Method for signal encoding of analytes in a sample

Abstract

The present invention relates to a method for sequentially signal-coding analytes in a sample, the use of a set of decoding oligonucleotides for sequentially signal-coding analytes in a sample, and a kit for sequentially signal-coding analytes in a sample.

Description

[Technical Field]

[0001] The present invention relates to a method for sequentially signal-coding analytes in a sample, the use of a set of decoding oligonucleotides for sequentially signal-coding analytes in a sample, and a kit for sequentially signal-coding analytes in a sample.

[0002] The present invention relates to the field of molecular biology, more particularly to the detection of analytes in a sample, preferably the detection of biomolecules such as nucleic acid molecules and / or proteins in a biological sample. [Background technology]

[0003] The analysis and detection of small amounts of analytes in biological and non-biological samples has become a routine procedure in clinical and analytical settings. Numerous analytical methods have been established for this purpose. Some of them use coding techniques that assign a specific readable code to a specific first analyte that is different from the code assigned to a specific second analyte.

[0004] One of the prior techniques in this field is so-called "single-molecule fluorescence in situ hybridization" (smFISH), which was essentially developed to detect mRNA molecules in a sample. In Lubeck et al. (2014), Single-cell in situ RNA profiling by sequential hybridization, Nat. Methods 11(4), pp. 360-361, the mRNA of interest is detected via a specific, directly labeled probe set. After one round of hybridization and detection, a set of mRNA-specific probes is eluted from the mRNA, and the same set of probes with a different (or the same) fluorescent label is used in the next round of hybridization and imaging to generate a gene-specific color-coding scheme over several rounds. This technique requires several different tagged probe sets for each transcript, and these probe sets must be denatured for each detection round.

[0005] A further development of this technology does not use directly labeled probe sets. Instead, the probe set oligonucleotides provide nucleic acid sequences that act as initiators for hybridization chain reactions (HCR), a technique that allows for signal amplification; see Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), pp. 342-357.

[0006] Another technique, called "multiplexed error-robust fluorescence in situ hybridization" (merFISH), is described by Chen et al. (2015) in RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells, Science 348(6233):aaa6090. There, mRNAs of interest are detected via specific probe sets that provide additional sequence elements for subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides four different sequence elements out of a total of 16 sequence elements. Following hybridization of the specific probe sets with the mRNAs of interest, so-called readout hybridizations are performed. In each readout hybridization, one of 16 fluorescently labeled oligonucleotides complementary to one of the sequence elements hybridizes. All readout oligonucleotides use the same fluorescent color. After imaging, the fluorescent signal is destroyed by irradiation, and the next round of readout hybridization is performed without a denaturation step. As a result, a binary code is generated for each mRNA species. Using only a single hybridization round to bind a specific probe set to the mRNA of interest, followed by 16 rounds of hybridization of a readout oligonucleotide labeled with a single fluorescent color, a unique signal signature of 16 rounds of four signals is created.

[0007] Further developments of this technique will improve throughput by using two different fluorescent colors, eliminating signal via disulfide cleavage between the readout oligonucleotide and the fluorescent label, and alternative hybridization buffers (see Moffitt et al. (2016), High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization, Proc. Natl. Acad. Sci. US A 113(39), pp. 11046-11051).

[0008] A technique called "intron-seqFISH" is described by Shah et al. (2018), "Dynamics and spatial genomics of the nascent transcriptome by intron-seqFISH," Cell 117(2), pp. 363-376. There, mRNAs of interest are detected via specific probe sets that provide additional sequence elements for subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides one of 12 possible sequence elements (representing the 12 "pseudocolors" used) per color-coding round. Each color-coding round consists of four sequential hybridizations. In each of these sequential hybridizations, three readout probes, each labeled with a different fluorophore, hybridize to corresponding elements of the mRNA-specific probe set. After imaging, the readout probes are removed with 55% formamide buffer, followed by the next hybridization. After five rounds of color-coding, each with four sequential hybridizations, the color-coding is complete.

[0009] European Patent No. 0 611 828 discloses the use of a bridging element to recruit a signal-generating element to a probe that specifically binds to an analyte. More specifically, it describes the detection of nucleic acids via a specific probe that recruits a bridging nucleic acid molecule. This bridging nucleic acid ultimately recruits a signal-generating nucleic acid. This document also describes the use of a bridging element with two or more binding sites for a signal-generating element, such as branched DNA, for signal amplification.

[0010] Player et al. (2001), "Single-copy gene detection using branched DNA (bDNA) in situ hybridization," J. Histochem. Cytochem. 49(5), pp. 603-611, describes a method in which a nucleic acid of interest is detected via a specific probe set that provides additional sequence elements. In a second step, preamplifier oligonucleotides hybridize to these sequence elements. These preamplifier oligonucleotides contain multiple binding sites for amplifier oligonucleotides that hybridize in subsequent steps. These amplifier oligonucleotides provide multiple sequence elements for labeled oligonucleotides. In this way, a branched oligonucleotide tree is constructed, resulting in signal amplification.

[0011] Further development of this method is described in Wang et al. (2012), "RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues," J. Mol. Diagn. 14(1), pp. 22-29, which uses a different mRNA-specific probe design. Here, two of the mRNA-specific oligonucleotides must hybridize closely to provide a sequence capable of recruiting the preamplifier oligonucleotide. This method increases the specificity of the method by reducing the number of false-positive signals.

