Amplification-free probe complex for detecting a target analyte

Abstract

This disclosure provides compositions, apparatus, and methods for detecting target analytes using amplification-free probe complexes. In some embodiments, the amplification-free probe complexes can be used to detect analytes on or inside cell samples.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 549,340, filed February 2, 2024, the contents of which are incorporated herein by reference in their entirety.

[0003] By referencing and incorporating into the sequence list

[0004] The contents of the electronic sequence list (ELEM_030_001WO_SeqList_ST26.xml; size: 5,420 bytes; and creation date: December 30, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure provides compositions, apparatus, and methods for detecting cellular target analytes using amplification-free probe complexes. In some embodiments, the amplification-free probe complex can be used to detect analytes on or inside a cell sample. Background Technology

[0006] Cells within a subject's tissues exhibit differences in composition, morphology, and function due to varying levels of intracellular analytes (e.g., gene and / or protein expression). Specific locations of cells within a tissue (e.g., cell position relative to neighboring cells or cell position relative to the tissue microenvironment), developmental stages, and pathological conditions can influence cell morphology, differentiation, fate, viability, proliferation, signal transduction, crosstalk with other cells in the tissue, and other cellular behaviors. Spatial heterogeneity has previously been investigated using techniques that provide data on a small number of analytes (such as proteins, nucleic acids, polysaccharides, or lipids) against the background of only an intact tissue or a portion of a tissue. However, previous methods are limited by the number of analytes that can be identified and imaged, as well as the time and labor involved. Therefore, there is a need for improved spatially resolved analytical methods that can be used to image multiple analytes within cells or tissues. Summary of the Invention

[0007] This disclosure provides an amplification-free probe complex comprising (i) at least one modified oligonucleotide; and (ii) at least one analyte-binding moiety capable of binding a target analyte, wherein the at least one modified oligonucleotide is attached to the at least one analyte-binding moiety. In some embodiments, the at least one modified oligonucleotide comprises two or more tandem copies of a sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide comprises a canonical nucleobase located immediately adjacent to the 5' end of each sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide comprises a base-free site located immediately adjacent to the canonical nucleobase at the 5' end.

[0008] In some embodiments of the amplification-free probe complex disclosed herein, the canonical nucleotide is positioned between one and ten nucleotides at the 5' end of each sequencing primer binding site. In some embodiments, the canonical nucleotide is located at and adjacent to the 5' end of each sequencing primer binding site.

[0009] This disclosure provides an amplification-free probe complex comprising (i) at least one modified oligonucleotide; and (ii) at least one analyte-binding moiety capable of binding a target analyte, wherein the at least one modified oligonucleotide is attached to the at least one analyte-binding moiety, and wherein the at least one modified oligonucleotide comprises at least one sequencing primer binding site and a barcode sequence. In some embodiments, the at least one modified oligonucleotide comprises two or more tandem copies of the sequencing primer binding site and a barcode sequence.

[0010] In some embodiments of the amplification-free probe complex disclosed herein, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the analyte-binding portion includes a lipid portion, wheat germ lectin, antibody, or biotin portion. In some embodiments, the target analyte is located inside and / or on the surface of the cell sample. In some embodiments, the cell sample includes whole single cells, multiple whole cells, intact tissue, sliced ​​cells, or sliced ​​tissue samples. In some embodiments, the cell sample includes fresh cell samples, fresh frozen cell samples, or formalin-fixed paraffin-embedded (FFPE) cell samples. In some embodiments, the cell sample includes fixed and permeabilized cell samples. In some embodiments, the analyte-binding portion includes a secondary antibody or a primary antibody.

[0011] This disclosure provides a method for detecting a target analyte, comprising: (a) providing a plurality of amplification-free probe complexes of the present disclosure; (b) providing a sample comprising a plurality of analytes, the plurality of analytes including at least one target analyte, wherein the sample is deposited on a support; (c) contacting the sample with the plurality of amplification-free probe complexes, wherein the contact is performed under conditions suitable for binding the analyte-binding portion to the target analyte; (d) contacting a plurality of modified oligonucleotides of the plurality of amplification-free probe complexes of step (c) with a plurality of sequencing primers under conditions suitable for each sequencing primer to bind to the sequencing primer binding site on the modified oligonucleotide to form a plurality of nucleic acid duplexes, wherein each nucleic acid duplex includes a sequencing primer binding site on the modified oligonucleotide that hybridizes to the sequencing primer; and (e) contacting the plurality of nucleic acid duplexes with the target analyte. Contact with multiple sequencing polymerases and multiple detectably labeled multivalent molecules, wherein each nucleic acid duplex binds to the sequencing polymerase and the nucleotide moiety of the multivalent molecule, thereby forming multiple detectably labeled complexes, wherein the nucleotide moiety of the detectably labeled multivalent molecule binds to the 3' end of the sequencing primer and is located opposite to the canonical nucleobase in the modified oligonucleotide, wherein the contact in step (e) is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the nucleotide moiety into the 3' end of the sequencing primer, and wherein the nucleotide moiety of the multivalent molecule binds to and is complementary to the nucleotide in the modified oligonucleotide, thereby producing multiple detectably labeled complexes; and (f) imaging the sample bound to the multiple detectably labeled complexes, thereby detecting the target analyte on or inside the cell sample.

[0012] This disclosure provides a method for detecting a target analyte, comprising: (a) providing a plurality of amplification-free probe complexes of the present disclosure; (b) providing a sample comprising a plurality of analytes, the plurality of analytes including at least one target analyte, wherein the sample is deposited on a support; (c) contacting the sample with the plurality of amplification-free probe complexes, wherein the contact is performed under conditions suitable for binding the analyte-binding portion to the target analyte; and (d) contacting a plurality of modified oligonucleotides of the plurality of amplification-free probe complexes of step (c) with a plurality of sequencing primers under conditions suitable for each sequencing primer to bind to the sequencing primer binding site on the modified oligonucleotide to form a plurality of nucleic acid duplexes, wherein each nucleic acid duplex contains (e) Sequencing primer binding sites on the modified oligonucleotide that hybridize with sequencing primers; (f) Contacting the plurality of nucleic acid duplexes with a plurality of sequencing polymerases and a plurality of detectably labeled multivalent molecules, wherein each nucleic acid duplex binds to the sequencing polymerase and the nucleotide portion of the multivalent molecule, thereby forming a plurality of detectably labeled complexes, wherein the nucleotide portion of the detectably labeled multivalent molecule binds to the 3' end of the sequencing primer and is located opposite to the barcode sequence in the modified oligonucleotide, and wherein the nucleotide portion of the multivalent molecule binds to the opposite nucleotide of the modified oligonucleotide and is complementary to it, thereby producing a plurality of detectably labeled complexes; and (f) Imaging the sample to detect the target analyte.

[0013] In some embodiments of the methods disclosed herein, the contact in step (e) is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of nucleotide moieties into the 3' end of the sequencing primer.

[0014] In some embodiments of the methods disclosed herein, each multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein each nucleotide arm contains a nucleotide portion. In some embodiments, the complementary nucleotide portion of the multivalent molecule is not incorporated into the 3' end of the sequencing primer. In some embodiments, the nucleotide portion of the multivalent molecule is complementary to the nucleotide at the 5' end of the modified oligonucleotide and adjacent to it at the sequencing primer binding site.

[0015] In some embodiments of the methods disclosed herein, the sample is a cell sample. In some embodiments, the target analyte is on and / or inside the cell sample. In some embodiments, the cell sample includes whole single cells, multiple whole cells, complete tissue, sliced ​​cells, or sliced ​​tissue samples. In some embodiments, the cell sample includes fresh cell samples, fresh frozen cell samples, or formalin-fixed paraffin-embedded (FFPE) cell samples. In some embodiments, the cell sample includes fixed and permeabilized cell samples.

[0016] This disclosure provides an amplification-free probe complex comprising: (i) at least one modified oligonucleotide; and (ii) at least one analyte-binding moiety capable of binding a cellular target analyte, wherein the at least one modified oligonucleotide is linked to the at least one analyte-binding moiety, wherein the at least one modified oligonucleotide comprises two or more tandem copies of a sequencing primer binding site, wherein the at least one modified oligonucleotide comprises an optional barcode sequence, wherein the at least one modified oligonucleotide comprises a canonical nucleobase located upstream of the start of each sequencing primer binding site, and wherein the at least one modified oligonucleotide comprises a base-free site located upstream of the canonical nucleobase.

[0017] In some embodiments of the amplification-free probe complex disclosed herein, the analyte-binding moiety may bind lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the analyte-binding moiety includes a lipid moiety, wheat germ lectin, antibody, or biotin moiety. In some embodiments, the analyte-binding moiety includes a secondary antibody or a primary antibody. In some embodiments, the analyte-binding moiety includes a polynucleotide containing a sequence complementary to the target nucleic acid.

[0018] This disclosure provides a method for detecting a target analyte on or inside a cell sample, comprising: (a) providing a plurality of amplification-free probe complexes of the present disclosure; (b) providing a cell sample deposited on a support, wherein the cell sample contains a plurality of analytes, the plurality of analytes including at least one target analyte; (c) contacting the cell sample with the plurality of amplification-free probe complexes, wherein the contact is performed under conditions suitable for binding an analyte-binding portion of the amplification-free probe complex to its homologous target analyte, wherein the target analyte is on or inside the cell sample; (d) contacting a plurality of modified oligonucleotides of the plurality of amplification-free probe complexes of step (c) with a plurality of sequencing primers under conditions suitable for each sequencing primer to bind to its homologous sequencing primer binding site on the modified oligonucleotide to form a plurality of nucleic acid duplexes, wherein each nucleic acid duplex contains a sequencing primer binding site on the modified oligonucleotide that hybridizes to the sequencing primer; (e) contacting the cell sample with the amplification-free probe complex; Multiple nucleic acid duplexes are contacted with multiple sequencing polymerases and multiple detectably labeled multivalent molecules, wherein each nucleic acid duplex binds to the sequencing polymerase and the nucleotide portion of the multivalent molecule, thereby forming multiple detectably labeled complexes, wherein the nucleotide portion of the multivalent molecule is bound to the 3' end of the sequencing primer and is located opposite the canonical nucleobase in the modified oligonucleotide, wherein each multivalent molecule contains a core connected to multiple nucleotide arms, wherein each nucleotide arm contains a nucleotide portion, wherein the contact in step (e) is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the nucleotide portion into the 3' end of the sequencing primer, and wherein the complementary nucleotide portion of the multivalent molecule is bound opposite the nucleotide in the given modified oligonucleotide, and the complementary nucleotide portion of the multivalent molecule is not incorporated into the 3' end of the sequencing primer; and (f) the cell sample bound to the multiple detectably labeled complexes is imaged, thereby detecting the target analyte on or inside the cell sample. Attached Figure Description

[0019] The features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of this disclosure are utilized, and in the accompanying drawings:

[0020] Figure 1Schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of multivalent molecules with a "starburst" or "helter-skelter" configuration. Middle (Class II): Schematic diagram of multivalent molecules with a dendritic macromolecular configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by the reaction of streptavidin with 4-arm or 8-arm PEG-NHS, biotin, and dNTPs. The nucleotide moiety is designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0021] Figure 2 A schematic diagram of an exemplary multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0022] Figure 3 This is a schematic diagram of an exemplary multivalent molecule comprising a dendritic core attached to multiple nucleotide arms.

[0023] Figure 4A A schematic diagram of an exemplary multivalent molecule is shown, comprising a core attached to multiple nucleotide arms, wherein each nucleotide arm includes a core attachment portion, a spacer, a linker, and a nucleotide portion.

[0024] Figure 4B This is a schematic diagram of an exemplary nucleotide arm of a multivalent molecule, wherein the nucleotide arm includes a core attachment portion, a spacer, a linker, and a nucleotide portion (e.g., a nucleotide unit).

[0025] Figure 5A A schematic diagram of an exemplary multivalent probe is shown, comprising a core attached to multiple nucleotide arms, wherein the nucleotide arms include a core attachment portion, a spacer, a linker, and a target-specific oligonucleotide probe.

[0026] Figure 5B This is a schematic diagram of an exemplary nucleotide arm of a multivalent probe that includes a core attachment portion, a spacer, a linker, and a target-specific oligonucleotide probe.

[0027] Figure 6 The chemical structures of exemplary spacers (top) and various exemplary connectors, including 11-atom connectors, 16-atom connectors, 23-atom connectors and N3 connectors (bottom), are shown.

[0028] Figure 7 The chemical structures of various exemplary connectors (including connectors 1 to 9) are shown.

[0029] Figure 8 The chemical structures of various exemplary connectors that join / attach to nucleotide moieties are shown.

[0030] Figure 9The chemical structures of various exemplary connectors that join / attach to nucleotide moieties are shown.

[0031] Figure 10 The chemical structures of various exemplary connectors that join / attach to nucleotide moieties are shown.

[0032] Figure 11 The chemical structures of various exemplary connectors that join / attach to nucleotide moieties are shown.

[0033] Figure 12 The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide portion is attached to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base.

[0034] Figure 13A This is a schematic diagram of an embodiment comprising an analyte-binding moiety attached to a modified oligonucleotide. The modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. The modified oligonucleotide may contain one or more base-free sites represented by hollow triangles. The modified oligonucleotide may contain one or more canonical nucleobases represented by solid arrows.

[0035] Figure 13B for Figure 13A The diagram shows a single amplification-free probe complex with multiple sequencing primers that hybridize with modified oligonucleotides. XXX indicates a modified nucleotide or modified nucleotide bond, hollow triangles represent base-free sites, and solid arrows indicate canonical nuclei.

[0036] Figure 14A This is a schematic diagram of an embodiment of an amplification-free probe complex comprising an analyte-binding portion (e.g., a secondary antibody) attached to a modified oligonucleotide. The modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. The modified oligonucleotide may contain one or more base-free sites represented by hollow triangles. The modified oligonucleotide may contain one or more canonical nucleobases represented by solid arrows.

[0037] Figure 14B for Figure 14A The diagram shows a single amplification-free probe complex with multiple sequencing primers that hybridize with modified oligonucleotides. XXX indicates a modified nucleotide or modified nucleotide bond, hollow triangles represent base-free sites, and solid arrows indicate canonical nuclei.

[0038] Figure 15AThis is a schematic diagram of an embodiment comprising an analyte-binding moiety attached to a modified oligonucleotide. The modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. The modified oligonucleotide may contain one or more base-free sites represented by hollow triangles. The modified oligonucleotide may contain one or more canonical nucleobases represented by solid arrows.

[0039] Figure 15B for Figure 15A The diagram shows a single amplification-free probe complex with multiple sequencing primers that hybridize with modified oligonucleotides. XXX indicates a modified nucleotide or modified nucleotide bond, hollow triangles represent base-free sites, and solid arrows indicate canonical nuclei.

[0040] Figure 16 This is a schematic diagram of an embodiment comprising an analyte-binding moiety attached to multiple modified oligonucleotides. Each modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. Each modified oligonucleotide may contain one or more base-free sites represented by hollow triangles. Each modified oligonucleotide contains one or more canonical nucleobases represented by solid arrows. The multiple modified oligonucleotides can hybridize with multiple sequencing primers, as shown in the figure.

[0041] Figure 17 This is a schematic diagram of an example of a branched, amplification-free probe complex comprising analyte binding moieties attached to a plurality of modified oligonucleotides. Each modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. Each modified oligonucleotide may contain one or more base-free sites represented by hollow triangles. Each modified oligonucleotide may contain one or more canonical nucleobases represented by solid arrows. The plurality of modified oligonucleotides can hybridize with a plurality of sequencing primers, as shown in the figure.

[0042] Figure 18AThis is a schematic diagram of an embodiment of a modified oligonucleotide comprising at least one sequencing primer binding site and an optional barcode sequence. The modified oligonucleotide may contain multiple sequencing primer binding sites. The modified oligonucleotide may contain a conjugate portion for attachment to an analyte binding moiety. The modified oligonucleotide may contain a linker portion, for example, to attach one end of the modified oligonucleotide to the conjugate portion. The modified oligonucleotide may contain a canonical nucleobase located upstream of the origin of each sequencing primer binding site. For example, the canonical nucleobase may be located upstream of the origin of each sequencing primer binding site. Canonical nucleobases in the modified oligonucleotide are indicated by solid arrows. The at least one modified oligonucleotide may contain a base-free site located upstream of the canonical nucleobase. For example, the base-free site may be located upstream of the canonical nucleobase. Base-free sites in the modified oligonucleotide are indicated by hollow triangles.

[0043] Figure 18B For multivalent molecules carrying sequencing polymerase and detectably labeled, such as Figure 18A The diagram illustrates the same modified oligonucleotide and sequencing primer. The sequencing primer hybridizes with the modified oligonucleotide, thereby forming a nucleic acid duplex. A sequencing polymerase binds to the nucleic acid duplex and to the complementary nucleotide portion (NT) of a multivalent molecule located opposite (e.g., complementary) to the nucleotide in the modified oligonucleotide, thereby forming a detectably labeled complex. The detectably labeled complex emits a detectable signal. The modified oligonucleotide may contain at least one base-free site that inhibits a second binding event of another detectably labeled multivalent molecule, such that a nucleotide portion of the detectably labeled multivalent molecule binds to a nucleic acid duplex and a sequencing polymerase to emit a signal associated with a detectably labeled complex.

[0044] Figure 19A This is a schematic diagram of an embodiment comprising an analyte-binding moiety attached to a modified oligonucleotide. The modified oligonucleotide may contain at least one modified nucleotide or modified nucleotide bond represented by XXX. The modified oligonucleotide may contain one or more target barcode sequences.

[0045] Figure 19B This is a schematic diagram of an embodiment comprising a portion of a modified oligonucleotide including at least one sequencing primer binding site, a target barcode sequence, and a linker and conjugate portion. XXX represents one or more modified nucleotides or modified nucleotide bonds.

[0046] Figure 20 This is a schematic diagram of an embodiment of a multiple amplification-free probe complex that binds to a cell sample. This multiple amplification-free probe complex can bind to the outer cell membrane, such as... Figure 20As shown in the diagram. Alternatively or additionally, the plurality of non-amplified probe complexes can enter cells and bind to the cell membranes of organelles (including, for example, the nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, peroxisomes, or lysosomes) located within the cell sample.

[0047] Figure 21 Tables illustrating several embodiments of barcode sequences that can be used to simultaneously detect and identify two or more cellular target analytes (e.g., cellular structures) by performing a single binding cycle and employing multicolor imaging. In some embodiments, Figure 21 The barcode sequences listed in the table can be used for cell staining. Figure 21 The sequences in the sequence from top to bottom are: CTACCCGTGGTG (SEQ ID NO: 1); TCAAAATGGGGT (SEQ ID NO: 2); CATCACTGTGGG (SEQ ID NO: 3); CCCTCAGTGTGG (SEQ ID NO: 4); AAACCTTGGTTT (SEQ ID NO: 5). Detailed Implementation

[0048] definition

[0049] The headings provided herein are not intended to limit any aspect of this disclosure, which is to be understood by referring to the entire specification.

[0050] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms related to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000) on bacterial-mediated plant transformation and regeneration. Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with the laboratory procedures and techniques described herein is that which is well-known and commonly used in the art.

[0051] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless explicitly and definitively limited to one referent, the singular forms “a” and “the” and any other word used in the singular shall include multiple referents.

[0052] It should be understood that the use of alternative terms (e.g., "or") is considered to refer to one or both of the alternatives or any combination thereof.

[0053] The term “and / or” as used herein shall be regarded as meaning that each of the specified features or components is explicitly disclosed with or without the other. For example, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). Similarly, “and / or” as used herein in terms such as “A, B and / or C” is intended to cover each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0054] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be substituted for or added to the listed items. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0055] As used herein, the terms “about” and “approximately” mean a value or composition within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” or “approximately” may mean within one or more standard deviations. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subranges may include the endpoints of the range and / or subranges.

[0056] As used herein, "corresponding to" or "corresponds to" means two or more entities whose identities are sufficiently related, such that the identity of one entity can be used to determine the identity, location, and / or other attributes of another entity. As a non-limiting example, if fluorophore color can be used to determine the identity of a barcode sequence, then it can be said that the barcode sequence corresponds to a specific target analyte or fluorophore color, and similarly, the barcode sequence can be used to determine the identity of a target analyte.

[0057] As used herein, the term "polymerase" and its variants include enzymes comprising a domain that binds a nucleotide (or nucleoside), wherein the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. Polymerases may have one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand substitution activity, and nucleotide binding and recognition. A polymerase can be any enzyme capable of catalyzing the polymerization of nucleotides (including their analogs) into a nucleic acid chain. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase contains other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand substitution activity. Polymerases may include, but are not limited to: naturally occurring polymerases and any subunits and truncated forms thereof, mutant polymerases, variant polymerases, recombinant, fusion, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases may include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing nucleotide-binding domains. In some embodiments, polymerases may be isolated from cells or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotes, eukaryotes, viruses, or bacteriophages. In some embodiments, polymerases may be post-translational modified proteins or functional fragments thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses, or bacteriophages. Polymerases may include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0058] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as used herein, and other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids may include recombinant and chemically synthesized forms. Nucleic acids may be isolated. Nucleic acids may include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids (PNAs) and non-naturally occurring nucleotide analogs), chimeric forms containing DNA and RNA, and combinations thereof. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may include polymers of nucleotides, wherein the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids contain naturally occurring nucleoside bonds, such as, but not limited to, phosphodiester bonds. Nucleic acids may lack phosphate ester groups. Nucleic acids may contain non-natural nucleoside bonds, including thiophosphate, thiophosphate, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids comprise one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0059] As used herein, the terms “operably linked” and “operably conjoined” or related terms refer to the juxtaposition of components. The juxtaposed components may be covalently linked together. For example, and not limited to, two nucleic acid components may be enzymatically linked together, wherein the bond conjoining the two components includes a phosphodiester bond. A first nucleic acid component and a second nucleic acid component may be linked together, wherein the first nucleic acid component may confer function to the second nucleic acid component. For example, and not limited to, a bond between a primer-binding sequence and a target sequence forms a nucleic acid library molecule having a primer-binding portion. In another non-limiting example, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) may be linked to a vector, wherein the bond allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. In some embodiments, the vector includes at least one host cell regulatory sequence, which includes a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signaling sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of transgene expression.

