sequences is located.
  • performing a sequencing reaction in step (e1) comprises: i) purifying the nucleic acid molecules that are bound to the target binding domains; ii) performing a template-switching reverse transcription reaction using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; iii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules. 19.
  • step (e 1 ) comprises: i) performing a template-switching reverse transcription reaction in situ within the biological sample using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; ii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • sequencing the cDNA molecules comprises: performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof. 21. The method of any one of embodiments 13-20, wherein the target nucleic acid sequence is in proximity to a splice junction. 22.
  • a nucleic acid probe comprising: a single-stranded target binding domain and a single-stranded identifier oligonucleotide; wherein the single-stranded target binding domain comprises: a reversible terminator at one terminus and a photocleavable linker at the other terminus; and a sequence that binds to a target nucleic acid sequence, wherein the single-stranded target binding domain and the single-stranded identifier oligonucleotide are linked together via the cleavable linker, wherein the reversible terminator comprises a photocleavable moiety such that when the reversible terminator is excited by light of a sufficient wavelength, the photocleavable moiety is cleaved,
  • nucleic acid probe of embodiment 24, wherein the reversible terminator comprises a cleavable moiety such that when the reversible terminator is cleaved, a terminal 3′-OH moiety is rendered accessible to a polymerase, preferably wherein the cleavable moiety is a photocleavable moiety, a chemically-cleavable moiety or an enzymatically- cleavable moiety.
  • the reversible terminator is (dU.I), (dU.VI).
  • nucleic acid probe of any one of the preceding embodiments wherein the single- stranded target binding domain is about 35 nucleotides in length. 29.
  • the single- stranded identifier oligonucleotide further comprises at least one of: i) a unique molecular identifier sequence (UMI); ii) a first amplification primer binding site; iii) a second amplification primer binding site; iv) at least one sequence specific to the cell in which the nucleic acid probe was bound; and v) at least one sequence specific to a region of interest in a biological sample.
  • UMI unique molecular identifier sequence
  • a plurality of the nucleic acid probes of any one of the preceding embodiments wherein the plurality of nucleic acid probes comprises at least two different species of nucleic acid probes, wherein the sequence of the single-stranded target binding domain of each species is distinct, such that the single-stranded target binding domain of each species binds to a distinct target nucleic acid molecule, wherein the single-stranded identifier oligonucleotide of a single species comprises at least one identifier sequence that is specific to the target binding domain of that single species, and wherein the at least one identifier sequences of different species are distinct. 32.
  • 33. A method comprising: (a) contacting a biological sample with a plurality of nucleic acid probes of any one of embodiments 24-32 such that the nucleic acid probes bind to one or more copies of the target nucleic acid sequence located within one or more nucleic acid molecules present within the biological sample; (b) illuminating a location of the biological sample with light of a sufficient wavelength to cleave the reversible terminators and photocleavable linkers of the nucleic acid probes bound to one or more copies of the target nucleic acid sequence in that location, thereby: releasing the identifier oligonucleotides of the nucleic acid probes bound to the one or more copies of the target nucleic acid sequence in that location, and rendering terminal 3′-OH moieties of the target binding domains bound to the one or more copies of the target nucleic acid sequence in
  • steps (b) – (e) are performed in two or more locations within the biological sample, thereby spatially detecting the one or more copies of the target nucleic acid sequence and sequencing the one or more nucleic acid molecules present in the two or more locations within the biological sample.
  • the plurality of probes is the plurality of probes of embodiment 31 or embodiment 32, and wherein the method comprises spatially detecting two or more target nucleic acid sequences and sequencing the one or more nucleic acid molecules within which at least one of the target nucleic acid sequences is located.
  • sequencing the amplification products in step (d)(ii) comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.
  • performing a sequencing reaction in step (e) comprises: i) purifying the nucleic acid molecules that are bound to the target binding domains; ii) performing a template-switching reverse transcription reaction using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; iii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • step (e) comprises: i) performing a template-switching reverse transcription reaction in situ within the biological sample using the target binding domains bound to the nucleic acid molecules as a primer for the reaction, thereby producing a plurality of cDNA molecules; ii) sequencing the cDNA molecules, thereby sequencing the one or more nucleic acid molecules.
  • sequencing the cDNA molecules comprises performing next-generation sequencing methods, sequencing by synthesis, massively parallel sequencing, or any combination thereof.

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Abstract

La présente divulgation concerne des compositions et des méthodes permettant la détection spatiale et l'identification de séquence combinées de molécules d'acide nucléique cibles dans un échantillon biologique, comprenant sans caractère limitatif, des échantillons de tissu, à l'aide de sondes d'acide nucléique comprenant un terminateur réversible.
PCT/US2024/020073 2023-03-17 2024-03-15 Sondes d'acide nucléique pour séquençage et analyse spatiale combinés Pending WO2024196728A2 (fr)

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