[0060] As used herein, the terms “linked,” “conjoined,” “attached,” “additional,” and variations thereof encompass any type of fusion, binding, attachment, or association between any combination of compounds or molecules that possesses sufficient stability to withstand use in a particular procedure. Such procedures may include, but are not limited to: nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of chain terminations); washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, but not limited to, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and so on. In some embodiments, such bonds occur intramolecularly, for example, by linking the ends of single-stranded or double-stranded linear nucleic acid molecules together to form a cyclic molecule. In some embodiments, such bonds may occur between combinations of different molecules or between molecules and non-molecules, including, but not limited to: bonds between nucleic acid molecules and solid surfaces; bonds between proteins and detectable reporter moieties; bonds between nucleotides and detectable reporter moieties; and so on. Suitable bonds are known in the art, and some examples of bonds can be found, for example but not limited to, the following literature: Hermanson, G., “Bioconjugate Techniques”, 2nd edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0061] As used herein, the term "primer" and related terms refer to oligonucleotides capable of hybridizing with DNA and / or RNA polynucleotide templates to form double-stranded molecules. Primers may include natural nucleotides and / or nucleotide analogs. Primers may be recombinant nucleic acid molecules. Primers may have any length, but typically range from about 4 to 50 nucleotides. Typical primers contain a 5' end and a 3' end. The 3' end of a primer may contain a 3' OH portion that acts as the initiation site for nucleotide polymerization in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer may lack a 3' OH portion, or may contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide or more along the length of the primer may be labeled with a detectable reporter portion (e.g., a fluorophore). Primers may be in solution (e.g., soluble primers) or may be immobilized to a support (e.g., a capture primer).

[0062] As used herein, the terms “template nucleic acid,” “template polynucleotide,” “template strand,” and other variations refer to the nucleic acid strand that serves as the basis for polymerase chain reaction or sequencing methods employing a templated strand extension step, etc. The template nucleic acid can be single-stranded or double-stranded, or it can have a single-stranded or double-stranded portion. The template nucleic acid can be obtained from naturally occurring sources, in recombinant forms, or chemically synthesized to include any type of nucleic acid analogue. The template nucleic acid can be linear, circular, or other suitable forms. In some embodiments, the template nucleic acid can be in a multiplicative form. The template nucleic acid can include an insert portion having an insert sequence. The template nucleic acid can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organelle (e.g., mitochondrial, chloroplast, or ribosome) recombinant molecules, cloned, amplified cDNA, RNA (such as precursor mRNA or mRNA), oligonucleotides, and whole-genome DNA obtained from fresh frozen paraffin-embedded tissue (FFPE), puncture biopsy, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library. It allows template nucleic acids to undergo nucleic acid analysis (including sequencing and composition analysis).

[0063] In some embodiments, any of the amplification primer sequence, sequencing primer sequence, barcode sequence, or spatial barcode sequence may be about 3 to 50 nucleotides in length, or about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length, or any range thereof. In some embodiments, any of the aforementioned sequences may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.

[0064] As used herein, the term "universal sequence" and related terms refer to a sequence common to two or more polynucleotide molecules in a nucleic acid molecule. For example, but not limited to, an adaptor having a universal sequence can operatively bind to multiple polynucleotides such that a co-bound population of molecules carries the same universal adaptor sequence. Non-limiting examples of universal adaptor sequences may include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble primers or immobilized capture primers).

[0065] As used herein, the term "selective binding" in the context of any binding agent (e.g., the oligonucleotides or oligonucleotide complexes of this disclosure) refers to a binding agent that specifically binds to a target (e.g., a target sequence) with high affinity and does not significantly bind to other unrelated targets or sequences. Those skilled in the art will understand that a binding agent that specifically binds to a target with high affinity but binds to non-target (off-target) sequences with appropriately low affinity can still be described as selectively binding to the target.

[0066] As used herein, the term "target polynucleotide" refers to any nucleic acid within a cell sample that has a sequence capable of binding at least one non-amplified probe complex. Target polynucleotides include, but are not limited to, RNA, cDNA, and DNA. In some embodiments, target polynucleotides comprise polynucleotides expressed within the cell sample, including, but not limited to, naturally occurring polynucleotides and recombinant polynucleotides.

[0067] As used herein, the term "target analyte" means any analyte that can be bound to the analyte-binding portion of the amplification-free probe complex disclosed herein. Exemplary target analytes may be located on the cell surface or inside the cell. Exemplary target analytes include, but are not limited to, polynucleotides, proteins, lipids, polysaccharides, etc. When used with respect to a primary antibody, the term "target analyte" means any analyte that can be bound to that primary antibody.

[0068] The terms "specific binding" or "specific binding activity" and related terms refer to two molecules that form a relatively stable complex under assay conditions, such as the analyte-binding moiety and the target analyte. The term also applies to cases where the antigen-binding domain of an antibody is specific to a particular epitope carried by many antigens; in such cases, an antibody carrying that antigen-binding domain will be able to bind to a variety of antigens carrying that epitope. Specific binding is characterized by high affinity and low to moderate capacity. As an example, when the affinity constant is approximately 1 × 10⁻⁶... -6 M, usually at least about 1×10 -7 M, usually at least about 1×10 -8 M, preferably at least about 1×10 -9 M or 1×10 -10 When M is smaller, binding is considered to be specific.

[0069] As used herein, the term "target sequence" refers to a sequence in a modified oligonucleotide or multivalent molecule that can bind a multivalent probe. In some embodiments, the multivalent probe comprises a core attached to a plurality of probe arms, wherein each probe arm comprises a polymer linked to a target-specific oligonucleotide probe. In some embodiments, the target sequence includes a barcode sequence, a sample index sequence, and / or a batch barcode sequence.

[0070] As used herein, with respect to nucleotide sequences, "upstream" refers to the sequence located at the 5' end of a particular reference feature within the nucleotide sequence. Similarly, "downstream" refers to the sequence located at the 3' end of a particular reference feature. "Immediately upstream" or "downstream" refers to a nucleotide or sequence located approximately 1 to 10 nucleotides (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) upstream or downstream of a reference feature.

[0071] As used herein, the term "sequencing read product" refers to a primer extension product generated by a sequencing reaction using sequencing primers, a sequencing polymerase, and multiple nucleotides that hybridize with a template molecule to be sequenced (e.g., a modified oligonucleotide or polynucleotide molecule). In some embodiments, the sequencing polymerase uses the 3' end of the sequencing primer as a start site to catalyze nucleotide incorporation and generate an extension product containing a sequence complementary to the template molecule. In some embodiments, the nucleotide incorporation reaction extends the sequencing primer by one nucleotide. In some embodiments, the number of nucleotide incorporation reactions performed determines the length of the sequencing read product. For example, performing eleven nucleotide incorporation reactions will generate a sequencing primer extended by eleven nucleotides. In some embodiments, one cycle of the sequencing reaction includes performing a single nucleotide incorporation reaction using a sequencing polymerase and nucleotides, thereby extending the sequencing primer by one nucleotide. In some embodiments, a cycle of the sequencing reaction includes: (i) binding a multivalent molecule to the 3' end of a first sequencing primer and a sequencing polymerase under conditions that inhibit polymerase-catalyzed nucleotide incorporation, wherein the multivalent molecule comprises a plurality of nucleotide arms attached to a core; (ii) removing the multivalent molecule and the sequencing polymerase while retaining a template molecule that hybridizes with the sequencing primer; and (iii) performing a nucleotide incorporation reaction using a second sequencing polymerase and nucleotides, thereby extending the sequencing primer by one nucleotide.

[0072] When used herein to refer to nucleic acid molecules, the terms “hybridize,” “hybridizing,” “hybridization,” or other related terms refer to the hydrogen bonding between two distinct nucleic acids to form a double-stranded nucleic acid. Hybridization can also include hydrogen bonding between two distinct regions of a single nucleic acid molecule to form a self-hybridized molecule with a double-stranded region. Hybridization can include Watson-Crick or Hoogstein binding to form a double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The two distinct regions of a double-stranded nucleic acid, or a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands do not need to hybridize across their entire length. Complementary base pairing can be standard AT or CG base pairing or other forms of base pairing interactions. Double-stranded nucleic acids may contain mismatched base-paired nucleotides.

[0073] When used herein to refer to nucleic acids, the terms “extend,” “extending,” “extension,” and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3' OH end of a nucleic acid chain, resulting in the extension of the nucleic acid chain. Nucleotide incorporation can be performed using native nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules known in the art can be used, including but not limited to primer extension catalyzed by DNA polymerase or RNA polymerase.

[0074] As used herein, the term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate ester group. Canonical or non-canonical nucleotides are used in accordance with this terminology. In some embodiments, phosphate esters include monophosphate, diphosphate, or triphosphate esters, or corresponding phosphate ester analogs. As used herein, the term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides may be unlabeled or partially labeled with a detectable reporter substance.

[0075] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants or analogues thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Husstein hydrogen bonds with suitable complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethylene adenine, N... 6 -Δ 2-Isopentenyladenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -Methyladenine, guanine (G), isoguanine, N 2 -Dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6 -Methylguanine; 7-denitro-purine, such as 7-denitro-adenine (7-denitro-A) and 7-denitro-guanine (7-denitro-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Further exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385–394, CRC Press, Boca Raton, Fla.

[0076] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016), and other sugar moieties (Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284: 2118-2124; and US Patent No. 5,558,991). In some embodiments, the sugar moiety includes ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or otherwise modified sugars.

[0077] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by intermediate O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group, which includes O, S, or BH3. In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, which includes phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide.

[0078] The terms "reporter moiety," "reporter moieties," or related terms refer to compounds that generate or cause the generation of a detectable signal. A reporter moiety is sometimes referred to as a "tag." Any suitable reporter moiety known in the art may be used, including but not limited to luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemical, mass spectrometry, Raman, hapten, affinity tags, atomic, or enzyme reporter moieties. A reporter moiety may generate a detectable signal caused by a chemical or physical change, such as heat, light, electricity, pH, salt concentration, enzyme activity, or proximity events. Proximity events may include two reporter moieties coming close to each other, associating with each other, or binding together. Those skilled in the art are well aware of selecting reporter moieties such that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties with spectrally distinct emission profiles or with minimal overlap spectral emission profiles may be selected. The reporter portion may be linked (e.g., operatively linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases) or supports (e.g., surfaces).

[0079] In some embodiments, the report portion (or label) includes a fluorescent label or fluorophore. Exemplary fluorescent portions that can serve as fluorescent markers or fluorophores include, but are not limited to, fluoresceins and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamide-fluorescein, fluorescein maleimide, SAMSA-fluorescein, aminothiourea fluorescein, hydrazine methylthioacetamide fluorescein; rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas red sulfonyl chloride, Texas red hydrazine; coumarins and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazine; BODIPY® and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FLC3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and its derivatives, such as Cascade Blue acetyl triazine, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and its derivatives, such as fluorescein iodoacetamide, fluorescein CH; anthocyanins and their derivatives, such as indolonyl anthocyanin dyes, benzoindolonyl anthocyanin dyes, pyridinyl anthocyanin dyes, thiazolonyl anthocyanin dyes, quinolinelonyl anthocyanin dyes, imidazolonyl anthocyanin dyes, Cy3, Cy5; lanthanide chelates and their derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT; europium chelates, terbium chelates; Alexa Fluor® dyes, DyLight® dyes, Atto TM Dyes, LightCycler® Red dye, CAL Flour dye, JOE and its derivatives, Oregon Green TMDyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, diphenylethylene, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as the fluorescent moieties described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edition, Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd edition, or derivatives thereof; or any combination thereof. Anthocyanin dyes may be present in sulfonated or non-sulfonated forms and consist of two pseudoindole, benzoindoline, pyridinium, thiazoline, and / or quinolinon groups separated by a polymethystylene bridge between two nitrogen atoms.Commercially available anthocyanin fluorophores include, for example, Cy3 (which may contain 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-yl}prop-1-en-1-yl)-3,3-dimethyl-3H-indol cation or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{ ...3,3-dimethyl-1,3-dihydro-2H-indol-2-yl}prop-1-en-1-yl)-3,3-dimethyl-3H-indol-cation or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3 [pyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfonyl-1,3-dihydro-2H-indole-2-ylidene]prop-1-en-1-yl)-3,3-dimethyl-3H-indole cation-5-sulfonate), Cy5 (which may contain 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-ylidene)pentyl -1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-cation or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonoindoline-2-ylidene)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-cation-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) -2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indole-2-ylidene)hept-1,3,5-trien-1-yl]-3H-indole cation or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfonyl-1,3-dihydro-2H-indole-2-ylidene)hept-1,3,5-trien-1-yl]-3H-indole cation-5-sulfonate), where "Cy" stands for 'cyanine', and the first number indicates the number of carbon atoms between the two pseudoindole groups. Cy2 is an oxazole derivative rather than a pseudoindole, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. The other fluorophores are described in WO 2024 / 124008, the contents of which are incorporated herein by reference in their entirety.

[0080] In some embodiments, the reporter portion may be a fluorescence resonance energy transfer (FRET) pair, enabling multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0081] When used herein to refer to nucleic acids, the terms “amplify,” “amplifying,” “amplification,” and other related terms include generating multiple copies of the original polynucleotide template molecule, wherein the copies contain sequences complementary to the template sequence or sequences identical to the template sequence. In some embodiments, the copies contain sequences substantially identical to or substantially identical to sequences complementary to the template sequence.

[0082] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or cellular samples for assays and / or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), peptides, carbohydrates, lipids, single cells, or multiple cells. Examples of cellular samples include, but are not limited to: saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, swabs, fluids from tissues or organs, and tissue samples (e.g., biopsy samples).

[0083] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0084] In some embodiments, the surface of the support may be substantially smooth. In some embodiments, the support may have a regular or irregular texture, including bumps, etched surfaces, holes, three-dimensional supports, or any combination thereof.

[0085] In some embodiments, the support comprises beads of any shape, including spherical, hemispherical, cylindrical, barrel-shaped, annular, disc-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or linear.

[0086] The support can be made of any material, including but not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic substrates are contemplated.

[0087] As used herein, the term "retention time" and related terms refer to the length of time a binding complex formed between a target nucleic acid binding component, polymerase, or conjugated or unconjugated nucleotide remains stable without any binding component dissociating from the binding complex. Retention time indicates the stability of the binding complex and the strength of the binding interaction. Retention time can be measured by observing the onset and / or duration of the binding complex, such as by observing a signal from the labeled component of the binding complex. For example, but not limited to, a labeled nucleotide or a labeled reagent containing one or more nucleotides may be present in the binding complex, thus allowing a signal from the label to be detected during the retention time of the binding complex. One non-limiting exemplary label is a fluorescent label. Such labels used for detection may also be referred to herein as "detectable labels".

[0088] No amplification probe complex

[0089] This disclosure provides compositions comprising at least one amplification-free probe complex and methods for detecting target analytes using the at least one amplification-free probe complex. In some embodiments, the target analyte may be localized inside a cell sample and / or on a cell membrane. In some embodiments, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the target analyte is localized at any location in the cell sample, including but not limited to the cytoplasm and nucleus. In some embodiments, the target analyte is localized on, but not limited to, any cell membrane or in the nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, peroxisomes, or lysosomes. In some embodiments, the cell sample includes, for example, but not limited to, a single cell, multiple cells, tissues, organs, tumors, or portions thereof. In some embodiments, the amplification-free probe complex does not undergo amplification, such as rolling circle amplification. In some embodiments, the amplification-free probe complex does not generate amplicones (e.g., multiply molecules).

[0090] This disclosure provides an amplification-free probe complex comprising: (i) at least one modified oligonucleotide; and (ii) at least one analyte-binding moiety capable of binding a target analyte. In some embodiments, the at least one modified oligonucleotide is attached to at least one analyte-binding moiety (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 and Figure 17 In some embodiments, the at least one modified oligonucleotide is attached to at least one analyte binding site via a conjugation portion and / or a linker. In some embodiments, the at least one modified oligonucleotide contains at least one primer binding site (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0091] Amplification-free probe complexes have numerous applications, including but not limited to the detection and imaging of target analytes on and / or inside cells (e.g., Figure 20 ).

[0092] In some embodiments, each amplification-free probe complex comprises at least one modified oligonucleotide (e.g., Figure 18A and Figure 18B In some embodiments, the at least one modified oligonucleotide comprises a nucleic acid, including recombinant and chemically synthesized forms. In some embodiments, the modified oligonucleotide comprises DNA, RNA, or DNA and RNA. In some embodiments, the modified oligonucleotide comprises a canonical nucleotide and / or a nucleotide analog. In some embodiments, the modified oligonucleotide comprises a polymer of nucleotides, wherein the nucleotides comprise natural or non-natural bases and / or sugars. In some embodiments, the modified oligonucleotide comprises a naturally occurring nucleoside internucleotide bond, such as, but not limited to, a phosphodiester bond. In some embodiments, the modified oligonucleotide comprises a 5' phosphate group or lacks a 5' phosphate group. In some embodiments, the modified oligonucleotide comprises a non-natural nucleoside internucleotide bond, including a thiophosphate, a thiol phosphate, or a peptide nucleic acid (PNA) bond.

[0093] In some embodiments, the modified oligonucleotide comprises a linear oligonucleotide (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B and Figure 16 ) or branched oligonucleotides (e.g., Figure 17 ).

[0094] In some embodiments, the at least one modified oligonucleotide includes a conjugation portion at one end to enable attachment to a cell-binding portion (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide includes a linker motif (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide comprises a modified nucleotide at one end, wherein the modified nucleotide confers nuclease resistance to the modified oligonucleotide (e.g., Figure 18A Exemplary modified nucleotides conferring nuclease resistance include, but are not limited to, at least one phosphate-thioester bond and / or at least one 2'-O-methylcytosine base. For example, a modified oligonucleotide may contain at least one phosphate-thioester bond at its 5' or 3' end, which makes the modified oligonucleotide resistant to nuclease degradation. In some embodiments, the modified oligonucleotide contains two to five or more consecutive phosphate-thioester diester bonds at its 5' end. In some embodiments, the oligonucleotide contains at least one ribonucleotide and / or at least one 2'-O-methyl, 2'-O-methoxyethyl (MOE), 2'-fluoronucleotide. In some embodiments, the 3' region of the oligonucleotide contains at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed elongation. Figure 18A In this context, one or more modified nucleotides or modified nucleotide bonds are designated as XXX.

[0095] In some embodiments, the modified oligonucleotide includes at least one sequencing primer binding site, wherein each sequencing primer binding site is located immediately adjacent to the canonical nucleobase and the no-base site. Figure 18A Each sequencing primer binding site can hybridize with a sequencing primer to form a nucleic acid duplex, which can be used for polymerase-catalyzed sequencing reactions by binding a detectably labeled multivalent molecule to the 3' end of the sequencing primer at a position opposite the canonical nucleobase. A base-free site inhibits a second sequencing reaction. The amplification-free probe complex is designed to restrict the sequencing reaction to a single binding event using a detectably labeled multivalent molecule. In some embodiments, the amplification-free probe complex comprises: (i) at least one modified oligonucleotide comprising at least one sequencing primer binding site, at least one canonical nucleobase, and at least one base-free site; and (ii) at least one analyte-binding portion capable of binding a target analyte. In some embodiments, the at least one modified oligonucleotide comprises a canonical nucleobase located upstream of the start of each sequencing primer binding site. In some embodiments, the canonical nucleobase is located upstream of the start of each sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide comprises a base-free site located upstream of the canonical nucleobase. In some embodiments, the base-free site may be located upstream of the canonical nucleobase.

[0096] In some embodiments, the at least one modified oligonucleotide contains one or more sequencing primer binding sites (e.g., Figure 18AIn some embodiments, the sequencing primer binding site includes a universal sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide contains two or more tandem copies of the sequencing primer binding site (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide comprises a multiplicative molecule having two or more tandem copies having a sequencing primer binding site (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide comprises a canonical nucleobase located upstream of the start of the respective sequencing primer binding site (e.g., Figure 18A In some embodiments, the canonical nucleobase is positioned upstream of the start of the binding site of each sequencing primer (e.g., Figure 18A For example, the at least one modified oligonucleotide comprises a canonical nucleobase located at one to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides at the 5' end of each sequencing primer binding site. Alternatively, the at least one modified oligonucleotide comprises a canonical nucleobase located at and immediately adjacent to the 5' end of each sequencing primer binding site, i.e., a 5' nucleotide located at and immediately adjacent to the 3' end of the bound sequencing primer on the complementary strand. In some embodiments, the at least one modified oligonucleotide comprises a base-free site located upstream (i.e., 5') of the canonical nucleobase (e.g., Figure 18A In some embodiments, the no-base site is located immediately adjacent to and adjacent to the 5' of the canonical nucleobase. In some embodiments, the no-base site is located immediately upstream of the canonical nucleobase (e.g., Figure 18A ).

[0097] In some embodiments, the modified oligonucleotide of any of the amplification-free probe complexes contains one or more sequencing primer binding sites. In some embodiments, each sequencing primer binding site includes a universal sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide contains two or more tandem copies of the sequencing primer binding site. In some embodiments, the two or more copies of the sequencing primer binding site may have the same sequence or different sequences.

[0098] In some embodiments, the amplification-free probe complex comprises at least one modified oligonucleotide containing at least one sequencing primer binding site, at least one target barcode sequence, and a conjugation motif (e.g., a barcode-encoded amplification-free probe complex). Figures 19A to 19B In some embodiments, the barcode-enabled amplification-free probe complex comprises similar... Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 and Figure 17 The linear or branched structure of the non-amplified probe complex shown in any of these embodiments. In some embodiments, the conjugation portion may be attached to the analyte-binding portion. In some embodiments, the target barcode sequence of the modified oligonucleotide comprises a short random sequence comprising a length of 3 to 20 nucleotides (e.g., NNN or NNNN) or any range therebetween. The short random sequence is designed, for example, to provide nucleotide diversity and color balance generated by a detectable signal during barcode sequencing.

[0099] In some embodiments, in the short random sequence, each base "N" at a given position is independently selected from A, G, C, T, or U. In some embodiments, the random sequence lacks a continuous repeating sequence with 2 or 3 identical nucleobases, such as AA, TT, CC, GG, UU, AAA, TTT, CCC, GGG, or UUU.

[0100] In some embodiments, the short random sequences in the modified oligonucleotide population include highly diverse sequences containing all four nucleotides (e.g., A, G, C, T, and / or U) in approximately equal proportions, which will be presented in each cycle of the sequencing run.

[0101] In some embodiments, short random sequences (e.g., NNN) include, but are not limited to: AGC, AGT, GAC, GAT, CAT, CAG, TAG, TAC. Those skilled in the art will recognize that more random sequences (e.g., 64 possible combinations) can be prepared, wherein each base "N" at a given position in the random sequence is independently selected from A, G, C, T, or U.

[0102] In some embodiments, a modified oligonucleotide is attached to an analyte-binding moiety. The analyte-binding moiety may bind, but is not limited to, lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the analyte-binding moiety includes a lipid moiety, a wheat germ lectin, an antibody, or a biotin moiety. In some embodiments, the analyte-binding moiety includes a polynucleotide containing a sequence complementary to a target nucleic acid. In some embodiments, the antibody includes a secondary antibody or a primary antibody. In some embodiments, the modified oligonucleotide may be attached to a primary antibody. In some embodiments, the modified oligonucleotide may be attached to a secondary antibody, and the secondary antibody may be attached to / associated with the primary antibody. In some embodiments, the primary antibody binds to a target analyte. In some embodiments, the primary and secondary antibodies may be cross-linked.

[0103] In some embodiments, the analyte binding portion may bind to a lipid membrane (e.g., a phospholipid membrane) containing a lipid portion. In some embodiments, the lipid portion includes a cholesterol portion, a lipid portion, a phospholipid portion, bile acids, thioethers (e.g., hexyl-S-triphenylmethylthiol), mercaptocholesterol, aliphatic chains (e.g., dodecyl glycol or undecyl residues), phospholipids (e.g., di-hexadecyl-racemic-glycerol or 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate triethylammonium), polyamines or polyethylene glycol chains, or adamantaneacetic acid, a palmityl portion, or an octadecylamine or hexylamino-carbonyl-hydroxycholesterol portion.

[0104] In some embodiments, the analyte-binding portion includes an antibody capable of binding a target analyte. In some embodiments, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the antibody includes primary and / or secondary antibodies. In some embodiments, the antibody includes an intact immunoglobulin, an antibody fragment, an antigen-binding portion of an antibody, or a single-chain antibody. The antibody may be a monoclonal or polyclonal antibody. The antibody is capable of specifically binding to the target analyte. In some embodiments, the target analyte includes an intact peptide or peptide fragment. The antibody may contain an antigen-binding region (e.g., a complementary site) that specifically binds to the target analyte. In some embodiments, the target analyte may be localized inside a cell sample or on the membrane of a cell sample, wherein the target analyte includes peptides, lipids, nucleic acids, or polysaccharides. In some embodiments, the target analyte includes peptides, enzymes, or lipids localized at any location in the cell sample (including, but not limited to, the cytoplasm and nucleus). In some embodiments, the target analyte includes polypeptides, enzymes, or lipids located in or on cellular structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0105] Antibodies or immunoglobulins are typically tetrameric molecules comprising two pairs of identical polypeptide chains, each pair including a light chain and a heavy chain. The amino moiety of each chain contains a variable region that associates with each other to form an antigen-binding region (e.g., a complementary site). Thus, a typical immunoglobulin can bind two antigens or two target analytes. The carboxyl moiety of the heavy chain contains a constant region that associates with each other to form an Fc region for effector function. The Fc moiety of the heavy chain defines the antibody class, which includes isotypes such as IgG, IgM, IgD, IgA, or IgE. The heavy chain and / or light chain can be prepared using recombinant techniques or by immunizing animals with a target antigen.

[0106] As used herein, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies. Such non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be generated recombinantly, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy and light chains. These and other methods for preparing, for example, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single-chain antibodies, and bifunctional antibodies are well known. Additionally, modified or derived antibodies, or antigen-binding fragments of antibodies, are considered to be within the scope of this disclosure. Fab, F(ab')2, Fd, and Fv fragments of antibodies that retain specific binding activity are included within the definition of antibody fragments.

[0107] In some embodiments, the antibody may be produced in a host species, including, for example, rabbit, mouse, rat, goat, sheep, guinea pig, chicken, hamster, donkey, camel, or horse. In some embodiments, the antibody comprises animal-free antibodies produced using recombinant DNA technology. In some embodiments, the antibody is humanized.

[0108] Antibody fragments typically comprise a portion of a complete immunoglobulin capable of binding to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-chain variable fragments (scFv), domain antibodies, VHH, and Fd. In some embodiments, an Fv fragment comprises a variable light chain region (VL) and a variable heavy chain region (VH). In some embodiments, a Fab fragment comprises a monovalent antibody fragment having a variable light chain region (VL), a constant light chain region (CL), a variable heavy chain region (VH), and a first constant region (CH1). In some embodiments, a Fab' fragment comprises a monovalent antibody fragment having at least a portion of a variable light chain region (VL), a constant light chain region (CL), a variable heavy chain region (VH), a first constant region (CH1), a hinge region, and a second constant region (CH2). In some embodiments, an F(ab')2 fragment comprises a bivalent antibody fragment having two Fab fragments connected via a disulfide bridge at the hinge region.

[0109] Single-chain antibodies (scFv) typically comprise a single polypeptide chain (e.g., a monovalent antibody molecule) having a variable light chain region (VL) and a variable heavy chain region (VH) connected via a polypeptide linker (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc.Natl.Acad.Sci.USA 85:5879-83). The amino terminus of a single-chain antibody contains either a variable light chain region (VL) or a variable heavy chain region (VH). In some embodiments, a single-chain antibody comprises an scFv-Fc antibody, which further comprises an antibody hinge region and at least a portion of an Fc region comprising CH2 and / or CH3 regions. In some embodiments, a single-chain antibody comprises an scFv-CH antibody, which further comprises an antibody hinge region and at least a portion of a CH3 region.

[0110] Antibodies and antigen-binding fragments of antibodies can be prepared using any suitable standard techniques, such as the manipulation and expression of DNA encoding the antibody or antigen-binding fragment. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (containing, for example, phage antibody libraries), or can be synthesized. Sequencing and chemical manipulation of DNA, or using molecular biology techniques such as placing one or more variable and / or constant domains in suitable conformations or introducing codons, can produce cysteine ​​residues, modify, add, or delete amino acids, and so on.

[0111] The sequences and structures of suitable antibodies and antibody fragments are known to those skilled in the art and are described, for example, in the KEGG drug database (www.genome.jp / kegg / drug / ) and the protein database (www.rcsb.org / ), as well as other sources known in the art.

[0112] In some embodiments, antibodies can be conjugated to at least one modified oligonucleotide using any well-known linking chemistry. For example, amplification-free probe complexes can be prepared by crosslinking the antibody and the amino groups on the modified oligonucleotide using glutaraldehyde. Lysine side-chain ε-amides are typically targeted to conjugate with the oligonucleotide. In some embodiments, maleimide-modified antibodies can react with thiol-modified oligonucleotides.

[0113] In some embodiments, a homobifunctional or heterobifunctional cross-linking agent may be introduced as a bridge to link the antibody and the modified oligonucleotide together.

[0114] In some embodiments, an amine-amine non-cleavable crosslinking agent comprising disuccinimidyl octanoate (DSS) can be used to attach antibodies to modified oligonucleotides. The DSS comprises an amine-reactive NHS ester at both ends of an 8-atom spacer arm.

[0115] In some embodiments, an MCC (4-[(2,5-dioxopyrrole-1-yl)methyl]cyclohexane-1-carboxamide) can be used to attach an antibody to at least one modified oligonucleotide, wherein the MCC is a thioether linker that links the antibody to the oligonucleotide via a thioether bond.

[0116] In some embodiments, copper-free click chemistry, such as dibenzocyclooctylene (DBCO), can be used to attach antibodies to at least one modified oligonucleotide. The DBCO-modified oligonucleotide can react with an azide-modified antibody to generate an antibody attached to the modified oligonucleotide.

[0117] In some embodiments, the amine-thiohydrogen crosslinker succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC) can be used to attach the antibody to at least one modified oligonucleotide. The thiol-modified oligonucleotide can react with an antibody having an amine-thiol linker to generate an antibody attached to the modified oligonucleotide.

[0118] In some embodiments, chemically unstable adapters (e.g., pH-sensitive adapters), such as hydrazone adapters, can be used to attach antibodies to at least one modified oligonucleotide.

[0119] In some embodiments, the modified oligonucleotide may be attached to the analyte binding site via a linker. In some embodiments, the linker comprises a polymer, including a polyether, polyamine, or polyamide. In some embodiments, the linker comprises triethylene glycol (e.g., a TEG linker) or polyethylene glycol (e.g., a PEG linker).

[0120] In some embodiments, the connector includes polymer spacer arms. In some embodiments, the spacer arms include C3, C6, C9, C12, or C18 spacer arms. In some embodiments, the connector includes a polymer, which includes polyether, polyamine, or polyamide. In some embodiments, the connector includes triethylene glycol (e.g., a TEG connector) or polyethylene glycol (e.g., a PEG connector).

[0121] This disclosure provides compositions comprising at least one amplification-free probe complex that binds to its homologous target analyte. In some embodiments, the at least one amplification-free probe complex binds to its homologous target analyte on or within a cell sample (e.g., Figure 20 ).

[0122] This disclosure provides compositions comprising at least one amplification-free probe complex and at least one sequencing primer. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer, thereby forming at least one nucleic acid double strand. In some embodiments, the sequencing primer comprises a 3' extendable end. In some embodiments, the sequencing primer comprises a 3' non-extendable (blocking) end, which can be converted into a 3' extendable end.

[0123] This disclosure provides a composition comprising at least one amplification-free probe complex, at least one sequencing primer, and at least one sequencing polymerase. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer. In some embodiments, the sequencing primer includes a 3' extendable end. In some embodiments, the sequencing primer includes a 3' non-extendable (blocking) end that can be converted into a 3' extendable end. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer, thereby forming at least one nucleic acid duplex. In some embodiments, the sequencing polymerase binds to at least one nucleic acid duplex.

[0124] This disclosure provides compositions comprising at least one amplification-free probe complex, at least one sequencing primer, at least one sequencing polymerase, and at least one multivalent molecule. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer. In some embodiments, the sequencing primer includes a 3' extendable end. In some embodiments, the sequencing primer includes a 3' non-extendable (blocking) end that can be converted to a 3' extendable end. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer, thereby forming at least one nucleic acid duplex. In some embodiments, the sequencing polymerase binds to at least one nucleic acid duplex. In some embodiments, the multivalent molecule includes at least one detectable label (e.g., a fluorophore). In some embodiments, the complementary nucleotide portion of the detectably labeled multivalent molecule binds to the sequencing polymerase and binds near the 3' end of the sequencing primer, opposite the canonical nucleobase in the modified oligonucleotide, thereby forming a detectably labeled complex. In some embodiments, the nucleotide portion of the multivalent molecule is complementary to the nucleotide in the modified oligonucleotide located at the 5' end and adjacent to the sequencing primer binding site.

[0125] This disclosure provides compositions comprising a target analyte homologous to it (e.g., a target analyte from a cell sample, e.g., ...). Figure 20The process involves at least one amplification-free probe complex, at least one sequencing primer, at least one sequencing polymerase, and at least one multivalent molecule. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer. In some embodiments, the sequencing primer includes a 3' extendable end. In some embodiments, the sequencing primer includes a 3' non-extendable (blocking) end that can be converted to a 3' extendable end. In some embodiments, the at least one amplification-free probe complex hybridizes with at least one sequencing primer, thereby forming at least one nucleic acid duplex. In some embodiments, the sequencing polymerase binds to at least one nucleic acid duplex. In some embodiments, the multivalent molecule includes at least one detectable label (e.g., a fluorophore). In some embodiments, the complementary nucleotide portion of the detectably labeled multivalent molecule binds to the sequencing polymerase and binds near the 3' end of the sequencing primer, opposite the canonical nucleobase in the modified oligonucleotide, thereby forming a detectably labeled complex. In some embodiments, the nucleotide portion of the multivalent molecule is complementary to the nucleotide in the modified oligonucleotide located at the 5' end and adjacent to the sequencing primer binding site.

[0126] Barcode sequences used for cell staining

[0127] This disclosure provides compositions comprising a plurality of amplification-free probe complexes, the plurality of amplification-free probe complexes including at least a first subgroup and a second subgroup of amplification-free probe complexes. In some embodiments, each amplification-free probe complex of the first and second subgroups comprises at least one modified oligonucleotide comprising at least one barcode sequence. In some embodiments, the first subgroup of the amplification-free probe complex comprises a barcode sequence having the same sequence. In some embodiments, the second subgroup of the amplification-free probe complex comprises a barcode sequence having the same sequence. In some embodiments, the barcode sequences of the first and second subgroups have different sequences. The barcode sequences may be designed to enable the simultaneous detection and identification of two or more target analytes by performing a single sequencing cycle and employing multicolor imaging. In some embodiments, the barcode sequences of the amplification-free probe complexes may be used for sequencing-based cell staining.

[0128] In some embodiments, the composition comprises: a plurality of modified oligonucleotides, including: at least a first subgroup and a second subgroup of modified oligonucleotides (e.g., a set including at least a first subgroup and a second subgroup of modified oligonucleotides). In some embodiments, each modified oligonucleotide in the first subgroup comprises a first barcode sequence corresponding to a first target analyte, wherein each modified oligonucleotide in the second subgroup comprises a second barcode sequence corresponding to a second target analyte. In some embodiments, the first barcode is at least 2 nucleotides in length. In some embodiments, the second barcode is at least 2 nucleotides in length. In some embodiments, the sequences of the first barcode and the second barcode comprise different sequences. In some embodiments, a nucleobase at a first position in the first barcode sequence comprises a nucleobase that generates a first color signal in a first sequencing cycle, and a nucleobase at the corresponding first position in the second barcode sequence comprises a nucleobase that generates a second color signal in the same first sequencing cycle. In some embodiments, a nucleobase at a second position in the first barcode sequence includes a nucleobase that generates a second color signal in the second sequencing cycle, and a nucleobase at a corresponding second position in the second barcode sequence includes a nucleobase that generates a first color signal in the same second sequencing cycle. In some embodiments, nucleobases at the first and / or second positions of the first and / or second barcodes are identified based on the first and / or second color signals. In some embodiments, the first color signal at a first corresponding position in the first barcode sequence in the first sequencing cycle identifies a first target analyte, and the first color signal at a second corresponding position in the second barcode sequence in the second sequencing cycle identifies a second target analyte (e.g., see...). Figure 21 (Table). In some embodiments, the sequencing cycle includes forming a detectably labeled complex that generates a color signal. In some embodiments, forming the detectably labeled complex includes hybridizing sequencing primers with modified oligonucleotides, thereby forming a nucleic acid duplex, and binding the nucleic acid duplex to a sequencing polymerase and a complementary nucleotide portion of a multivalent molecule opposite to the nucleotide in the modified oligonucleotide, thereby forming the detectably labeled complex.

[0129] This disclosure provides compositions comprising: a plurality of barcoded oligonucleotides comprising at least a first modified oligonucleotide subgroup, a second modified oligonucleotide subgroup, and a third modified oligonucleotide subgroup (e.g., a set comprising at least a first modified oligonucleotide subgroup, a second modified oligonucleotide subgroup, and a third modified oligonucleotide subgroup). In some embodiments, each modified oligonucleotide in the first subgroup comprises a first barcode sequence corresponding to a first target analyte. In some embodiments, the first barcode is at least 3 nucleotides in length. In some embodiments, each modified oligonucleotide in the second subgroup comprises a second barcode sequence corresponding to a second target analyte. In some embodiments, the second barcode is at least 3 nucleotides in length. In some embodiments, each modified oligonucleotide in the third subgroup comprises a third barcode sequence corresponding to a third target analyte. In some embodiments, the third barcode is at least 3 nucleotides in length. In some embodiments, the first, second, and third barcode sequences comprise different sequences. In some embodiments, a nucleobase at a first position in the first barcode sequence comprises a nucleobase that generates a first color signal in a first sequencing cycle. In some embodiments, a nucleobase at a corresponding first position in the second and third barcode sequences includes a nucleobase that generates a second color signal in the same first sequencing cycle. In some embodiments, a nucleobase at a corresponding second position in the first and third barcode sequences includes a nucleobase that generates a second color signal in the second sequencing cycle. In some embodiments, a nucleobase at a corresponding second position in the second barcode sequence includes a nucleobase that generates a first color signal in the same second sequencing cycle. In some embodiments, a nucleobase at a corresponding third position in the first and second barcode sequences includes a nucleobase that generates a second color signal in the third sequencing cycle. In some embodiments, a nucleobase at a corresponding third position in the third barcode sequence includes a nucleobase that generates a first color signal in the same third sequencing cycle. In some embodiments, a first color signal at a first corresponding position in the first barcode sequence in the first sequencing cycle identifies a first target analyte. In some embodiments, a first color signal at a second corresponding position in the second barcode sequence in the second sequencing cycle identifies a second target analyte. In some embodiments, a first color signal at a third corresponding position in the third barcode sequence in the third sequencing cycle identifies a third target analyte (e.g., see...). Figure 21(Table). In some embodiments, a second color signal in a first sequencing cycle identifies a second target analyte and a third target analyte. In some embodiments, a second color signal in a second sequencing cycle identifies a first target analyte and a third target analyte. In some embodiments, a second color signal in a third sequencing cycle identifies a first target analyte and a second target analyte. In some embodiments, the sequencing cycle includes forming a detectably labeled complex (which generates a color signal). In some embodiments, forming a detectably labeled complex includes hybridizing sequencing primers with a modified oligonucleotide, thereby forming a nucleic acid duplex, and binding the nucleic acid duplex to a sequencing polymerase and a complementary nucleotide portion of a multivalent molecule opposite to the nucleotide in the modified oligonucleotide, thereby forming a detectably labeled complex.

[0130] Detection method using amplification-free probe complex

[0131] This disclosure provides a method for detecting a target analyte using at least one amplification-free probe complex. In some embodiments, the target analyte may be localized inside a cell sample or on a cell membrane. In some embodiments, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides localized at any location in the cell sample, including but not limited to the cytoplasm and nucleus. In some embodiments, the target analyte includes lipids, peptides, nucleic acids, or polysaccharides localized in or on cell structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, peroxisomes, and lysosomes. In some embodiments, the cell sample includes, for example, but not limited to, a single cell, multiple cells, tissue, organ, tumor, or part thereof.

[0132] In some embodiments, a method for detecting a target analyte includes step (a): providing a plurality of amplification-free probe complexes as described herein. In some embodiments, each amplification-free probe complex comprises (i) at least one modified oligonucleotide; and (ii) at least one analyte-binding moiety capable of binding a cellular target analyte, wherein the at least one modified oligonucleotide is attached to the at least one analyte-binding moiety (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 and Figure 17 In some embodiments, the at least one modified oligonucleotide contains at least one primer binding site (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0133] In some embodiments, in step (a) of the method for detecting a target analyte, the at least one modified oligonucleotide comprises a nucleic acid, including recombinant and chemically synthesized forms. In some embodiments, the modified oligonucleotide comprises DNA, RNA, or DNA and RNA. In some embodiments, the modified oligonucleotide comprises a canonical nucleotide and / or a nucleotide analog. In some embodiments, the modified oligonucleotide comprises a polymer of nucleotides, wherein the nucleotides comprise natural or non-natural bases and / or sugars. In some embodiments, the modified oligonucleotide comprises a naturally occurring nucleoside internucleotide bond, such as, but not limited to, a phosphodiester bond. In some embodiments, the modified oligonucleotide comprises a 5' phosphate group or lacks a 5' phosphate group. In some embodiments, the modified oligonucleotide comprises a non-natural nucleoside internucleotide bond, including a thiophosphate, a thiol phosphate, or a peptide nucleic acid (PNA) bond.

[0134] In some embodiments, in step (a) of the method for detecting a target analyte, the modified oligonucleotide includes linear oligonucleotides (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B and Figure 16 ) or branched oligonucleotides (e.g., Figure 17 ).

[0135] In some embodiments, in step (a) of the method for detecting a target analyte, the at least one modified oligonucleotide includes a conjugation portion at one end to enable attachment to a cell-binding portion (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide includes a linker motif (e.g., Figure 18A In some embodiments, the at least one modified oligonucleotide comprises a modified nucleotide at one end, wherein the modified nucleotide confers nuclease resistance to the modified oligonucleotide (e.g., Figure 18A Exemplary modified nucleotides conferring nuclease resistance include, but are not limited to, at least one thiophosphate bond and / or at least one 2'-O-methylcytosine base.

[0136] In some embodiments, the at least one modified oligonucleotide contains one or more sequencing primer binding sites (e.g., Figure 18A In some embodiments, the one or more sequencing primer binding sites include universal sequencing primer binding sites. In some embodiments, the at least one modified oligonucleotide contains two or more tandem copies of the sequencing primer binding site (e.g., Figure 18AIn some embodiments, the at least one modified oligonucleotide comprises a multiplicative molecule having a sequencing primer binding site having two or more tandem copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies). Figure 18A In some embodiments, the at least one modified oligonucleotide comprises a canonical nucleobase located upstream of the start of the respective sequencing primer binding site (e.g., Figure 18A For example, the at least one modified oligonucleotide comprises a canonical nucleotide located at the 5' end of each sequencing primer binding site. In some embodiments, the canonical nucleotide is located immediately upstream of the start of each sequencing primer binding site (e.g., Figure 18A For example, the at least one modified oligonucleotide comprises a canonical nucleobase located at a position within 1 to 10 nucleotides, optionally adjacent to the 5' end of each sequencing primer binding site. In some embodiments, the at least one modified oligonucleotide comprises a base-free site located upstream of the canonical nucleobase (e.g., Figure 18A For example, the abase-free site is located at the 5' end of the canonical nucleobase and within 1 to 10 nucleotides. In some embodiments, the abase-free site is located immediately upstream of the canonical nucleobase (e.g., Figure 18A For example, the baseless site is located at and adjacent to the 5' of the canonical nucleobase.

[0137] In some embodiments, in step (a) of the method for detecting a target analyte, a modified oligonucleotide is attached to the analyte-binding portion. In some embodiments, the analyte-binding portion may bind lipids, peptides, nucleic acids, or polysaccharides. In some embodiments, the analyte-binding portion includes a lipid portion, a wheat germ lectin, an antibody, or a biotin portion. In some embodiments, the antibody includes a secondary antibody or a primary antibody. In some embodiments, the analyte-binding portion comprises a polynucleotide containing a sequence complementary to the target analyte comprising a nucleic acid.

[0138] In some embodiments, in step (a) of the method for detecting a target analyte, the analyte binding portion includes a lipid portion comprising a cholesterol portion, a lipid portion, a phospholipid portion, a bile acid, a thioether (e.g., hexyl-S-triphenylmethylthiol), thiocholesterol, an aliphatic chain (e.g., dodecyl glycol or undecyl residue), a phospholipid (e.g., di-hexadecyl-racemic-glycerol or 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate triethylammonium), a polyamine or polyethylene glycol chain, or adamantaneacetic acid, a palmityl portion, or an octadecylamine or hexylamino-carbonyl-hydroxycholesterol portion.

[0139] In some embodiments, in step (a) of the method for detecting a target analyte, the analyte binding portion includes an antibody. In some embodiments, the antibody includes a primary antibody and / or a secondary antibody. In some embodiments, a modified oligonucleotide may be attached to the primary antibody. In some embodiments, a modified oligonucleotide may be attached to a secondary antibody, and the secondary antibody may be attached to / associated with the primary antibody. In some embodiments, the primary antibody binds to the target analyte. In some embodiments, the primary and secondary antibodies may be cross-linked.

[0140] In some embodiments, antibodies comprise complete immunoglobulins, antibody fragments, antigen-binding portions of antibodies, or single-chain antibodies. Antibodies can be monoclonal or polyclonal. Antibodies are capable of specifically binding to a target analyte. In some embodiments, the target analyte comprises a complete polypeptide or peptide fragment. Antibodies may contain an antigen-binding region (e.g., a complementary site) that specifically binds to the target analyte. In some embodiments, the target analyte may be localized within a cell sample and / or on the cell membrane. In some embodiments, the target analyte comprises polypeptides, lipids, nucleic acids, or polysaccharides. In some embodiments, the target analyte comprises polypeptides, enzymes, or lipids localized at any location in the cell sample (including, but not limited to, the cytoplasm and nucleus). In some embodiments, the target analyte comprises polypeptides, enzymes, or lipids localized in or on cell structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0141] In some embodiments, in step (a) of the method for detecting a target analyte, the antibody comprises an immunoglobulin. In some embodiments, the immunoglobulin comprises a tetrameric molecule of two pairs of identical polypeptide chains, each pair comprising a light chain and a heavy chain. The amino moiety of each of the heavy and light chains comprises a variable region that associates with each other to form an antigen-binding region (e.g., a complementary site). Thus, a typical immunoglobulin can bind two antigens or two target analytes. The carboxyl moiety of the heavy chain comprises a constant region that associates with each other to form an Fc region for effector function. The Fc moiety of the heavy chain may define the class of antibody, including isotypes such as IgG, IgM, IgD, IgA, or IgE. The heavy chain and / or light chain may be prepared using recombinant techniques or by immunizing animals with a target antigen.

[0142] In some embodiments, the antibody may be produced in a host species, including, for example, rabbit, mouse, rat, goat, sheep, guinea pig, chicken, hamster, donkey, camel, or horse. In some embodiments, the antibody may include animal-free antibodies produced using recombinant DNA technology.

[0143] In some embodiments, in step (a) of the method for detecting a target analyte, the antibody comprises an antibody fragment containing a portion of a complete immunoglobulin capable of binding an antigen. Examples of antibody fragments include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, and Fd. In some embodiments, the Fv fragment comprises a variable light chain region (VL) and a variable heavy chain region (VH). In some embodiments, the Fab fragment comprises a monovalent antibody fragment having a variable light chain region (VL), a constant light chain region (CL), a variable heavy chain region (VH), and a first constant region (CH1). In some embodiments, the Fab' fragment comprises a monovalent antibody fragment having at least a portion of a variable light chain region (VL), a constant light chain region (CL), a variable heavy chain region (VH), a first constant region (CH1), a hinge region, and a second constant region (CH2). In some embodiments, the F(ab')2 fragment comprises a bivalent antibody fragment having two Fab fragments connected via a disulfide bridge at the hinge region.

[0144] In some embodiments, in step (a) of the method for detecting a target analyte, the antibody comprises a single-chain variable fragment (scFv) comprising a single polypeptide chain (e.g., a monovalent antibody molecule) having a variable light chain region (VL) and a variable heavy chain region (VH) connected via a polypeptide linker (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc.Natl.Acad.Sci.USA 85:5879-83). The amino terminus of the single-chain antibody comprises a variable light chain region (VL) or a variable heavy chain region (VH). In some embodiments, the single-chain antibody comprises an scFv-Fc antibody, which further comprises an antibody hinge region and at least a portion of an Fc region comprising CH2 and / or CH3 regions. In some embodiments, the single-chain antibody comprises an scFv-CH antibody, which further comprises an antibody hinge region and at least a portion of a CH3 region.

[0145] In some embodiments, in step (a) of the method for detecting a target analyte, the antibody may be conjugated to at least one modified oligonucleotide using any well-known linking chemistry. For example, an amplification-free probe complex may be prepared by crosslinking the antibody and the amino groups on the modified oligonucleotide using glutaraldehyde. Lysine side-chain ε-amides are typically targeted to conjugate with the oligonucleotide. In some embodiments, maleimide-modified antibodies may react with thiol-modified oligonucleotides. In some embodiments, homobifunctional or heterobifunctional crosslinking agents may be introduced as bridges to link the antibody and the modified oligonucleotide together.

[0146] In some embodiments, in step (a) of the method for detecting a target analyte, an amine-amine non-cleavable crosslinking agent comprising disuccinimidyl octanoate (DSS) may be used to attach the antibody to the modified oligonucleotide. The DSS comprises an amine-reactive NHS ester at both ends of an 8-atom spacer arm.

[0147] In some embodiments, in step (a) of the method for detecting a target analyte, an MCC (4-[(2,5-dioxopyrrole-1-yl)methyl]cyclohexane-1-carboxamide) can be used to attach an antibody to at least one modified oligonucleotide, wherein the MCC is a thioether linker that links the antibody to the oligonucleotide via a thioether bond.

[0148] In some embodiments, in step (a) of the method for detecting a target analyte, copper-free click chemistry, such as dibenzocyclooctylene (DBCO), can be used to attach an antibody to at least one modified oligonucleotide. The modified oligonucleotide modified with DBCO can react with an azide-modified antibody to generate an antibody attached to the modified oligonucleotide.

[0149] In some embodiments, in step (a) of the method for detecting a target analyte, an amine-thiocyanate crosslinking agent, succinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), may be used to attach an antibody to at least one modified oligonucleotide. The thiol-modified oligonucleotide may react with an antibody having an amine-thiol linker to generate an antibody attached to the modified oligonucleotide.

[0150] In some embodiments, in step (a) of the method for detecting a target analyte, a chemically unstable adapter (e.g., a pH-sensitive adapter) such as, for example, a hydrazone adapter can be used to attach an antibody to at least one modified oligonucleotide.

[0151] In some embodiments, in step (a) of the method for detecting a target analyte, a modified oligonucleotide may be attached to the analyte binding site via a linker. In some embodiments, the linker comprises a polymer, including a polyether, a polyamine, or a polyamide. In some embodiments, the linker comprises triethylene glycol (e.g., a TEG linker) or polyethylene glycol (e.g., a PEG linker).

[0152] In some embodiments, in step (a) of the method for detecting a target analyte, the connector includes a polymer spacer arm. In some embodiments, the spacer arm includes a C3, C6, C9, C12, or C18 spacer arm. In some embodiments, the connector includes a polymer, which includes a polyether, a polyamine, or a polyamide. In some embodiments, the connector includes triethylene glycol (e.g., a TEG connector) or polyethylene glycol (e.g., a PEG connector).

[0153] In some embodiments, the method for detecting a target analyte includes step (b): providing a cell sample. In some embodiments, the cell sample is deposited (e.g., seeded) on a support. In some embodiments, the cell sample contains multiple analytes, including at least one target analyte.

[0154] In some embodiments, in step (b) of the method for detecting target analytes, the cell sample contains multiple target analytes, including peptides, lipids, nucleic acids, and / or polysaccharides. In some embodiments, the target analytes may be localized to the cell sample (e.g., the outer cell membrane). In some embodiments, the target analytes may be localized to the interior of the cell sample (e.g., the nuclear membrane, nucleolar membrane, mitochondrial membrane, chloroplast membrane, Golgi apparatus membrane, endoplasmic reticulum membrane, peroxisome membrane, or lysosomal membrane). In some embodiments, the target analytes may be localized both inside and on the cell sample.

[0155] In some embodiments, in step (b) of the method for detecting a target analyte, the cell sample includes whole cells, multiple whole cells, complete tissue, or sliced ​​cell samples. In some embodiments, the cell sample includes fresh cell samples, freshly frozen cell samples, sliced ​​cell samples, or FFPE cell samples. In some embodiments, the cell sample may be fixed and / or permeabilized. In some embodiments, the cell sample includes expanded cell samples that have been cultured in simple or complex cell culture media.

[0156] In some embodiments, in step (b) of the method for detecting a target analyte, the cell sample comprises an intracellular matrix and / or a cross-linked matrix, the cross-linked matrix comprising a hydrogel, a swellable hydrogel, or a cross-linked matrix. In some embodiments, the cell sample comprises an intracellular matrix wherein the cell sample is infused with a swellable polyelectrolyte hydrogel (e.g., see U.S. Patent No. 10,309,879 and Chen 2015 Science 347:543, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the immobilized and permeabilized cell sample may be infused with sodium acrylate, acrylamide, and a cross-linking agent, such as N,N'-methylenebisacrylamide. In some embodiments, the cell sample may be infused with an ammonium persulfate (APS) initiator and a tetramethylethylenediamine (TEMED) accelerator to achieve polymerization within the cell sample. In some embodiments, the cell sample may be infused with a protease (e.g., proteinase K) for proteolysis and incubation in a digestion buffer. In some embodiments, the gel within the cell sample may be swollen by adding water. In some embodiments, the cell sample lacks an intracellular matrix.

[0157] In some embodiments, in step (b) of the method for detecting a target analyte, a cell sample may be deposited (seeded) onto a support. In some embodiments, the support comprises a planar or non-planar support. In some embodiments, the support comprises a solid or semi-solid support. In some embodiments, the support comprises a porous, semi-porous, or non-porous support. The support may be made of any material such as glass, plastic, or polymeric materials. In some embodiments, the surface of the support may be coated with one or more compounds to create a passivation layer on the support. In some embodiments, the passivation layer forms a porous or semi-porous layer.

[0158] In some embodiments, in step (b) of the method for detecting a target analyte, a cell sample may be deposited (seeded) onto a support having a coating that promotes the proliferation, migration, differentiation, and / or adhesion of cultured cells or live ex vivo cell or tissue samples. Examples of coatings are described herein. In some embodiments, a cell sample may be deposited onto a support lacking fixed capture primers capable of binding target polynucleotides from the cell sample. In some embodiments, a cell sample may be deposited (e.g., seeded) onto an uncoated support. In some embodiments, in step (b) of the method for detecting a target analyte, a cell sample may be deposited (e.g., seeded) onto a support lacking fixed capture primers capable of binding target polynucleotides from the cell sample. Alternatively, the support may comprise a plurality of fixed capture primers capable of binding target polynucleotides from the cell sample.

[0159] In some embodiments, the method for detecting a target analyte includes step (c): contacting a cell sample with a plurality of non-amplification probe complexes, wherein the contact is performed under conditions suitable for binding the analyte-binding portion of the non-amplification probe complex to its homologous target analyte.

[0160] In some embodiments, the plurality of amplification-free probe complexes can bind to cell samples (e.g., Figure 20 In some embodiments, the plurality of amplification-free probe complexes can bind to the outer cell membrane (e.g., Figure 20 In some embodiments, the plurality of amplification-free probe complexes can enter cells and bind to the cell membranes of organelles located within the cell sample, including, for example, the nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, or lysosomes.

[0161] In some embodiments, the method for detecting a target analyte includes step (d): contacting multiple modified oligonucleotides of a plurality of amplified probe complexes from step (c) with multiple sequencing primers under conditions suitable for each sequencing primer to bind to its homologous (i.e., complementary) sequencing primer binding site on each modified oligonucleotide, to form multiple nucleic acid duplexes, wherein each nucleic acid duplex contains a sequencing primer binding site that hybridizes with the sequencing primer (e.g., Figure 13B , Figure 14B , Figure 15B , Figure 16 , Figure 17 and Figure 18A In some embodiments, the sequencing primer hybridizes with the modified oligonucleotide at a position immediately downstream (3') of the canonical nucleobase. For example, the sequencing primer hybridizes with a sequencing primer binding site located at the 3' of the canonical nucleobase, or alternatively at the 3' and adjacent to it, between 1 and 10 nucleotides. In some embodiments, the sequencing primer hybridizes with the modified oligonucleotide at a position two bases downstream (i.e., 3') of a baseless site on the modified oligonucleotide. In some embodiments, the sequencing primer includes a 3' extendable end. In some embodiments, the sequencing primer includes a 3' non-extendable (blocking) end, which can be converted into a 3' extendable end.

[0162] In some embodiments, the method for detecting a target analyte includes step (e): contacting the plurality of nucleic acid duplexes with a plurality of sequencing polymerases and a plurality of multivalent molecules. In some embodiments, individual multivalent molecules include a core attached to a plurality of nucleotide arms, wherein each nucleotide arm is attached to a nucleotide moiety (e.g., Figures 1 to 4A and Figure 4B In some embodiments, each multivalent molecule is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. In some embodiments, the core of the multivalent molecule is labeled with a fluorophore. In some embodiments, the fluorophore attached to a given core of the multivalent molecule corresponds to a nucleotide base of a nucleotide arm (e.g., adenine, guanine, cytosine, thymine, or uracil), thereby allowing identification of complementary nucleobases in the modified oligonucleotide. In some embodiments, at least one of the nucleotide arms of the multivalent molecule includes a linker and / or a nucleotide base attached to a fluorophore. In some embodiments, the fluorophore attached to a given nucleotide base corresponds to a nucleotide base of a nucleotide arm (e.g., adenine, guanine, cytosine, thymine, or uracil), thereby allowing identification of complementary nucleobases in the modified oligonucleotide.

[0163] In some embodiments, individual sequencing polymerases are capable of binding nucleic acid duplexes (e.g., Figure 18BIn some embodiments, individual sequencing polymerases are capable of binding to a nucleic acid duplex and to the complementary nucleotide portion of a multivalent molecule opposite to the nucleotide (e.g., containing canonical nucleobases) in a given modified oligonucleotide. Figure 18B This forms multiple detectably labeled complexes. In some embodiments, the detectably labeled complexes generate color signals as described herein. In some embodiments, each detectably labeled complex comprises: a modified oligonucleotide that hybridizes with a sequencing primer to form a nucleic acid duplex; a sequencing polymerase bound to the nucleic acid duplex; and a nucleotide portion of a multivalent molecule bound to the 3' end of the sequencing primer and located opposite the canonical nucleobase in the modified oligonucleotide.

[0164] In some embodiments, the detectably labeled complex can be formed in less than about 30 minutes, or less than about 20 minutes, or less than about 10 minutes, or less than about 5 minutes.

[0165] In some embodiments, the contact in step (e) is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of nucleotide moieties into the 3' end of the sequencing primer. In some embodiments, the contact in step (e) includes contacting the plurality of nucleic acid duplexes with a plurality of sequencing polymerases, a plurality of multivalent molecules, and a plurality of non-catalytic cations. In some embodiments, the non-catalytic cations include strontium, barium, and / or calcium. In some embodiments, in step (e), the complementary nucleotide moieties of the multivalent molecules bind opposite to the nucleotides in a given modified oligonucleotide, and the complementary nucleotide moieties of the multivalent molecules are not incorporated into the 3' end of the sequencing primer. In some embodiments, the binding of the complementary nucleotide moieties of the multivalent molecules in step (e) does not lead to primer extension. In some embodiments, the binding of the complementary nucleotide moieties of the multivalent molecules in step (e) does not lead to nucleic acid amplification.

[0166] In some embodiments, the modified oligonucleotide is designed to include at least one base-free site that inhibits a second binding event of another detectably labeled polyvalent molecule, such that in the contact of step (e), a nucleotide portion of a detectably labeled polyvalent molecule binds to a nucleic acid duplex and a sequencing polymerase to emit a signal associated with a detectably labeled complex. In some embodiments, the signal can be imaged.

[0167] In some embodiments, the detectably labeled complex is stable and does not dissociate until subjected to conditions that induce dissociation. In some embodiments, the detectably labeled complex has a residence time of more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. The detectably labeled complex has a residence time greater than about 0.1 seconds to 0.25 seconds, about 0.25 seconds to 0.5 seconds, about 0.5 seconds to 0.75 seconds, about 0.75 seconds to 1 second, about 1 second to 2 seconds, about 2 seconds to 3 seconds, about 3 seconds to 4 seconds, or about 4 seconds to 5 seconds, and / or the method is performed at or above 15°C, at or above 20°C, at or above 25°C, at or above 35°C, at or above 37°C, at or above 42°C, at or above 55°C, at or above 60°C, at or above 72°C, or at or above 80°C, or within the range defined by any of the foregoing. In some embodiments, the detectably labeled complex remains stable until subjected to conditions that cause dissociation between any of the sequencing polymerase, the modified oligonucleotide, the sequencing primer, and / or the nucleotide motif of the multivalent molecule. For example, dissociation conditions include contacting the detectably labeled complex with any or any combination of detergent, EDTA, and / or water.

[0168] In some embodiments, during the contact in step (e), each multivalent molecule comprises a plurality of nucleotide arms, wherein each nucleotide arm carries at least one nucleotide moiety. In some embodiments, the nucleotide moiety of a single multivalent molecule may form two or more detectably labeled complexes comprising affinity complexes.

[0169] In some embodiments, the first nucleotide portion of the multivalent molecule may form a first detectably labeled complex on a first non-amplified probe complex, and the second nucleotide portion of the same multivalent molecule may form a second detectably labeled complex on a second non-amplified probe complex, wherein the first non-amplified probe complex and the second non-amplified probe complex bind to the same cell sample. In some embodiments, the first detectably labeled complex and the second detectably labeled complex form an affinity complex.

[0170] In some embodiments, the first nucleotide portion of the multivalent molecule may form a first detectably labeled complex on a first region of the first amplification-free probe complex, and the second nucleotide portion of the same multivalent molecule may form a second detectably labeled complex on a second region of the same amplification-free probe complex, wherein the first amplification-free probe complex binds to the cell sample. In some embodiments, the first detectably labeled complex and the second detectably labeled complex form an affinity complex.

[0171] In some embodiments, the method for detecting a target analyte includes step (f): imaging a cell sample that is conjugated to the detectably labeled complex of step (e) (e.g., Figure 20 ).

[0172] In some embodiments, the imaging in step (f) includes using an image larger than 1.0 mm. 2 An optical imaging system with a field of view (FOV). In some embodiments, the detectably labeled complex of step (e) can be formed in less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm. 2 And it is used to scan large areas (> 10 mm) 2 The cycle time can be less than 5 minutes.

[0173] In some embodiments, in the method for detecting the target analyte in step (f), the modified oligonucleotide without amplification probe complex hybridizes with a plurality of sequencing primers in the presence of a hybridization solution. In some embodiments, the hybridization solution comprises a saline-sodium citrate (SSC) solution. In some embodiments, the hybridization solution comprises a 2X, 3X, 4X, 5X, 7X, 8X, 9X, or 10X SSC solution. In some embodiments, the hybridization solution comprises a 2X, 3X, or 5X SSC solution. In some embodiments, the hybridization solution comprises acetonitrile. In some embodiments, the hybridization solution comprises dimethyl sulfoxide (DMSO). In some embodiments, the hybridization solution comprises Denhardt's solution containing Ficoll (e.g., Ficoll 400), polyvinylpyrrolidone, and bovine serum albumin (BSA). In some embodiments, the hybridization solution comprises betaine. In some embodiments, the hybridization solution comprises trehalose. In some embodiments, the hybridization solution comprises guanidine isothiocyanate (GITC). In some embodiments, the hybridization solution comprises any one or a combination of two or more of a pH buffer, chelating agent, detergent, denaturant, crowding agent, and / or blocking agent. In some embodiments, the hybridization solution comprises a sodium salt (e.g., NaCl). In some embodiments, the pH buffer comprises HEPES (e.g., 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) or MES (e.g., 2-(N-morpholino)ethanesulfonic acid). In some embodiments, the chelating agent comprises EDTA (ethylenediaminetetraacetic acid) and / or EGTA (ethylene glycol tetraacetic acid). In some embodiments, the detergent comprises Tween-20, Triton-X 100, and / or SDS. In some embodiments, the denaturing agent comprises formamide, 2-pyrrolidone, urea, and / or ethylene carbonate. In some embodiments, the blocking agent comprises dextran sulfate and / or polyethylene glycol (PEG), such as, but not limited to, 1KPEG, 2KPEG, 4KPEG, 5KPEG, and / or PEG200. In some embodiments, the blocking agent comprises BSA.

[0174] Barcode sequences used in cell staining methods

[0175] This disclosure provides amplification-free probe complexes comprising barcode sequences and methods for sequencing these barcode sequences. The disclosure provides a variety of amplification-free probe complexes comprising subgroups of modified oligonucleotides. In some embodiments, the subgroups comprise modified oligonucleotides, wherein the modified oligonucleotides of different subgroups comprise different barcode sequences. For example, all amplification-free probe complexes of one subgroup comprise the same barcode sequence, which is different from the barcode sequences of other subgroups. The barcode sequences can be used to simultaneously detect and identify two or more target analytes by performing a single sequencing cycle and employing multicolor imaging. In some embodiments, the barcode sequences of the amplification-free probe complexes can be used in sequencing-based cell staining methods.

[0176] In some embodiments, in a method for sequencing barcodes, sequencing includes step (a): sequencing a plurality of modified oligonucleotides by performing at least two sequencing cycles, including sequencing the same corresponding nucleotide position of all barcodes in a plurality of subsets of the modified oligonucleotides in each sequencing cycle. In some embodiments, a first barcode of a modified oligonucleotide in a first subset generates a first color signal in a particular sequencing cycle, and barcodes of modified oligonucleotides in other subsets generate a second color signal in the same particular sequencing cycle. In some embodiments, the first color signal and the second color signal are distinguishable in the same sequencing cycle. In some embodiments, the first color signal of any barcode in any sequencing cycle identifies the identity of the corresponding nucleotide in that barcode, and thereby identifies the target analyte corresponding to the barcode sequence of the non-amplified probe complex. In some embodiments, the plurality of modified oligonucleotides are on and / or inside a cell sample.

[0177] In some embodiments, sequencing includes step (b): imaging a first color signal and a second color signal in a specific sequencing cycle, and identifying a target analyte corresponding to a barcode sequence from the amplified probe complex that generated the first color signal. In some embodiments, the first color signal and the second color signal are generated on and / or inside a cell sample.

[0178] In some embodiments, in steps (a) and (b), imaging a first color signal and a second color signal generated in a single sequencing cycle can simultaneously identify at least two different target analytes. For example, the plurality of amplified probe complexes includes a first subgroup and a second subgroup, the first subgroup comprising a first barcode sequence and a first analyte-binding portion binding a first analyte, and the second subgroup comprising a second barcode sequence and a second analyte-binding portion binding a second analyte. In this example, but not limited to, the first and second barcode sequences are different, and the first and second analyte-binding portions are different. Imaging the first and second color signals allows imaging of the location of the subgroup of the amplified probe complex containing the first and second barcode sequences, corresponding to the first and second analyte-binding portions binding to the first and second analytes in a single sequencing cycle.

[0179] In some embodiments, in steps (a) and (b), at least two different cellular structures can be simultaneously identified by imaging a first color signal and a second color signal generated in a single sequencing cycle. In some embodiments, cellular structures include, but are not limited to, any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0180] In some embodiments, in steps (a) and (b), multiple subgroups of the modified oligonucleotide are located on and / or inside the cell sample.

[0181] In some embodiments, in steps (a) and (b), multiple sequencing primers containing the same sequence may be used to sequence all modified oligonucleotides substantially simultaneously to detect and identify target analytes.

[0182] In some embodiments, in steps (a) and (b), multiple sequencing primers containing different sequences can be used to sequence all modified oligonucleotides substantially simultaneously to detect and identify target analytes.

[0183] In some embodiments, in steps (a) and (b), modified oligonucleotides can be sequenced in separate batches using multiple sequencing primers with different sequences to detect and identify target analytes.

[0184] In some embodiments, in steps (a) and (b), only a portion of the barcode length needs to be sequenced to identify different target analytes. In some embodiments, the full length of the barcode can be sequenced to detect and identify different target analytes. In some embodiments, the barcode can be 2 to 20 nucleotides in length.

[0185] This disclosure provides a method for sequencing barcodes, comprising the step (a): sequencing a plurality of modified oligonucleotides comprising a plurality of subgroups of modified oligonucleotides by performing at least two sequencing cycles. In some embodiments, in each sequencing cycle, sequencing comprises sequencing the same corresponding nucleotide position of all barcodes in the plurality of subgroups of modified oligonucleotides. In some embodiments, a first barcode of a modified oligonucleotide in a first subgroup generates a first color signal in a particular sequencing cycle, and barcodes of modified oligonucleotides in other subgroups generate a second color signal in the same particular sequencing cycle. In some embodiments, the first color signal and the second color signal are distinguishable in the same sequencing cycle. In some embodiments, the first color signal of any barcode in any sequencing cycle identifies a target analyte corresponding to its homologous barcode. In some embodiments, the plurality of subgroups of modified oligonucleotides are on and / or inside a cell sample.

[0186] In some embodiments, the method for sequencing a barcode includes step (b): imaging a first color signal and a second color signal in a specific sequencing cycle, and identifying a target analyte corresponding to the barcode sequence that generated the first color signal. In some embodiments, the first color signal and the second color signal are generated on and / or inside a cell sample.

[0187] In some embodiments, at least two different target analytes can be identified simultaneously by imaging a first color signal and a second color signal generated in a single sequencing cycle.

[0188] In some embodiments, multiple subgroups of the modified oligonucleotide are located on and / or inside the cell sample.

[0189] In some embodiments, multiple sequencing primers containing the same sequence can be used to sequence all barcodes substantially simultaneously to detect and identify target analytes.

[0190] In some embodiments, multiple sequencing primers containing different sequences can be used to sequence all barcodes substantially simultaneously to detect and identify target analytes.

[0191] In some embodiments, multiple sequencing primers with different sequences can be used to sequentially sequence barcodes (e.g., sequencing in separate batches) to detect and identify target analytes.

[0192] In some embodiments, only a portion of the barcode length needs to be sequenced to identify different target analytes. In some embodiments, the full length of the barcode can be sequenced to detect and identify different target analytes. In some embodiments, the barcode can be 2 to 20 nucleotides in length.

[0193] In some embodiments, the target analyte is located inside the cell sample and / or on the cell membrane. In some embodiments, the target analyte includes peptides, lipids, nucleic acids, or polysaccharides. In some embodiments, the target analyte includes peptides, enzymes, or lipids located at any location in the cell sample (including but not limited to the cytoplasm and nucleus). In some embodiments, the target analyte includes peptides, enzymes, or lipids located in or on cell structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0194] In some embodiments, simultaneously sequencing two or more subgroups of modified oligonucleotides located within a cell sample and imaging the color signals generated by sequencing can be used to simultaneously identify two or more cellular target analytes. In some embodiments, simultaneous sequencing and multicolor imaging can be used for cell staining.

[0195] This invention provides a method for sequencing barcodes, the method comprising the step (a): providing a plurality of modified oligonucleotides, comprising at least: a first subgroup of modified oligonucleotides containing a plurality of first barcodes corresponding to a first target analyte; and a second subgroup of modified oligonucleotides containing a plurality of second barcodes corresponding to a second target analyte. In some embodiments, the first barcodes are 2 to 20 nucleotides in length. In some embodiments, the second barcodes are 2 to 20 nucleotides in length. In some embodiments, the plurality of modified oligonucleotides are located on and / or inside a cell sample.

[0196] In some embodiments, the method of sequencing the barcode includes step (b): performing a first sequencing cycle, wherein sequencing includes sequencing the first nucleobase positions of the first barcode and the second barcode in a first and second subgroup of the modified oligonucleotides substantially simultaneously. In some embodiments, sequencing is performed using a plurality of sequencing primers having the same sequence. In some embodiments, the first nucleobase position of the first barcode generates a first color signal, and the first nucleobase position of the second barcode generates a second color signal. In some embodiments, the first color signal and the second color signal are distinguishable from each other in the first sequencing cycle. In some embodiments, the first color signal identifies a first target analyte. In some embodiments, the second color signal identifies a second target analyte. In some embodiments, the first sequencing cycle is performed on and / or inside a cell sample.

[0197] In some embodiments, the first sequencing cycle of step (b) may be performed using any sequencing method described herein or known in the art, including but not limited to two-stage sequencing using multivalent molecules, binding sequencing, or sequencing using chain terminator nucleotides.

[0198] In some embodiments, the method for sequencing barcodes includes step (c): performing a second sequencing cycle. In some embodiments, sequencing includes sequencing the second nucleotide positions of the first barcode and the second barcode in a first and second subgroup of modified oligonucleotides substantially simultaneously. In some embodiments, the second nucleotide position of the first barcode generates a second color signal. In some embodiments, the second nucleotide position of the second barcode generates a first color signal. In some embodiments, the first color signal and the second color signal are distinguishable from each other in the second sequencing cycle. In some embodiments, the first color signal identifies a second target analyte. In some embodiments, the second color signal identifies a first target analyte. In some embodiments, the second sequencing cycle is performed on and / or inside a cell sample.

[0199] In some embodiments, the second sequencing cycle in step (c) may be performed using any sequencing method described herein or known in the art, including but not limited to two-stage sequencing using multivalent molecules, binding sequencing, or sequencing using chain terminator nucleotides.

[0200] In some embodiments, the method for sequencing a barcode further includes step (d): imaging a first color signal and a second color signal in a first sequencing cycle and identifying a first target analyte. In some embodiments, the first color signal and the second color signal are generated on and / or in a cell sample.

[0201] In some embodiments, the method for sequencing a barcode further includes step (e): imaging a first color signal and a second color signal in a second sequencing cycle and identifying a second target analyte.

[0202] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a first sequencing cycle and identifying a first target analyte corresponding to the first color signal, and identifying a second target analyte corresponding to the second color signal.

[0203] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a second sequencing cycle and identifying a second target analyte corresponding to the first color signal, and identifying a first target analyte corresponding to the second color signal.

[0204] In some embodiments, the first color signal and the second color signal are generated on and / or in the cell sample.

[0205] In some embodiments, at least two different target analytes can be identified simultaneously by imaging a first color signal and a second color signal generated in a single sequencing cycle.

[0206] In some embodiments, the first target analyte and the second target analyte can be identified by performing no more than two sequencing cycles.

[0207] In some embodiments, the first barcode and the second barcode can be identified by sequencing the full length of the first barcode and the second barcode.

[0208] In some embodiments, the method includes sequencing a first nucleobase position of the first barcode from step (b) and sequencing a second nucleobase position of the first barcode from step (c). In some embodiments, the first and second nucleobase positions are consecutive nucleobase positions in the first barcode. In some embodiments, the first and second nucleobase positions are non-consecutive nucleobase positions in the first barcode. For example, the first and second non-consecutive nucleobase positions may have a gap of 1 to 10 nucleobase positions.

[0209] In some embodiments, the method includes sequencing the first nucleobase position of the second barcode in step (b) and sequencing the second nucleobase position of the second barcode in step (c). In some embodiments, the first and second nucleobase positions are consecutive nucleobase positions in the second barcode. In some embodiments, the first and second nucleobase positions are non-consecutive nucleobase positions in the second barcode. For example, the first and second non-consecutive nucleobase positions may have a gap of 1 to 10 nucleobase positions.

[0210] In some embodiments, the first and second target analytes are located inside the cell sample and / or on the cell membrane. In some embodiments, the first and / or second target analytes comprise peptides, lipids, nucleic acids, or polysaccharides. In some embodiments, the first and / or second target analytes comprise peptides, enzymes, or lipids located anywhere in the cell sample (including but not limited to the cytoplasm and nucleus). In some embodiments, the first and / or second target analytes comprise peptides, enzymes, or lipids located in or on cell structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0211] In some embodiments, simultaneously sequencing at least two subgroups of modified oligonucleotides located within a cell sample and imaging the color signals generated by sequencing can be used to simultaneously identify two or more cellular target analytes. In some embodiments, simultaneous sequencing and multicolor imaging can be used for sequencing-based cell staining.

[0212] This invention provides a method for sequencing barcodes, the method comprising the step (a): providing a plurality of modified oligonucleotides, comprising at least: a first subgroup of modified oligonucleotides containing a plurality of first barcodes corresponding to a first target analyte; a second subgroup of modified oligonucleotides containing a plurality of second barcodes corresponding to a second target analyte; and a third subgroup of modified oligonucleotides containing a plurality of third barcodes corresponding to a third target. In some embodiments, the plurality of modified oligonucleotides are located within a cell sample. In some embodiments, the first barcode is 4 to 20 nucleotides in length. In some embodiments, the second barcode is 4 to 20 nucleotides in length. In some embodiments, the third barcode is 4 to 20 nucleotides in length. In some embodiments, the first, second, and / or third barcodes are 3 to 20 nucleotides in length. In some embodiments, the first, second, and / or third barcodes are 2 to 20 nucleotides in length.

[0213] In some embodiments, the method of sequencing the barcode includes step (b): performing a first sequencing cycle, wherein the sequencing cycle includes sequencing the first nucleotide positions of the first, second, and third barcodes in a first, second, and third subgroup of the modified oligonucleotides substantially simultaneously. In some embodiments, the first sequencing cycle includes using a plurality of sequencing primers having the same sequence. In some embodiments, the first nucleotide position of the first barcode generates a first color signal, and the first nucleotide positions of the second and third barcodes generate a second color signal. In some embodiments, the first color signal and the second color signal are distinguishable from each other in the first sequencing cycle. In some embodiments, the first color signal identifies a first target analyte. In some embodiments, the first sequencing cycle occurs on and / or inside a cell sample.

[0214] In some embodiments, the first sequencing cycle of step (b) may be performed using any sequencing method described herein or known in the art, including but not limited to, two-stage sequencing using multivalent molecules, binding sequencing, or sequencing using chain terminator nucleotides.

[0215] In some embodiments, the method of sequencing the barcode includes step (c): performing a second sequencing cycle, wherein sequencing includes sequencing the second nucleotide positions of the first, second, and third barcodes in a first, second, and third subgroup of the modified oligonucleotides substantially simultaneously. In some embodiments, the second nucleotide position of the first barcode generates a second color signal. In some embodiments, the second nucleotide position of the second barcode generates a first color signal. In some embodiments, the second nucleotide position of the third barcode generates a second color signal. In some embodiments, the first color signal and the second color signal are distinguishable from each other in the second sequencing cycle. In some embodiments, the first color signal identifies a second target analyte. In some embodiments, the second sequencing cycle occurs on and / or inside a cell sample.

[0216] In some embodiments, the second sequencing cycle in step (c) may be performed using any sequencing method described herein or known in the art, including but not limited to two-stage sequencing using multivalent molecules, binding sequencing, or sequencing using chain terminator nucleotides.

[0217] In some embodiments, the method of sequencing the barcode includes step (d): performing a third sequencing cycle, wherein sequencing includes sequencing the third nucleotide positions of the first, second, and third barcodes in the first, second, and third subgroups of the modified oligonucleotides substantially simultaneously. In some embodiments, the third nucleotide position of the first barcode generates a second color signal. In some embodiments, the third nucleotide position of the second barcode generates a second color signal. In some embodiments, the third nucleotide position of the third barcode generates a first color signal. In some embodiments, the first color signal and the second color signal are distinguishable from each other in the third sequencing cycle. In some embodiments, the first color signal identifies a third target analyte. In some embodiments, the third sequencing cycle occurs on and / or inside a cell sample.

[0218] In some embodiments, the third sequencing cycle of step (d) may be performed using any sequencing method described herein or known in the art, including but not limited to two-stage sequencing using multivalent molecules, binding sequencing, or sequencing using chain terminator nucleotides.

[0219] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a first sequencing cycle and identifying a first target analyte.

[0220] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a second sequencing cycle and identifying a second target analyte.

[0221] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a third sequencing cycle and identifying a third target analyte.

[0222] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a first sequencing cycle and identifying a first target analyte corresponding to the first color signal, and identifying a second target analyte and a third target analyte corresponding to the second color signal.

[0223] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal in a second sequencing cycle and identifying a second target analyte corresponding to the first color signal, and identifying a first target analyte and a third target analyte corresponding to the second color signal.

[0224] In some embodiments, the method for sequencing a barcode further includes: imaging a first color signal and a second color signal generated inside a cell sample in a third sequencing cycle and identifying a third target analyte corresponding to the first color signal, and identifying a first target analyte and a second target analyte corresponding to the second color signal.

[0225] In some embodiments, the first color signal and the second color signal generated in any of the first sequencing cycle, the second sequencing cycle and / or the third sequencing cycle are generated on and / or inside the cell sample.

[0226] In some embodiments, at least two different target analytes can be identified simultaneously by imaging a first color signal and a second color signal generated in a single sequencing cycle.

[0227] In some embodiments, the first target analyte, the second target analyte, and the third target analyte can be identified by performing no more than three sequencing cycles.

[0228] In some embodiments, the first barcode, the second barcode, and the third barcode can be identified by sequencing the full length of the first barcode, the second barcode, and the third barcode.

[0229] In some embodiments, the method includes sequencing a first nucleobase position of the first barcode from step (b) and sequencing a second nucleobase position of the first barcode from step (c). In some embodiments, the first and second nucleobase positions are consecutive nucleobase positions in the first barcode. In some embodiments, the first and second nucleobase positions are non-consecutive nucleobase positions in the first barcode. For example, the first and second non-consecutive nucleobase positions may have a gap of 1 to 10 nucleobase positions.

[0230] In some embodiments, the method includes sequencing the first nucleobase position of the second barcode in step (b) and sequencing the second nucleobase position of the second barcode in step (c). In some embodiments, the first and second nucleobase positions are consecutive nucleobase positions in the second barcode. In some embodiments, the first and second nucleobase positions are non-consecutive nucleobase positions in the second barcode. For example, the first and second non-consecutive nucleobase positions may have a gap of 1 to 10 nucleobase positions.

[0231] In some embodiments, the method includes sequencing the first nucleobase position of the third barcode in step (b) and sequencing the second nucleobase position of the third barcode in step (c). In some embodiments, the first and second nucleobase positions are consecutive nucleobase positions in the third barcode. In some embodiments, the first and second nucleobase positions are non-consecutive nucleobase positions in the third barcode. For example, the first and second non-consecutive nucleobase positions may have a gap of 1 to 10 nucleobase positions.

[0232] In some embodiments, the first, second, and third target analytes are located inside the cell sample and / or on the cell membrane. In some embodiments, the target analytes include peptides, lipids, nucleic acids, or polysaccharides. In some embodiments, the first, second, and third target analytes include peptides, enzymes, or lipids located anywhere in the cell sample (including but not limited to the cytoplasm and nucleus). In some embodiments, the first, second, and third target analytes include peptides, enzymes, or lipids located in or on cell structures, including but not limited to any cell membrane, nucleus, nucleolus, mitochondria, chloroplasts, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules, actin cytoskeleton, spindle apparatus, flagella, peroxisomes, and lysosomes.

[0233] In some embodiments, simultaneously sequencing two or more subgroups of modified oligonucleotides located within a cell sample and imaging the color signals generated by sequencing can be used to simultaneously identify two or more cellular target analytes. In some embodiments, simultaneous sequencing and multicolor imaging can be used for cell staining.

[0234] In some embodiments, the first sequencing cycle in step (b), the second sequencing cycle in step (c), and the third sequencing cycle in step (d) may be performed using any sequencing method, including binding sequencing, sequencing using chain terminator nucleotides, or sequencing using multivalent molecules.

[0235] Two-stage method for nucleic acid sequencing

[0236] This disclosure provides a two-stage method for sequencing any of the modified oligonucleotides of the amplification-free probe complexes described herein (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 , Figure 17 , Figures 18A to 18B and Figures 19A to 19BIn some embodiments, the two-stage sequencing method can be performed on and / or inside a cell sample. In some embodiments, the two-stage sequencing method can be used to sequence the canonical nucleobases of any of the modified oligonucleotides described herein (e.g., Figures 18A to 18B In some embodiments, a two-stage sequencing approach may be used to sequence the target barcode sequence (or a portion thereof) of any of the modified oligonucleotides described herein (e.g., Figures 19A to 19B ).

[0237] In some embodiments, the first stage typically includes binding a multivalent molecule to a polymerase complex to form a multivalent binding polymerase complex, and detecting the multivalent binding polymerase complex. In some embodiments, the second stage includes nucleotide incorporation and extension of sequencing primers. In some embodiments, a sequencing cycle includes the completion of both the first and second stages.

[0238] In some embodiments, the first stage includes step (a): contacting (i) a first plurality of sequencing polymerases, (ii) a plurality of modified oligonucleotides localized within a cell sample, and (iii) a plurality of nucleic acid sequencing primers, wherein the contact is performed under conditions suitable for forming the first plurality of sequencing polymerase complexes, each complex containing a first sequencing polymerase that binds to a nucleic acid duplex, wherein the nucleic acid duplex contains a portion of the modified oligonucleotide that hybridizes with the sequencing primer. In some embodiments, the sequencing primers contain either a 3' extendable end or a 3' non-extendable end. In some embodiments, the first sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0239] In some embodiments, the sequencing method includes step (b): contacting a first plurality of polymerase complexes with a plurality of multivalent molecules to form a plurality of multivalent binding polymerase complexes (e.g., binding complexes) within a cell sample. In some embodiments, each of the plurality of multivalent molecules includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide moiety (e.g., Figures 1 to 3 and Figures 4A to 4B In some embodiments, the contact in step (b) is performed under conditions suitable for binding the complementary nucleotide moiety of the multivalent molecule to at least two of the polymerase complexes in the first plurality of polymerase complexes, thereby forming a plurality of multivalent-binding polymerase complexes. In some embodiments, the conditions are suitable for inhibiting the polymerase-catalyzed incorporation of the complementary nucleotide moiety into the primers of the plurality of multivalent-binding polymerase complexes. In some embodiments, the contact in step (b) is performed in the presence of at least one non-catalyzed cation that inhibits polymerase-catalyzed nucleotide incorporation. In some embodiments, the at least one non-catalyzed cation includes strontium, barium, and / or calcium.

[0240] In some embodiments, in the method of step (b), at least one of the plurality of multivalent molecules is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore.

[0241] In some embodiments, in the method of step (b), each nucleotide arm of the multivalent molecule comprises: (i) a core attachment portion, (ii) a spacer containing a PEG portion, (iii) a linker, and (iv) a nucleotide portion, wherein the core is attached to a plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide portion (e.g., Figure 4B In some embodiments, the labeled multivalent molecule includes a fluorophore attached to the core, spacers, linkers, and / or nucleotide moieties of the multivalent molecule.

[0242] In some embodiments, in the method of step (b), the plurality of multivalent molecules comprises at least one multivalent molecule having a plurality of nucleotide arms (e.g., Figures 1 to 3 and Figure 4A Each nucleotide arm is attached with a nucleotide analog (e.g., a nucleotide analog portion), wherein the nucleotide analog includes a chain termination portion (e.g., a blocking portion) at the 2' and / or 3' positions of the sugar. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule containing a plurality of nucleotide arms, each nucleotide arm being attached with a nucleotide portion lacking a chain termination portion.

[0243] In some embodiments, the sequencing method includes step (c): detecting the plurality of multivalent binding polymerase complexes. In some embodiments, detection includes detecting multivalent molecules bound to polymerase complexes in the first plurality of polymerase complexes, wherein complementary nucleotide portions of the multivalent molecules bind to primers, but incorporation of the complementary nucleotide portions is suppressed. In some embodiments, the multivalent molecules are labeled with a detectable reporter portion to allow detection. In some embodiments, the detectable reporter portion includes a fluorophore that generates a color signal. In some embodiments, the detection in step (c) includes imaging the plurality of multivalent binding polymerase complexes.

[0244] In some embodiments, the method for sequencing further includes step (d): identifying the nucleobases of the complementary nucleotide moiety bound to the first plurality of polymerase complexes, thereby determining the sequence of the modified oligonucleotide. In some embodiments, the multivalent molecule is labeled with a detectable reporter portion corresponding to a specific nucleotide moiety attached to a nucleotide arm to allow identification of the complementary nucleotide moiety bound to the first plurality of polymerase complexes (e.g., the nucleotide bases adenine, guanine, cytosine, thymine, or uracil).

[0245] In some embodiments, modified oligonucleotides containing canonical nucleobases and abase-free sites (e.g., Figures 18A to 18B Sequencing includes performing a first stage of a two-stage sequencing method (e.g., steps (a) to (d)) and optionally a second stage (e.g., steps (e) to (k) as described below).

[0246] In some embodiments, modified oligonucleotides containing target barcode sequences are sequenced (e.g., Figures 19A to 19B This includes performing a first stage of a two-stage sequencing method (e.g., steps (a) to (d)) and a second stage (e.g., steps (e) to (k)).

[0247] In some embodiments, the second stage of the two-stage sequencing method typically includes nucleotide incorporation. In some embodiments, the sequencing method includes step (e): dissociating the plurality of multivalent binding polymerase complexes and removing the first plurality of sequencing polymerases and the multivalent molecules they bind, and retaining the plurality of nucleic acid duplexes inside the cell sample.

[0248] In some embodiments, the sequencing method includes step (f): contacting a plurality of nucleic acid duplexes retained inside a cell sample in step (e) with a second plurality of sequencing polymerases, wherein the contact is performed under conditions suitable for binding the second plurality of sequencing polymerases to the plurality of nucleic acid duplexes, thereby forming a second plurality of polymerase complexes, each complex containing a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerases include recombinant mutant sequencing polymerases.

[0249] In some embodiments, the plurality of first sequencing polymerases in step (a) have an amino acid sequence that is 100% identical to the amino acid sequence of the plurality of second sequencing polymerases in step (f). In some embodiments, the plurality of first sequencing polymerases in step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases in step (f).

[0250] In some embodiments, the method for sequencing further includes step (g): contacting a second plurality of polymerase complexes with a plurality of nucleotides, wherein the contact is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second polymerase complexes, thereby forming a plurality of nucleotide-polymerase complexes. In some embodiments, the contact in step (g) is performed under conditions suitable for polymerase-catalyzed incorporation of complementary nucleotides that promote binding into primers of the nucleotide-polymerase complex. In some embodiments, incorporating nucleotides into the 3' end of the primers in step (g) includes a primer extension reaction. In some embodiments, the contact in step (g) is performed in the presence of at least one catalytic cation that promotes nucleotide incorporation. In some embodiments, the at least one catalytic cation includes magnesium and / or manganese. In some embodiments, the plurality of nucleotides includes natural nucleotides (e.g., non-analogous nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides includes removable or non-removable 2' and / or 3' chain termination portions. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, the plurality of nucleotides are unlabeled nucleotides. In some embodiments, the plurality of nucleotides includes a plurality of nucleotides labeled with a detectable reporter portion. The detectable reporter portion may include a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base with a linker that may be cleaved / removed from the base or may not be removable from the base. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of the nucleotide base.

[0251] In some embodiments, when the plurality of nucleotides in step (g) contains labeled nucleotides, the method for sequencing further includes step (h): detecting complementary nucleotides in a primer incorporated into a nucleotide-polymerase complex. In some embodiments, the plurality of nucleotides are partially labeled with a detectable reporter to allow detection. In some embodiments, when the plurality of nucleotides in step (g) contains unlabeled nucleotides, the detection in step (h) is omitted.

[0252] In some embodiments, when the plurality of nucleotides in step (g) comprises labeled nucleotides, the method for sequencing further comprises step (i): identifying the bases of complementary nucleotides in primers incorporated into the nucleotide-polymerase complex. In some embodiments, the identification of the incorporated complementary nucleotides in step (i) may be used to confirm the identity of the complementary nucleotides of the multivalent molecules bound to the first plurality of polymerase complexes in step (d). In some embodiments, the identification in step (i) may be used to determine the sequence of the modified oligonucleotide. In some embodiments, when the plurality of nucleotides in step (g) comprises unlabeled nucleotides, the identification in step (i) is omitted.

[0253] In some embodiments, when the plurality of nucleotides in step (g) include 2' and / or 3' chain-terminating nucleotides, the method for sequencing further includes step (j): removing the chain-terminating portion from the incorporated nucleotides.

[0254] In some embodiments, the method for sequencing further includes step (k): repeating steps (a) through (j) at least once. In some embodiments, the sequence of the modified oligonucleotide can be determined by detecting and identifying multivalent molecules that bind to the sequencing polymerase but are not incorporated into the 3' end of the primer in steps (c) and (d). In some embodiments, the sequence of the modified oligonucleotide can be determined (or confirmed) by detecting and identifying nucleotides incorporated into the 3' end of the primer in steps (h) and (i).

[0255] Formation of affinity complex

[0256] In some embodiments, in either of a two-stage sequencing method, a first plurality of polymerase complexes are bound to a plurality of multivalent molecules to form at least one affinity complex, the method comprising the steps of: (1) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a modified oligonucleotide, thereby forming a first binding complex, wherein the first nucleotide portion of the first multivalent molecule binds to the first sequencing polymerase; and (2) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same modified oligonucleotide, thereby forming a second binding complex, wherein the second nucleotide portion of the first multivalent molecule binds to the second sequencing polymerase, including the first binding complex and the second binding complex of the same multivalent molecule forming an affinity complex. In some embodiments, the modified oligonucleotide comprises at least one universal site for binding sequencing primers, a canonical nucleobase, and a base-free tandem repeat sequence. In some embodiments, the modified oligonucleotide comprises at least one universal site for binding sequencing primers and a target barcode sequence tandem repeat sequence. The first and second sequencing primers may bind to the sequencing primer binding site along the modified oligonucleotide. Exemplary multivalent molecules in Figures 1 to 3and Figures 4A to 4B As shown in the image.

[0257] Formation of affinity complexes and detection and identification

[0258] In some embodiments, in any of the two-stage sequencing methods, the method includes binding a first plurality of polymerase complexes to a plurality of multivalent molecules to form at least one affinity complex, and the method includes the steps of: (1) contacting a plurality of sequencing polymerases and a plurality of sequencing primers with different portions of a modified oligonucleotide to form at least a first polymerase complex and a second polymerase complex on the same modified oligonucleotide; (2) contacting the plurality of multivalent molecules with at least the first polymerase complex and the second polymerase complex on the same modified oligonucleotide under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first polymerase complex and the second polymerase complex, wherein at least a first nucleotide portion of the single multivalent molecule binds to the first polymerase complex, the first polymerase complex comprising a first sequencing primer that hybridizes to a first portion of the modified oligonucleotide, thereby forming a first binding complex (e.g., a first ternary complex). (3) Detecting the first and second binding complexes on the same modified oligonucleotide, and (4) Identifying the first nucleotide portion in the first binding complex to determine the sequence of the first portion of the modified oligonucleotide, and identifying the second nucleotide portion in the second binding complex to determine the sequence of the second portion of the modified oligonucleotide.

[0259] In some embodiments, the modified oligonucleotide comprises a tandem repeat sequence of at least one universal site for binding sequencing primers, a canonical nucleobase, and a base-free site. In some embodiments, the modified oligonucleotide comprises a tandem repeat sequence of at least one universal site for binding sequencing primers and a target barcode sequence. The plurality of sequencing primers can bind to the sequencing primer binding site along the modified oligonucleotide. Exemplary multivalent molecules in Figures 1 to 3 and Figures 4A to 4B As shown in the image.

[0260] Combining sequencing methods

[0261] In some embodiments, any of the modified oligonucleotides of the probe-free complex described herein (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 , Figure 17 , Figures 18A to 18B and Figures 19A to 19B Sequencing can be performed by performing a sequencing-by-binding (SBB) reaction. In some embodiments, the SBB reaction occurs on and / or inside a cell sample. In some embodiments, the sequencing-by-binding (SBB) procedure employs unlabeled chain-terminating nucleotides. In some embodiments, the sequencing-by-binding (SBB) cycle includes the following steps: (a) sequentially contacting an initiated modified oligonucleotide (e.g., a modified oligonucleotide annealed to multiple sequencing primers) with at least two separate mixtures under ternary complex stable conditions, wherein each of the at least two separate mixtures contains a polymerase and a nucleotide, whereby the sequential contact results in the initiated modified oligonucleotide contacting, under ternary complex stable conditions, nucleotide homologs of a first, second, and third base type in the modified oligonucleotide; (b) examining the at least two separate mixtures to determine whether a ternary complex has formed; and (c) Identifying the next correct nucleotide of the initiated modified oligonucleotide, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of a first, second, or third base type, and wherein if a ternary complex is not present in step (b), the next correct nucleotide is extrapolated as a homolog of a fourth base type; (d) Adding the next correct nucleotide to the primer of the initiated modified oligonucleotide after step (b), thereby producing an extended primer; and (e) Repeating steps (a) through (d) at least once for the initiated modified oligonucleotide containing the extended primer. Exemplary binding sequencing methods are described in U.S. Patent Nos. 10,246,744 and 10,731,141 (the contents of which are incorporated herein by reference in their entirety).

[0262] Methods for sequencing using nucleotide analogs

[0263] In some embodiments, any of the modified oligonucleotides of the probe-free complex described herein (e.g., Figures 13A to 13B , Figures 14A to 14B , Figures 15A to 15B , Figure 16 , Figure 17 , Figures 18A to 18B and Figures 19A to 19B Sequencing can be performed using a sequencing reaction with at least one nucleotide analog containing a 2' or 3' chain termination portion.

[0264] In some embodiments, the chain termination sequencing method includes step (a): contacting a sequencing polymerase with (i) a modified oligonucleotide and (ii) a sequencing primer, wherein the contact is performed under conditions suitable for binding the sequencing polymerase to the modified oligonucleotide hybridizing with the sequencing primer, wherein the modified oligonucleotide hybridizing with the sequencing primer forms a nucleic acid duplex. In some embodiments, the sequencing polymerase includes a recombinant mutant sequencing polymerase capable of binding and incorporating nucleotide analogs. In some embodiments, the sequencing primer includes a 3' extendable end. In some embodiments, the sequencing primer includes a 3' non-extendable end that can be converted into a 3' extendable end.

[0265] In some embodiments, the method for sequencing includes step (b): contacting a sequencing polymerase with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to a sequencing polymerase (which binds to a nucleic acid duplex) and suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase is contacted with the plurality of nucleotides in the presence of at least one catalytic cation including magnesium and / or manganese. In some embodiments, the plurality of nucleotides includes at least one nucleotide analog having a chain-terminating portion at a 2' or 3' position of a sugar. In some embodiments, the chain-terminating portion may be removed from the 2' or 3' position of the sugar to convert the chain-terminating portion to an OH or H group. In some embodiments, the plurality of nucleotides includes at least one nucleotide lacking a chain-terminating portion. In some embodiments, at least one nucleotide is labeled with a detectable reporter portion (e.g., a fluorophore). In some embodiments, the plurality of nucleotides includes a plurality of labeled nucleotides.

[0266] In some embodiments, the method for sequencing includes step (c): incorporating at least one nucleotide into the 3' end of an extendable primer under conditions suitable for incorporating at least one nucleotide. In some embodiments, the conditions suitable for the nucleotide-binding polymerase and the conditions suitable for incorporating the nucleotide may be the same or different. In some embodiments, the conditions suitable for incorporating the nucleotide include the addition of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the at least one nucleotide binds to the sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, step (c) of incorporating the nucleotide into the 3' end of the sequencing primer includes a primer extension reaction.

[0267] In some embodiments, the method for sequencing includes step (d): detecting and identifying nucleotides incorporated into the 3' end of the sequencing primer.

[0268] In some embodiments, the method for sequencing includes step (e): removing the chain termination portion from the incorporated nucleotide and generating a plurality of sequencing primers having an extendable 3' end.

[0269] In some embodiments, the method for sequencing includes step (f): repeating steps (a) through (e) at least once.

[0270] In some embodiments, in step (b), the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion comprises a fluorophore. In some embodiments, the fluorophore may be attached to a nucleotide base. In some embodiments, the fluorophore may be attached to a nucleotide base using a linker that can be cleaved / removed from the base. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of the nucleotide base.

[0271] Cell samples

[0272] In some embodiments, in any of the compositions or methods described herein, the cell sample includes cells, multiple cells, cell sections, intact tissue, organs, tissue sections, intact tumors, or tumor sections. In some embodiments, the cell sample includes fresh cell samples, fresh-frozen cell samples, sliced ​​cell samples, or FFPE cell samples. In some embodiments, the cell sample includes one or more live or non-live cells. In some embodiments, the cell sample may be obtained from viruses, fungi, prokaryotes, or eukaryotes. In some embodiments, the cell sample may be obtained from animals, fungi, plants, or bacteria. In some embodiments, the animal is a mammal or insect. In some embodiments, the cell sample includes one or more virus-infected cells. In some embodiments, the cell sample includes a biofilm, i.e., a microbial aggregate adhered together. In some embodiments, the cell sample may be obtained from any organism, including humans, apes, great apes, dogs, felines, cattle, horses, rats, pigs, goats, wolves, frogs, fish, plants, insects, fungi, yeast, or bacteria. In some embodiments, the cell sample may be obtained from any organ, including the head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs. In some embodiments, the cell sample includes cells that can form an adhesive monolayer when cultured on a support (e.g., a coated or uncoated support). In some embodiments, the cell sample includes human cells that can form adherent cells (e.g., an adherent monolayer) when cultured on a support, including, for example, HeLa, HEK293, HUVEC, HCT116, A549, PC3, HepG2, MCF7, and U2OS. In some embodiments, the cell sample includes human stem cells. In some embodiments, the cell sample includes cells that have differentiated in vitro into one or more cell types, including but not limited to neurons, glial cells, myocytes, epithelial cells, T cells, B cells, NK cells, and so on.

[0273] Cell samples cultured on supports

[0274] In some embodiments, in any of the compositions or methods described herein, a cell sample may be cultured on a support (e.g., a flow-through cell). In some embodiments, the method includes culturing a cell sample on a support under conditions suitable for expanding the cell sample for 2 to 10 passages (e.g., 2 to 10 cell passages) or more. The cultured cell sample may generate cell colonies. In some embodiments, the method includes culturing cells of the cell sample to confluent or non-confluent states. In some embodiments, the method includes culturing cells of the cell sample and then inducing terminal differentiation of the cells using methods known in the art. In some embodiments, the method includes culturing the cell sample on a support in a simple or complex cell culture medium. Suitable cell culture media are known to those skilled in the art who will be able to select a medium based on cell type and culture conditions. Exemplary cell culture media include, but are not limited to, D-MEM high glucose (e.g., from Thermo Fisher Scientific®, catalog number 11965118), fetal bovine serum (e.g., 10% FBS; e.g., from Thermo Fisher Scientific, catalog number A3160402), MEM non-essential amino acids (e.g., 0.1 mM MEM, e.g., from Thermo Fisher Scientific, catalog number 11140050), L-glutamine (e.g., 6 mM L-glutamine, e.g., from Thermo Fisher Scientific, catalog number A2916801), MEM sodium pyruvate (e.g., 1 mM sodium pyruvate, e.g., from Thermo Fisher Scientific™, catalog number 11360070), and antibiotics (e.g., 1% penicillin-streptomycin-glutamine, e.g., from Thermo Fisher®, catalog number 10378016). In some embodiments, the method includes culturing cell samples at humidity and temperature suitable for culturing cells on a support. Exemplary, non-limiting suitable conditions include approximately 37°C and a humidified atmosphere containing approximately 5% to 10% carbon dioxide in the air. Cell samples can be cultured under suitable aeration conditions (e.g., with oxygen and / or nitrogen).

[0275] In some embodiments, "simple cell culture medium" or related terms refer to a cell culture medium that typically lacks components that support cell growth and / or proliferation in a culture. Simple cell culture medium can be used, for example, to wash, suspend, or dilute cell samples. Simple cell culture medium can be mixed with certain components to prepare a cell culture medium that supports cell growth and / or proliferation in a culture medium. Simple cell culture medium contains any one or any combination of two or more of buffers, phosphate compounds, sodium compounds, potassium compounds, calcium compounds, magnesium compounds, and / or glucose. In some embodiments, simple cell culture medium contains PBS (phosphate-buffered saline), DPBS (Dulbecco's phosphate-buffered saline), HBSS (Hank's balanced salt solution), DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum Essential Medium), and / or EBSS. In some embodiments, cell samples may be placed in simple cell culture medium before or during any of the steps in the nucleic acid methods described herein.

[0276] In some embodiments, "composite cell culture medium" or related terms refer to a cell culture medium that can be used to support the growth and / or proliferation of cells in a medium without supplements or additives. Composite cell culture media may include any combination of two or more of buffer systems (e.g., HEPES), inorganic salts, amino acids, proteins, peptides, carbohydrates, fatty acids, lipids, purines and their derivatives (e.g., hypoxanthine), pyrimidines and their derivatives, and / or trace elements. Composite cell culture media include fluids obtained from fluids or tissue extracts. Composite cell culture media include artificial cell culture media. In some embodiments, composite cell culture media may be serum-containing media, such as composite cell culture media comprising fluids such as fetal bovine serum, plasma, serum, lymph, human placental cord serum, and amniotic fluid. In some embodiments, composite cell culture media may be serum-free media, which are generally (but not necessarily) defined cell culture media. In some embodiments, composite cell culture media may be chemically defined media, which generally (but not necessarily) include recombinant peptides and ultrapure inorganic and / or organic compounds. In some embodiments, the composite cell culture medium may be a protein-free medium, including, for example, MEM (minimum essential medium) and RPMI-1640 (Roswell Park Memorial Institute). In some embodiments, the composite cell culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium). In some embodiments, the composite cell culture medium comprises DMEM (Dulbecco's Modified Iscove's Medium). In some embodiments, cell samples may be placed in the composite cell culture medium before or during any of the steps in the nucleic acid methods described herein.

[0277] In some embodiments, cell samples may be deposited (e.g., seeded) onto a support. In some embodiments, the support comprises a planar or non-planar support. In some embodiments, the support comprises a solid or semi-solid support. In some embodiments, the support comprises a porous, semi-porous, or non-porous support. The support may be made of any material such as glass, plastic, or polymeric materials. In some embodiments, the surface of the support may be coated with one or more compounds to create a passivation layer on the support. In some embodiments, the passivation layer forms a porous or semi-porous layer.

[0278] In some embodiments, cell samples may be deposited (e.g., seeded) onto a support passivated with a coating that promotes the proliferation, migration, differentiation, and / or adhesion of cultured cells or live ex vivo cell or tissue samples. In some embodiments, cell samples may be deposited onto a support lacking immobilized capture primers capable of binding target polynucleotides from the cell sample. In some embodiments, the support may be coated with dissolved basement membrane matrix. Suitable methods for coating the support with dissolved basement membrane matrix are known to those skilled in the art. In some embodiments, the dissolved basement membrane matrix is ​​secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as MATRIGEL®. In some embodiments, the support may be coated with a gel matrix, including, for example, collagen gel, alginate gel, or lactate gel. In some embodiments, the support may be coated with one or more animal-derived proteins, including, for example, agglutinins, short proteoglycans, collagen (e.g., type I, II, III, or IV collagen), fibronectin, elastin, laminin, laminin / fibronectin, laminin / poly-D-lysine, laminin / poly-D-ornithine, hyalin, osteopontin, gelatin (e.g., porcine), fibrinogen, fibrinogen, plasminogen, plasmin, tendinin, hyaluronic acid proteoglycan, keratin sulfate proteoglycan, heparan sulfate proteoglycan, chondroitin sulfate proteoglycan, syndecan-1 (e.g., proteoglycan), and IGF-binding proteins. In some embodiments, the support may be coated with one or more compounds that generate an electrically coated surface. In some embodiments, the support is coated with polyamino acids, including, but not limited to, polylysine compounds (e.g., poly-L-lysine (PLL) or poly-D-lysine (PDL)), arginine compounds, polyarginine compounds, polyornithine compounds, or amino-terminated compounds (e.g., amino-terminated PEG). The support may be coated with unbranched compounds, branched compounds, or mixtures of unbranched and branched compounds. In some embodiments, the support may be coated with modified peptides, including, for example, but not limited to, cationic antimicrobial peptides or bisurface antimicrobial peptides. In some embodiments, the support may be coated with a polycyclic peptide antibiotic comprising the thioether amino acid lanethionine or methyllanethionine and / or the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. In some embodiments, the support may be coated with at least one small peptide, such as melitrix venom peptide. In some embodiments, the support may be coated with compounds that promote integrin-mediated cell adhesion. For example, but not limited to, the support may be coated with the tripeptide arginyl-glycyl-aspartic acid (Arg-Gly-Asp; also known as RGD).In some embodiments, the support may be coated with short peptides of extracellular matrix proteins, including, but not limited to, Arg-Gly-Asp (RGD), RGD-coupled alginate, Ile-Lys-Val-Ala-Val (IKVAV), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV), Val-Ala-Pro-Gly (VAPG), Phe-Gly-Leu (FGL), fibronectin domains, and laminin. In some embodiments, the support may be coated with amines or polymers having -NH2 groups that promote cell adhesion, including, for example, polyethyleneimine (PEI) or polydopamine (PDA). In some embodiments, the support may be coated with any compound known in the art that can be used to promote the proliferation, migration, differentiation, and / or adhesion of cultured cells or ex vivo living cell or tissue samples. In some embodiments, cell samples may be deposited (e.g., seeded) onto an uncoated support.

[0279] Cell fixation

[0280] In some embodiments, in any of the compositions or methods described herein, the cell sample includes a fixed cell sample. In some embodiments, the cell sample may be treated with a fixation reagent (e.g., a fixing reagent) that preserves the cells and their contents to inhibit degradation and cell lysis. For example, and not limited to, the fixation reagent may preserve RNA carried by the cell sample. In some embodiments, the fixation reagent inhibits the loss of nucleic acids in the cell sample.

[0281] In some embodiments, the fixation agent can cross-link RNA to prevent RNA from escaping from the cell sample. In some embodiments, the cross-linking fixation agent includes any combination of aldehydes, formaldehyde, paraformaldehyde, formalin, glutaraldehyde, imine esters, N-hydroxysuccinimide esters (NHS), and / or glyoxal (bifunctional aldehydes).

[0282] In some embodiments, the fixative comprises at least one alcohol, including methanol or ethanol. In some embodiments, the fixative comprises at least one ketone, including acetone. In some embodiments, the fixative comprises acetic acid, glacial acetic acid, and / or picric acid. In some embodiments, the fixative comprises mercuric chloride. In some embodiments, the fixative comprises a zinc salt, including zinc sulfate or zinc chloride. In some embodiments, the fixative may denature the peptide.

[0283] In some embodiments, the fixative comprises 4% w / v paraformaldehyde relative to water / PBS. In some embodiments, the fixative comprises 10% formaldehyde at 35% neutral pH. In some embodiments, the fixative comprises 2% v / v glutaraldehyde relative to water / PBS. In some embodiments, the fixative comprises 25% of a 37% formaldehyde solution, 70% picric acid, and 5% acetic acid.

[0284] In some embodiments, cell samples may be fixed on a support with 4% paraformaldehyde for about 30 to 60 minutes or any range therebetween and washed with PBS.

[0285] In some embodiments, the cell sample may be stained, destained (e.g., removed staining), or unstained.

[0286] Cell permeation

[0287] In some embodiments, in any of the compositions or methods described herein, the cell sample comprises a permeabilized cell sample. In some embodiments, the method comprises treating the cell sample with a permeabilizing agent that alters the cell membrane to allow reagents (such as multivalent molecules, nucleotides, chain terminator nucleotides, sequencing polymerases, sequencing primers, rolling circle amplification (RCA) reagents, polymerase chain reaction (PCR) reagents, sequencing reagents, etc.) to permeate into the cell. For example, but not limited to, the permeabilizing agent removes membrane lipids from the cell membrane. In some embodiments, the cell sample may be treated with a permeabilizing agent comprising any combination of organic solvents, detergents, chemical compounds, crosslinking agents, and / or enzymes. In some embodiments, the organic solvents include acetone, ethanol, and methanol. In some embodiments, the detergent comprises saponins, Triton X-100, Tween-20, sodium dodecyl sulfate (SDS), N-lauroyl sarcosinate sodium salt solution, or nonionic polyoxyethylene surfactants (e.g., NP40). In some embodiments, the crosslinking agent comprises paraformaldehyde. In some embodiments, the enzyme comprises trypsin, pepsin, or a protease (e.g., proteinase K). In some embodiments, alkaline or acidic conditions may be used to permeate cells with a protease. In some embodiments, the permeation reagent includes water and / or PBS.

[0288] For example, but not limited to, fixed cells can be permeabilized with 70% ethanol for about 30 to 60 minutes, and the permeabilization reagent can be replaced with PBS-T (e.g., PBS with 0.05% Tween-20). In some embodiments, cells can be post-fixed with 3% paraformaldehyde and 0.1% glutaraldehyde for about 30 to 60 minutes or any range therebetween, and washed with PBS-T, for example, multiple times.

[0289] Target analytes

[0290] In some embodiments, the analyte-binding portion conjugated to the modified oligonucleotide described herein may selectively bind to a target analyte located on and / or within the cell. In some embodiments, the modified oligonucleotide (and the conjugated analyte-binding portion) may enter the cell. In some embodiments, the modified oligonucleotide (and the conjugated analyte-binding portion) may be located in the cytoplasm of the cell. In some embodiments, the analyte-binding portion of the modified oligonucleotide may bind to or enter organelles, including, for example, the nucleus, nucleolus, mitochondria, Golgi apparatus, endoplasmic reticulum, flagella, cilia, or chloroplasts.

[0291] In some embodiments, the analyte binding portion binds to the target analyte, including naturally occurring molecules, recombinant molecules, modified molecules, and / or synthetic molecules. In some embodiments, the target analyte includes peptides, including but not limited to polypeptides, proteins, protein fragments, and enzymes. In some embodiments, the target analyte includes cell surface receptors, including but not limited to ion channel receptors (e.g., ligand-gated ion channel receptors), G protein-coupled receptors, enzyme-linked receptors, and intracellular cell receptors. In some embodiments, the target analyte includes differentiation clusters (CDs), including CDs that act as cell receptors, cell ligands, cell signaling, and cell adhesion. In some embodiments, the target analyte includes immunoglobulin molecules, including but not limited to antibodies, antibody fragments, and single-chain antibodies. In some embodiments, the target analyte includes carbohydrates, including but not limited to monosaccharides, disaccharides, oligosaccharides, polysaccharides, homopolysaccharides, and heteropolysaccharides. In some embodiments, the target analyte includes lipids, including but not limited to triglycerides, phospholipids, steroids, fatty acyls, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides (e.g., derived from the condensation of ketoacyl subunits), sterol lipids, and isoprenol lipids (e.g., derived from the condensation of isoprenol subunits). In some embodiments, the target analyte includes nucleic acids, including but not limited to polynucleotides, oligonucleotides, DNA, cDNA, and RNA. In some embodiments, the target analyte includes glycosylated molecules, including but not limited to glycoproteins and glycolipids.

[0292] In some embodiments, the analyte-binding moiety may bind to cell membrane proteins. In some embodiments, the analyte-binding moiety may bind to PMCA (plasma membrane Ca2+ ATPase), wherein the analyte-binding moiety includes calmodulin, RASSF1 (Ras-associated factor 1), calcineurin A, α-1 myotrophin, or caloxin. In some embodiments, the analyte-binding moiety may bind to ezrin, wherein the analyte-binding moiety includes DAG1 (dystrophin), SLC9A3R1 (Na(+) / H(+) exchange regulator cofactor NHR-RF1), RDX (root protein), ARHGAP18 (rho GTPase activator protein 18), or SDC4 (multiligand proteoglycan-4). In some embodiments, the analyte-binding moiety may bind to ZO1 (zonula occludens-1), wherein the analyte-binding moiety includes DbpA (DNA-binding protein A), occludin, afadin, or α-catenin. In some embodiments, the analyte-binding moiety may bind to claudin-1, wherein the analyte-binding moiety includes occludedin, ZO-1, ZO-2, or ZO-3 (zonula occludens-1, occludin, or occludin-3), and their homologues and orthologs.

[0293] In some embodiments, the analyte-binding moiety may bind to G-actin (globulin) or F-actin (filamentous actin). In some embodiments, the analyte-binding moiety includes a phalloidin that can bind to F-actin.

[0294] In some embodiments, the analyte binding portion may bind to proteins located on the nuclear membrane, including but not limited to SUN2 (protein 2 containing Sad1 and UNC84 domains), TMPO (thymopoietin), SUN1 (protein 1 containing Sad1 and UNC84 domains), LEMD2 (LEM domain nuclear envelope protein 2), LMNB1 (lamin B), TOR1AIP1 (torsin 1A interacting protein 1), LBR (lamin B receptor), LMNB2 (lamin B2), and type A lamin, as well as their homologues and orthologs.

[0295] In some embodiments, the analyte-binding moiety may bind to proteins located in the cell nucleus. In some embodiments, the analyte-binding moiety may bind to the following: histones, TAF15 (TAT box-binding protein-associated factor 15), SMARCAD1 (SWI / SNF-associated matrix-associated actin-dependent chromatin regulator, containing DEAD / H box 1 subfamily A), SRRM2 (serine / arginine repeat matrix 2), RBM25 (RNA-binding motif protein 25), PML (PML nuclear scaffold), SMN2 (motor neuron 2 survival gene, centromere copy), and MKI67 (proliferation marker Ki-67), as well as their homologs and orthologs.

[0296] In some embodiments, the analyte-binding moiety may bind to proteins located in the cytoplasm. In some embodiments, the analyte-binding moiety may bind to: ATXN2 (ataxia protein 2), G3BP2 (G3BP stress granule assembly factor 2), AIMP1 (aminoacyl-tRNA synthetase complex interacting multifunctional protein 1), SERBP1 (SERPINE1 mRNA-binding protein 1), CCDC43 (protein 43 containing a coiled-coil domain), ATXN2L (ataxia protein 2-like), AMPD2 (adenosine monophosphate deaminase 2), and RABGAP1 (RAB GTPase activating protein 1). In some embodiments, the analyte-binding moiety may bind to tubulin, for example, including colchicine, vinblastine, pironetin, maytansine, taxane, laulimalide, peloluside, cevipabulin, or rhizosin.

[0297] In some embodiments, the analyte-binding moiety may bind to the Golgi apparatus. In some embodiments, the analyte-binding moiety may bind to Golgi protein 97, for example, when the analyte-binding moiety includes FIP1 / RCP (family interacting protein) (Rab-coupled protein). In some embodiments, the analyte-binding moiety may bind to TGN 46 (reverse Golgi network 46), wherein the analyte-binding moiety includes PKD (protein kinase D) or OSBP (oxidative sterol-binding protein), as well as its homologues and orthologs.

[0298] In some embodiments, the analyte-binding moiety may bind to the endoplasmic reticulum. In some embodiments, the analyte-binding moiety may bind to the following: HSP90B1 (heat shock protein 90β family member 1), CANX (calcinin), KTN1 (kinasein 1), PDIA3 (protein disulfide isomerase family A member 3), RCN1 (reticulocalcinin 1), RRBP1 (ribosome-binding protein 1), SEC61B (SEC61 transloson subunit β), and CY51A1 (cytochrome P450 family 51 subfamily A member 1), as well as their homologs and orthologs.

[0299] In some embodiments, the analyte-binding moiety may bind to mitochondria. In some embodiments, the analyte-binding moiety may bind to: CS (citrate synthase), LRPPRC (a leucine-rich pentapeptide repeat sequence), SLC25A24 (a member of solute carrier family 25), TIMM44 (a translocase of mitochondrial inner membrane 44), GCDH (glutaryl-CoA dehydratase), and TRAP1 (TNF receptor-associated protein 1), as well as their homologs and orthologs.

[0300] In some embodiments, the analyte binding portion may bind to a cytokine. In some embodiments, the cytokine includes proteins expressed on the surface of immune cells and non-immune cells. In some embodiments, the cytokine includes human cytokines. Cytokines may include, but are not limited to, interleukins, interferons, tumor necrosis factor, transforming growth factor, chemokines, lymphokines, monokines, and colony-stimulating factors.

[0301] In some embodiments, the analyte binding portion may bind to interleukins, including but not limited to IL1α (hemopoietin-1), IL-1β (catabolite), IL-1RA (IL-1 receptor antagonist), IL-18 (interferon-γ inducible factor), IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF (leukemia inhibitory factor), OSM (oncokinase-M), IL-10, IL-20, IL-14, IL-16, IL-17, IL-17A, IL-17F, IL-21, IL-23, IL-22, and IL-35, as well as their homologues and orthologs.

[0302] In some embodiments, the analyte binding portion may bind to an interferon, including but not limited to IFN-α, IFN-β, and IFN-γ, as well as their homologues and orthologs.

[0303] In some embodiments, the analyte binding portion may bind to tumor necrosis factor, which includes, but is not limited to, CD154 (CD40L or TRAP), LT-β, ​​TNF-α (causcin), TNF-β (LT-α), 4-1BBL, APRIL (TALL-2), CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL (Apo2L), TWEAK (Apo3L), and TRANCE (OPGL), as well as their homologues and orthologs.

[0304] In some embodiments, the analyte binding portion may bind to transforming growth factors, including but not limited to TGF-β1, TGF-β2, and TGF-β3, as well as their homologues and orthologs.

[0305] In some embodiments, the analyte binding portion may bind to a chemokine, which includes, but is not limited to, XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14 and CX3CL1, as well as their homologues and orthologs.

[0306] In some embodiments, the analyte binding portion may bind to angiogenic factors, including but not limited to VEGF-A, VEGF-B, VEGF-C, and VEGF-D, as well as their homologues and orthologs.

[0307] In some embodiments, the analyte binding portion may bind to platelet-derived growth factors, including but not limited to PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB, as well as their homologues and orthologs.

[0308] In some embodiments, the analyte binding portion may bind to cytokines, including but not limited to erythropoietin (EPO), M-CSF (macrophage colony-stimulating factor), and GM-CSF (granulocyte-macrophage colony-stimulating factor), as well as their homologues and orthologs.

[0309] In some embodiments, the analyte binding portion may bind to a peptide hormone. In some embodiments, the peptide hormone includes human peptide hormones. In some embodiments, the analyte binding portion may bind to a peptide hormone, including, but not limited to, adrenocorticotropic hormone (ACTH), adopine, amylin, angiotensin, anti-Müllerian hormone (AMH), atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), human chorionic gonadotropin-1 (CSH1), human chorionic gonadotropin-2 (CSH2), corticotropin-releasing hormone (CRH), gastrin, ghrelin, glucagon, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and gonadotropin-releasing hormone 1. (GNRH1), gonadotropin-releasing hormone 2 (GNRH2), growth hormone, growth hormone-releasing hormone (GHRH), follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), parathyroid hormone-like hormone (PTHLH), melanocyte-concentrating hormone (PMCH), prolactin, renin, resistin (RETN), somatostatin, growth hormone (GH1), thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, and vasoactive intestinal peptide (VIP), as well as their homologues and orthologs.

[0310] In some embodiments, the analyte binding portion may comprise an antibody or antibody fragment, including but not limited to single-chain antibodies, microantibodies, single-chain variable fragments (scFv), Fab fragments, F(ab)2 fragments, F(ab')2 fragments, domain antibodies, VHH antibodies, one or more isolated complementarity-determining regions (CDRs), T cell receptors or antigen-binding domains of synthetic T cell receptors, etc.

[0311] In some embodiments, the analyte binding portion comprises a lectin, including but not limited to concanavalin A, wheat germ lectin, or other lectins known in the art.

[0312] In some embodiments, the analyte binding portion comprises hemagglutinin or other phage or virus cell surface or cell-binding proteins.

[0313] In some embodiments, the analyte binding portion comprises peptide hormones or peptide signaling molecules, including but not limited to endocrine or paracrine signaling peptides and bacterial signaling peptides.

[0314] In some embodiments, the analyte binding portion comprises a surface-bound peptide, such as an amphiphilic peptide or an antimicrobial peptide, examples of which include, but are not limited to, melittin, coliformin, antibiotic antimicrobial agents, brevinin, esculentin, ranacyclin, and the like.

[0315] In some embodiments, the analyte binding portion comprises a polynucleotide containing a sequence complementary to a target analyte comprising a nucleic acid.

[0316] Target analytes may include molecules from any source, including molecules carried in air, water, soil, or food. In some embodiments, target analytes may be isolated from any organism, including viruses, fungi, prokaryotes, or eukaryotes. In some embodiments, target analytes may be isolated from any organism, including humans, apes, great apes, dogs, felines, cattle, horses, rats, pigs, goats, wolves, frogs, fish, plants, insects, or bacteria. In some embodiments, target analytes may be isolated from any biological fluid, including blood, urine, serum, lymph, tumors, saliva, anal secretions, vaginal secretions, amnion samples, sweat, semen, environmental samples, or culture samples. In some embodiments, target analytes may be isolated from any organ, including the head, neck, brain, mammary gland, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs. In some embodiments, the target analyte includes naturally occurring molecules, recombinant molecules, modified molecules, and / or synthetic molecules. In some embodiments, the target analyte may be prepared using recombinant nucleic acid technology, which includes, but is not limited to, any combination of vector cloning, transgenic host cell preparation, host cell culture, and / or PCR amplification.

[0317] Sequencing polymerase

[0318] In some embodiments, in any of the compositions or methods described herein, a plurality of sequencing polymerases may be used to perform any of the polymerase-catalyzed sequencing reactions described herein. In some embodiments, the sequencing polymerase is capable of binding to and incorporating a complementary nucleotide opposite to a nucleotide in a template molecule (e.g., a modified oligonucleotide). In some embodiments, the sequencing polymerase is capable of binding to a complementary nucleotide portion of a multivalent molecule opposite to a nucleotide in a template molecule (e.g., a modified oligonucleotide). In some embodiments, the plurality of sequencing polymerases includes a recombinant mutant polymerase.

[0319] Examples of suitable polymerases for sequencing nucleotides and / or multivalent molecules include, but are not limited to: Klenow DNA polymerase; Thermophila aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaea; Yellowstone subterranean archaea provisional species; Hadesarchaea archaea; Euryarchaeota archaea; Thermoplasmata archaea; Thermococcus polymerases, such as Thermococcus litoralis, bacteriophage T7 DNA polymerase; human α, δ, and ε DNA polymerases; bacteriophage polymerases, such as T4, RB69, and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; Escherichia coli DNA polymerase III α and ε; 9°N polymerase; reverse transcriptases, such as HIV M-type or O-type reverse transcriptase; avian myeloblastoma virus reverse transcriptase; Moloney murine leukemia virus (MMLV) reverse transcriptase; or telomerase. Other non-limiting examples of DNA polymerases include those polymerases from various archaea genera (such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Steteria, Sulfolobus, Thermococcus, and Vulcanisaeta, etc., or variants thereof), including such polymerases as known in the art, such as 9°N, VENT™, DEEP VENT™, THERMINATOR™, Pfu, KOD, Pfx, Tgo, and RB69 polymerases. The phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi™ from Expedeon™), a variant EquiPhi29™ DNA polymerase (e.g., from Thermo Fisher Scientific®), or a chimeric QualiPhi™ DNA polymerase (e.g., from 4basebio™). Additional polymerases are described in U.S. Patent No. 11,859,241, the contents of which are incorporated herein by reference in their entirety.

[0320] Nucleotides and chain-terminating nucleotides

[0321] In some embodiments, in any of the compositions or methods described herein, the sequencing method described herein may employ at least one nucleotide. In some embodiments, the nucleotide may be used in a polymerase-catalyzed sequencing method.

[0322] In some embodiments, each nucleotide comprises a base, a sugar, and at least one phosphate ester group. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate ester groups (e.g., 1 to 10 phosphate ester groups). The plurality of nucleotides may include at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides may include a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, at least one nucleotide of the plurality of nucleotides is not a nucleotide analog. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a nucleotide analog.

[0323] In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide of the plurality of nucleotides is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group, the substituted side group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, the analogue comprising phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide.

[0324] In some embodiments, at least one nucleotide of the plurality of nucleotides includes a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion may inhibit the polymerase-catalyzed incorporation of subsequent nucleotide portions or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain-terminating portion is attached to the 3' hydroxyl position of the sugar, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain-terminating portion may be removed / cleaved from the 3' hydroxyl position to generate a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion includes alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thio, disulfide, carbonate, urea, silyl, or acetal groups. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide, for example by reaction of the chain-terminating portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetrakis(triphenylphosphine)-palladium(O) (Pd(PPh3)4) using piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thio, or disulfide groups of the chain-terminating portion may be cleaved with, for example, phosphine or thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the carbonate group of the chain-terminating portion may be cleaved with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain-terminating portion of the urea and silyl group can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0325] In some embodiments, at least one nucleotide of the plurality of nucleotides includes a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion includes an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion includes a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating portion azide, azide group, and an azidemethyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenylphosphine) (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0326] In some embodiments, the nucleotide comprises a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof.

[0327] In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base with a linker that can be cleaved / removed from the base. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of the nucleotide base.

[0328] In some embodiments, the nucleotide includes a cleavable linker on the nucleotide base. In some embodiments, the cleavable linker on the nucleotide base includes a cleavable portion comprising: alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide group, amine group, amide group, ketone group, isocyanate group, phosphate group, thioyl, disulfide group, carbonate group, urea group, silyl group, or acetal group. In some embodiments, the cleavable linker on the base can be cleaved / removed from the base by reacting the cleavable portion with a chemical agent, pH change, light, or heat. In some embodiments, the cleavable portions of alkyl, alkenyl, alkynyl, and allyl can be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the cleavable portions of aryl and benzyl can be cleaved with H2Pd / C. In some embodiments, cleavable amines, amides, ketones, isocyanates, phosphates, thiocyanates, or disulfides can be cleaved with phosphine or thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, cleavable carbonates can be cleaved with potassium carbonate (K₂CO₃) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, cleavable ureas and silyl groups can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0329] In some embodiments, the cleavable linker on the nucleotide base includes a cleavable portion comprising an azide, an azide group, or an azide methyl group. In some embodiments, the cleavable portion of the azide, azide group, and azide methyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenyl)phosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent comprises 4-dimethylaminopyridine (4-DMAP).

[0330] In some embodiments, the chain termination portion (e.g., at the 2' and / or 3' positions of the sugar) and the cleavable linker on the nucleotide base have the same or different cleavable portions. In some embodiments, the chain termination portion (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter portion linked to the base can be chemically cleaved / removed using the same chemical agent. In some embodiments, the chain termination portion (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter portion linked to the base can be chemically cleaved / removed using different chemical agents.

[0331] Multivalent molecules containing nucleotide moieties

[0332] In some embodiments, in any of the compositions or methods described herein, sequencing may employ at least one multivalent molecule. In some embodiments, the multivalent molecule may be used in a polymerase-catalyzed sequencing method.

[0333] In some embodiments, a single multivalent molecule comprises multiple nucleotide arms attached to a core and has any conformation, including starburst, random, or bottlebrush conformations (e.g., Figure 1 An exemplary multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms comprising (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide portion, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide portion. In some embodiments, the nucleotide portion comprises a base, a sugar, and at least one phosphate ester group, and the linker is attached to the nucleotide portion via a base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein two linker chains have 2 to 6 subunits. In some embodiments, the linker also comprises an aromatic portion. An exemplary nucleotide arm is shown in Figure 5. An exemplary multivalent molecule in Figure 1 As shown in Figure 4. An exemplary spacer is shown in... Figure 6 The exemplary connector shown (at the top) is in Figure 6 (Bottom) and Figure 7 As shown in the diagram. An exemplary nucleotide attached to the adapter is shown in... Figures 8 to 11 As shown in the figure. An exemplary biotinylated nucleotide arm in Figure 12 As shown in the image.

[0334] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the plurality of nucleotide arms have the same type of nucleotide motif selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0335] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, each arm containing a nucleotide moiety. The nucleotide moiety comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may comprise one type of multivalent molecule having a nucleotide moiety selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein each multivalent molecule in the mixture contains a nucleotide moiety selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0336] In some embodiments, the nucleotide moiety comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, at least one nucleotide moiety is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group, the substituted side group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate ester group substituted with an analog, the analog comprising phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide.

[0337] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide moiety having a chain-terminating portion (e.g., a blocking portion) at the 2' position, the 3' position, or both the 2' and 3' positions of the sugar. In some embodiments, the nucleotide moiety comprises a chain-terminating portion (e.g., a blocking portion) at the 2' position, the 3' position, or both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion may inhibit the polymerase-catalyzed incorporation of subsequent nucleotide moieties or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain-terminating portion is attached to the 3' hydroxyl position, wherein the sugar comprises a ribose or deoxyribose moiety. In some embodiments, the chain-terminating portion may be removed / cleaved from the 3' hydroxyl position to generate a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion includes alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thio, disulfide, carbonate, urea, silyl, or acetal groups. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide portion, for example by reacting the chain-terminating portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetrakis(triphenylphosphine)-palladium(O) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thio, and disulfide groups of the chain-terminating portion may be cleaved with phosphine or thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating carbonate portion can be cleaved using potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating urea and silyl groups can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0338] In some embodiments, the nucleotide moiety comprises a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion comprises a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating portion azide, azide group, and an azidemethyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine moiety or a derived triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenylphosphine) (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0339] In some embodiments, the nucleotide portion comprises a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof.

[0340] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the nucleotide arms include spacers, linkers, and nucleotide moieties, and wherein the core, linkers, and / or nucleotide moieties are labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of the nucleotide moieties (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of nucleotide bases.

[0341] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide portion attached to a detectable reporter portion. In some embodiments, the detectable reporter portion is attached to a nucleotide base. In some embodiments, the detectable reporter portion includes a fluorophore. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of the nucleotide portion (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0342] In some embodiments, the core of the multivalent molecule includes an avidin-like or streptavidin-like moiety, and the core attachment portion includes biotin. In some embodiments, the core includes a streptavidin-type or avidin-type moiety (including avidin protein), and any derivative, analog, or other non-natural form of avidin that can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces averdin), and derived forms such as N-acylavidin, such as N-acetyl, N-phthaloyl, and N-succinylavidin, and commercially available products EXTRAVIDIN™, CAPTAVIDIN™, NEUTRAVIDIN™, and NEUTRAALITE AVIDIN™.

[0343] Any of the methods described herein for sequencing nucleic acid molecules may include forming a binding complex, wherein the binding complex comprises (i) a polymerase, a template molecule (e.g., a modified oligonucleotide) that forms a double strand with the primer, and a nucleotide, or the binding complex comprises (ii) a polymerase, a template molecule (e.g., a modified oligonucleotide) that forms a double strand with the primer, and a nucleotide portion of a multivalent molecule. In some embodiments, the binding complex has a residence time greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. In some embodiments, the binding complex has a residence time greater than about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, and / or the method is performed at or may be performed at temperatures of 15°C or higher, 20°C or higher, 25°C or higher, 35°C or higher, 37°C or higher, 42°C or higher, 55°C or higher, 60°C or higher, 72°C or higher, or 80°C, or within the range defined by any of the foregoing. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that lead to dissociation between the polymerase, template molecule, primer, and / or nucleotide moieties or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with any one or any combination of a detergent, EDTA, and / or water. In some embodiments, this disclosure provides a method in which the binding complex is formed within a cell sample, wherein the cell sample is deposited on a surface exhibiting a contrast-to-noise ratio greater than 20 during a detection step. In some embodiments, this disclosure provides a method in which contact is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide moiety is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide moiety is not complementary to the next base of the template nucleic acid.

[0344] Multivalent probes containing target-specific oligonucleotide probes

[0345] In some embodiments, in any of the compositions or methods described herein, sequencing may employ at least one multivalent probe comprising a core connected to a plurality of probe arms, wherein each probe arm comprises a polymer arm linked to a target-specific oligonucleotide probe (e.g., Figures 5A to 5B In some embodiments, multivalent probes can be used for hybridization sequencing methods.

[0346] In some embodiments, in the hybridization sequencing method, the multivalent probe comprises: (1) a core; and (2) a plurality of probe arms comprising (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a adapter, and (iv) a target-specific oligonucleotide probe. In some embodiments, the core is attached to the plurality of probe arms, wherein the spacer is attached to the adapter, and wherein the adapter is attached to the target-specific probe. In some embodiments, the adapter is attached to the target-specific probe via a base of one of the nucleotides in the oligonucleotide probe. In some embodiments, the adapter comprises an aliphatic chain or an oligoethylene glycol chain, wherein two adapter chains have 2 to 6 subunits. In some embodiments, the adapter further comprises an aromatic portion.

[0347] In some embodiments, the target-specific probe may be attached to the end of the probe arm, or the target-specific probe may be attached to the internal region of the probe arm. In some embodiments, the structure of the multivalent probe is as follows: Figure 5A As shown in the figure. In some embodiments, the structure of the probe arm comprising the target-specific oligonucleotide probe is shown in Figure 5B As shown in the image.

[0348] In some embodiments, the spacer includes Figure 6 The structure shown (at the top). In some embodiments, the connector includes Figure 6 (Bottom) and Figure 7 Any structure shown in the diagram.

[0349] In some embodiments, the core of the multivalent probe includes an avidin-like or streptavidin-like moiety, and the core attachment portion includes biotin. In some embodiments, the core includes a streptavidin-type or avidin-type moiety (including avidin protein), and any derivative, analog, or other non-natural form of avidin that can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces averdin), and derived forms such as N-acylavidin, such as N-acetyl, N-phthaloyl, and N-succinylavidin, and commercially available products EXTRAVIDIN™, CAPTAVIDIN™, NEUTRAVIDIN™, and NEUTRAALITE AVIDIN™.

[0350] In some embodiments, each multivalent probe includes a core linked to multiple probe arms, wherein each probe arm contains the same target-specific sequence. For example, the core may be linked to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more probe arms, wherein each probe arm contains the same target-specific sequence. In some embodiments, probe arms containing the same target-specific sequence may selectively hybridize with the same target sequence at multiple regions on the same template molecule (e.g., a modified oligonucleotide), or may selectively hybridize with the same target sequence on different template molecules (e.g., modified oligonucleotides).

[0351] In some embodiments, each multivalent probe includes a core linked to multiple probe arms, wherein each probe arm contains a different target-specific sequence. For example, the core may be linked to probe arms having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different target-specific sequences. In some embodiments, probe arms containing different target-specific sequences may selectively hybridize with different target sequences on the same template molecule, or may selectively hybridize with different target sequences on different template molecules.

[0352] In some embodiments, the target-specific probe of the multivalent probe comprises an oligonucleotide of any range or longer, including 10 to 20 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, or longer.

[0353] In some embodiments, each multivalent probe may be labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. In some embodiments, the core of the multivalent probe may be labeled with at least one fluorophore, wherein a fluorophore attached to a given core of the multivalent probe corresponds to a specific target-specific probe sequence of the probe arm. In some embodiments, the adapter of the multivalent probe may be labeled with at least one fluorophore, wherein a fluorophore attached to a given adapter of the multivalent probe corresponds to a specific target-specific probe sequence of the probe arm. In some embodiments, the spacer of the multivalent probe may be labeled with at least one fluorophore, wherein a fluorophore attached to a given spacer of the multivalent probe corresponds to a specific target-specific probe sequence of the probe arm. In some embodiments, the target-specific oligonucleotide of the multivalent probe may be labeled with at least one fluorophore, wherein a fluorophore attached to a given target-specific oligonucleotide of the multivalent probe corresponds to a specific target-specific probe sequence of the probe arm.

[0354] In some embodiments, sequencing includes: (1) contacting a plurality of template molecules (e.g., modified oligonucleotides) with a plurality of first detectably labeled multivalent probes (e.g., first fluorophore-labeled multivalent probes) under conditions suitable for selectively hybridizing the target-specific probes of each first detectably labeled multivalent probe with a first target sequence region of a template molecule, thereby forming a first plurality of detectably labeled template-probe complexes, wherein the target-specific probes of the first detectably labeled multivalent probes comprise a first probe sequence; (2) detecting and imaging the fluorescence signal and color emitted by the first plurality of detectably labeled multivalent probes selectively hybridizing with the first target sequence region of the template molecule (e.g., the first plurality of template-probe complexes); and (3) extracting the first template-probe complex from the first template-probe complex. (3) The template molecule is removed from the first plurality of detectably labeled multivalent probes and the template molecule is retained; (4) Under conditions suitable for selectively hybridizing the target-specific probes of each second detectably labeled multivalent probe with the second target sequence region of the same template molecule, the template molecule retained in step (3) is contacted with a plurality of second detectably labeled multivalent probes (e.g., second fluorophore-labeled multivalent probes), thereby forming a second plurality of detectably labeled template-probe complexes, wherein the target-specific probes of the second detectably labeled multivalent probes contain a second probe sequence; and (5) the fluorescence signal and color emitted by the second plurality of detectably labeled multivalent probes selectively hybridizing with the second target sequence of the template molecule (e.g., the second plurality of template-probe complexes) are detected and imaged. In some embodiments, the plurality of template molecules are on and / or inside the cell sample.

[0355] In some embodiments, steps (4) to (5) may be repeated at least once. In some embodiments, repeating steps (4) to (5) includes contacting the template molecule with a plurality of third detectably labeled multivalent probes under conditions suitable for selectively hybridizing the target-specific probes of each third detectably labeled multivalent probe with a third target sequence region of the same template molecule, thereby forming a third plurality of detectably labeled template-probe complexes, wherein the target-specific probes of the third detectably labeled multivalent probes comprise a third probe sequence. In some embodiments, any of the first target sequence region, the second target sequence region, and the third target sequence region of the template molecule may overlap or not overlap. In some embodiments, the first probe sequence, the second probe sequence, and the third probe sequence are not identical.

[0356] In some embodiments, the hybridization sequencing cycle includes steps (1) to (2). In some embodiments, the hybridization sequencing cycle includes steps (1) to (3).

[0357] In some embodiments, images of sequential hybridization sequencing cycles using multivalent probes can be used to decode target sequences of template molecules on and / or inside cell samples.

[0358] In some embodiments, during a given hybridization sequencing cycle, the template molecule may be contacted with a detectably labeled multivalent probe of one type containing the same target-specific probe sequence.

[0359] In some embodiments, during a given hybridization sequencing cycle, a template molecule may be contacted with a mixture of different types of detectably labeled multivalent probes, each multivalent probe carrying a different target-specific probe sequence that can selectively hybridize with its homologous target sequence.

[0360] In some embodiments, in a given hybridization sequencing cycle, a mixture of different types of detectably labeled multivalent probes can selectively hybridize with different target sequences on the same template molecule.

[0361] In some embodiments, in a given hybridization sequencing cycle, a mixture of different types of detectably labeled multivalent probes can selectively hybridize with different target sequences on different template molecules.

[0362] In some embodiments, at least two hybridization sequencing cycles may be performed using the plurality of detectably labeled multivalent probes containing target-specific probes, as described herein.

[0363] In some embodiments, 2 to 50 hybridization sequencing cycles may be performed using the plurality of detectably labeled multivalent probes containing target-specific probes, as described herein.

[0364] Flow pool

[0365] In some embodiments, in any of the compositions or methods described herein, cell samples may be deposited onto a solid support (e.g., a flow cell). In some embodiments, cell samples are deposited onto a flow cell having walls (e.g., a top wall or first wall, and a bottom wall or second wall) and a gap therebetween, wherein the gap may be fluid-filled, and the flow cell is positioned in a fluorescence optical imaging system. When using conventional imaging systems, the thickness of the cell sample may require focusing the imaging system on a first and a second surface of the flow cell, respectively. To improve imaging of sequencing reactions of amplified probe complexes on and / or within the cell sample, the flow cell may be placed in a high-performance fluorescence imaging system comprising two or more tube lenses designed to provide optimal imaging performance for the first and second surfaces of the flow cell at two or more fluorescence wavelengths. In some embodiments, the high-performance imaging system further includes a focusing mechanism configured to refocus the optics between acquiring images of the first and second surfaces of the flow cell. In some embodiments, the high-performance imaging system is configured to image two or more fields of view on at least one of the first or second flow cell surfaces.

[0366] Automation mode

[0367] Any combination of the steps described herein can be performed in an automated mode using fluid dispensing systems known in the art, including cell deposition (e.g., cell seeding), culturing cell samples on a flow cell, cell fixation, cell permeabilization, reverse transcription, contacting the cell sample with any of the amplification-free probe complexes described herein, and sequencing.

[0368] In some embodiments, this disclosure provides apparatus and methods for a nucleic acid workflow of growing / culturing cell samples on a flow-through cell and processing cultured cell samples on a flow-through cell.

[0369] In some embodiments, cell samples may be deposited on a flow cell, wherein the flow cell may be coated with a reagent that promotes cell adhesion to the flow cell. The flow cell with the cell sample adhered thereto may be placed on a sequencing device equipped with a flow cell holder / scaffold fluidly connected to an automated fluid delivery system and configured on a fluorescence microscope. In some embodiments, the sequencing device may be configured with at least one fluid delivery device, at least one fluid device (e.g., a microfluidic device), at least one imaging device, and / or at least one sensor to detect signals from the sequencing reaction.

[0370] In some embodiments, an automated fluid dispensing system can be used to deliver simple and / or complex cell culture media to cell samples on a flow-through cell. In some embodiments, cell samples can be cultured / amplified on the flow-through cell for 2 to 10 generations (e.g., cell passage) or longer. In some embodiments, cell samples can be amplified to confluent or non-confluent states.

[0371] In some embodiments, an automated fluid dispensing system can be used to deliver fixation reagents to an amplified cell sample on a flow cell, and the cell sample can be incubated under conditions suitable for cell fixation.

[0372] In some embodiments, an automated fluid dispensing system can be used to deliver permeation reagents to a fixed cell sample on a flow cell, and the cell sample can be incubated under conditions suitable for cell permeation.

[0373] In some embodiments, an automated fluid dispensing system may be used to deliver reagents for reverse transcription of RNA within a fixed and permeabilized cell sample under conditions suitable for generating multiple cDNAs within the cell sample. In some embodiments, the reverse transcription reaction may be omitted.

[0374] In some embodiments, an automated fluid dispensing system may be used to deliver reagents for hybridizing amplification-free probe complexes with cell samples.

[0375] In some embodiments, an automated fluid dispensing system can be used to deliver reagents for rolling circle amplification under conditions suitable for generating multiple multiply molecules within a cell sample.

[0376] In some embodiments, an automated fluid dispensing system can be used to deliver sequencing reagents for one or more sequencing cycles of the template molecule or modified oligonucleotide described herein, under conditions suitable for generating multiple sequencing reads within a cellular sample. In some embodiments, the individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm. 2 And it is used to scan large areas (> 10 mm) 2 The cycle time can be less than 5 minutes.

[0377] In some embodiments, an automated fluid dispensing system can be used to deliver reagents for removing the plurality of sequencing read products from the template molecule and retaining the template molecule on or inside the cell sample.

[0378] In some embodiments, an automated fluid dispensing system can be used to deliver sequencing reagents for one or more sequencing cycles of template molecules under conditions suitable for generating multiple sequencing reads within a cellular sample (e.g., independent batch sequencing or repeated sequencing). In some embodiments, the individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm. 2 And it is used to scan large areas (> 10 mm) 2 The cycle time can be less than 5 minutes.

[0379] By incorporating via reference

[0380] Throughout this application, various publications, patents, and / or patent applications have been cited. The disclosures of these publications, patents, and / or patent applications are hereby incorporated, in their entirety, by reference in order to provide a more comprehensive description of the current state of the art to which this disclosure pertains.

[0381] Equivalent solution

[0382] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are now described. Other features, objects, and advantages of this disclosure will become apparent from this specification and claims. In the specification and appended claims, the singular form includes plural indicators unless the context clearly requires otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in this specification are incorporated herein by reference.

Claims

1. A probe-free complex comprising: (i) at least one modified oligonucleotide; and (ii) It can bind to at least one analyte-binding moiety of a target analyte, wherein the at least one modified oligonucleotide is attached to the at least one analyte-binding moiety. The at least one modified oligonucleotide contains two or more tandem copies of the sequencing primer binding site. The at least one modified oligonucleotide comprises a canonical nucleobase located at the 5' end immediately adjacent to the 5' end of each sequencing primer binding site, and The at least one modified oligonucleotide contains a base-free site located at the 5' of the canonical nucleobase.

2. The amplification-free probe complex according to claim 1, wherein the canonical nucleobase is located between one and ten nucleotides at the 5' end of each sequencing primer binding site.

3. The amplification-free probe complex according to claim 1, wherein the canonical nucleobase is 5' end and adjacent to the 5' end of each sequencing primer binding site.

4. A probe-free complex comprising: (i) at least one modified oligonucleotide; and (ii) It can bind to at least one analyte-binding moiety of a target analyte, wherein the at least one modified oligonucleotide is attached to the at least one analyte-binding moiety. The at least one modified oligonucleotide contains at least one sequencing primer binding site and a barcode sequence.

5. The amplification-free probe complex of claim 4, wherein the at least one modified oligonucleotide comprises two or more tandem copies of the primer binding site and the barcode sequence.

6. The amplification-free probe complex according to any one of claims 1 to 5, wherein the target analyte comprises lipids, peptides, nucleic acids, or polysaccharides.

7. The amplification-free probe complex according to any one of claims 1 to 5, wherein the analyte binding portion comprises a lipid portion, a wheat germ agglutinin, an antibody portion, or a biotin portion.

8. The amplification-free probe complex according to any one of claims 1 to 7, wherein the target analyte is inside the cell sample and / or on the surface of the cell sample.

9. The amplification-free probe complex of claim 8, wherein the cell sample comprises a whole single cell, multiple whole cells, a complete tissue, a slice of cell, or a slice of tissue.

10. The amplification-free probe complex of claim 8, wherein the cell sample comprises a fresh cell sample, a fresh frozen cell sample, or a formalin-fixed paraffin-embedded (FFPE) cell sample.

11. The amplification-free probe complex of claim 8, wherein the cell sample comprises a fixed and permeabilized cell sample.

12. The amplification-free probe complex according to any one of claims 1 to 11, wherein the analyte binding portion comprises a secondary antibody or a primary antibody.

13. A method for detecting a target analyte, comprising: a) Provide a plurality of probe-free complexes according to any one of claims 1 to 3 or 6 to 12; b) Provide a sample containing multiple analytes, said multiple analytes including at least one target analyte, wherein said sample is deposited on a support; c) Contacting the sample with the plurality of amplification-free probe complexes, wherein the contact is performed under conditions suitable for binding the analyte-binding portion to the target analyte; d) Contacting the multiple modified oligonucleotides of the multiple amplification-free probe complexes of step (c) with multiple sequencing primers under conditions suitable for each sequencing primer to bind to the sequencing primer binding site on the modified oligonucleotide to form multiple nucleic acid duplexes, wherein each nucleic acid duplex contains a sequencing primer binding site on the modified oligonucleotide that hybridizes with the sequencing primer. e) Contacting the plurality of nucleic acid duplexes with a plurality of sequencing polymerases and a plurality of detectably labeled multivalent molecules, wherein each nucleic acid duplex binds to the sequencing polymerase and the nucleotide moiety of the multivalent molecule, thereby forming a plurality of detectably labeled complexes, wherein the nucleotide moiety of the detectably labeled multivalent molecule binds to the 3' end of the sequencing primer and is located opposite to the canonical nucleobase in the modified oligonucleotide, wherein the contact in step (e) is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the nucleotide moiety into the 3' end of the sequencing primer, and wherein the nucleotide moiety of the multivalent molecule binds to and is complementary to the nucleotide in the modified oligonucleotide, thereby producing a plurality of detectably labeled complexes; and f) Imaging of samples bound to the plurality of detectably labeled complexes, thereby detecting target analytes on or inside cell samples.

14. A method for detecting a target analyte, comprising: a) Provide a plurality of probe-free complexes according to any one of claims 4 to 12; b) Provide a sample containing multiple analytes, said multiple analytes including at least one target analyte, wherein said sample is deposited on a support; c) Contacting the sample with the plurality of amplification-free probe complexes, wherein the contact is performed under conditions suitable for binding the analyte-binding portion to the target analyte; d) Contacting the plurality of modified oligonucleotides of the plurality of amplification-free probe complexes of step (c) with a plurality of sequencing primers under conditions suitable for each sequencing primer to bind to the sequencing primer binding site on the modified oligonucleotide to form a plurality of nucleic acid duplexes, wherein each nucleic acid duplex contains a sequencing primer binding site on the modified oligonucleotide that hybridizes with the sequencing primer. e) Contacting the plurality of nucleic acid duplexes with a plurality of sequencing polymerases and a plurality of detectably labeled multivalent molecules, wherein each nucleic acid duplex binds to a nucleotide moiety of the sequencing polymerase and the multivalent molecule, thereby forming a plurality of detectably labeled complexes, wherein the nucleotide moiety of the detectably labeled multivalent molecule binds to the 3' end of the sequencing primer and is located opposite to the barcode sequence in the modified oligonucleotide, and wherein the nucleotide moiety of the multivalent molecule binds to and is complementary to the nucleotide in the modified oligonucleotide, thereby producing a plurality of detectably labeled complexes; and f) Image the sample to detect the target analyte.

15. The method of claim 14, wherein the contact in step (e) is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the nucleotide moiety into the 3' end of the sequencing primer.

16. The method according to any one of claims 13 to 15, wherein each multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide portion.

17. The method according to any one of claims 13 to 15, wherein the complementary nucleotide portion of the multivalent molecule is not incorporated into the 3' end of the sequencing primer.

18. The method according to any one of claims 13 to 17, wherein the nucleotide portion of the multivalent molecule is complementary to the nucleotide in the modified oligonucleotide located at and adjacent to the sequencing primer binding site at 5'.

19. The method according to any one of claims 13 to 18, wherein the sample is a cell sample.

20. The method of claim 19, wherein the target analyte is on and / or inside the cell sample.

21. The method of claim 19 or 20, wherein the cell sample comprises a whole single cell, a plurality of whole cells, a complete tissue, a slice of cell, or a slice of tissue.

22. The method according to claim 19 or 20, wherein the cell sample comprises a fresh cell sample, a fresh frozen cell sample, or a formalin-fixed paraffin-embedded (FFPE) cell sample.

23. The method of claim 19 or 20, wherein the cell sample comprises a fixed and permeabilized cell sample.

Images