[0012] Choi et al. (2010), Programmable in situ amplification for multiplexed imaging of mRNA expression, Nat. Biotechnol. 28(11), pp. 1208-1212, discloses a method known as "HCR hybridization chain reaction." The target mRNA is detected via a specific probe set that provides an additional sequence element. The additional sequence element is an initiator sequence for initiating the hybridization chain reaction. Essentially, the hybridization chain reaction is based on metastable oligonucleotide hairpins that self-assemble into polymers after the first hairpin is opened via the initiator sequence.

[0013] A further development of this technique, similar to the RNAscope technique, is the use of so-called split initiator probes, which must hybridize closely to form the initiator sequence of the HCR, thereby reducing the number of false positive signals; see Choi et al. (2018), Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145(12).

[0014] Mateo et al. (2019), Visualizing DNA folding and RNA in embryos at single-cell resolution, Nature Vol. 568, p. 49ff., discloses a method called Optical Reconstruction of Chromatin Architecture (ORCA), which aims to visualize chromosome lines. Summary of the Invention [Problem to be solved by the invention]

[0015] However, the methods known in the art have a number of drawbacks: in particular, they are inflexible, expensive, complicated, time-consuming and very often give inaccurate results. In particular, the coding capacity of existing methods is low and does not meet the requirements of modern molecular biology and medicine.

[0016] Against this background, the underlying object of the present invention is to provide a method that can reduce or avoid the drawbacks of the prior art methods.

[0017] The present invention meets these and other needs.

[0018] The present invention provides a method for sequentially signal-coding analytes in a sample, the method comprising the steps of: (1) providing a set of analyte-specific probes, each analyte-specific probe comprising: a binding element (S) that specifically interacts with one of the encoded analytes; an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) unique to the set of analyte-specific probes; (2) incubating a set of analyte-specific probes with the sample, thereby allowing specific binding of the analyte-specific probes to the encoded analytes; (3) removing unbound probe from the sample; (4) providing a set of decoding oligonucleotides, each decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a portion of the unique identifier sequence; - a translator element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; (5) incubating the set of decoding oligonucleotides with the sample, thereby allowing specific hybridization of the decoding oligonucleotides to the unique identifier sequences; (6) removing unbound decoding oligonucleotides from the sample; (7) providing a set of signal oligonucleotides, each signal oligonucleotide comprising: a second connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a portion of the nucleotide sequence of the translator element (c); a signal element; (8) Incubating a set of signal oligonucleotides with the sample, thereby allowing specific hybridization of the signal oligonucleotides to the translator element (c).

[0019] The inventors have recognized that this novel method provides the essential steps required to set up a process that allows for the specific quantitative and / or spatial detection or counting of different analytes or different single analyte molecules in a sample in parallel through specific hybridization. The technique allows for the differentiation of a larger number of analytes than the available different signals. In contrast to other state-of-the-art methods, the oligonucleotides that provide the detectable signal do not interact directly with the sample-specific nucleic acid sequence, but are mediated by a so-called "decoding oligonucleotide." This mechanism decouples the dependency between the analyte-specific oligonucleotide and the signal oligonucleotide, thus dramatically increasing the coding capacity.

[0020] Another subject of the invention is the use of a set of decoding oligonucleotides for sequentially signal-coding analytes in a sample, each decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a portion of a nucleotide sequence that is unique to the set of analyte-specific probes (unique identifier sequence); a translator element (c), which comprises a nucleotide sequence allowing specific hybridization of a signal oligonucleotide.

[0021] A further subject of the present invention is a kit for sequential signal coding of analytes in a sample, comprising: a set of analyte-specific probes, each analyte-specific probe comprising: a binding element (S) that specifically interacts with one of the encoded analytes; an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) unique to the set of analyte-specific probes; and a set of decoding oligonucleotides, each decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a portion of the unique identifier sequence; a translator element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide, and preferably a set of signal oligonucleotides, each signal oligonucleotide comprising: a second connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a portion of the nucleotide sequence of the translator element (c); - a signal element; and a set including:

[0022] The use of decoding oligonucleotides dramatically reduces the number of different signal oligonucleotides required, while allowing for much greater flexibility, thereby increasing the encoding capacity that can be achieved. The use of decoding oligonucleotides results in a sequential signal-encoding technique that is more flexible, cheaper, simpler, faster, and / or more accurate than other methods. In particular, the present invention provides a significant increase in encoding capacity compared to prior art methods.

[0023] The use of decoding oligonucleotides breaks the dependency between target-specific probes and signal oligonucleotides. When two different molecular tags are used, rather than separating target-specific probes and signal generation as in state-of-the-art methods, two distinct signals can be generated for only a specific target. Each of these molecular tags can be used only once. Multiple readouts of the same molecular tag do not increase information about the target. To create a coding scheme, either the target-specific probe set must be changed after each round (SeqFISH) or multiple molecular tags must be present in the same probe set (merFISH, intron-SeqFISH, etc.). These limitations in the art are highly relevant and reduce flexibility, coding capacity, precision, and reproducibility, while increasing the cost of experiments.

[0024] According to the present invention, an "analyte" is an entity that is specifically detected as present or absent in a sample and, if present, coded for. An analyte can be any type of entity, including a protein or nucleic acid molecule (RNA or DNA) of interest. An analyte provides at least one site for specific binding with an analyte-specific probe. As used herein, the term "analyte" may be interchanged with "target." An "analyte" according to the present invention includes a complex of interest, e.g., at least two individual nucleic acid, protein, or peptide molecules. In one embodiment of the present invention, an "analyte" excludes a chromosome. In another embodiment of the present invention, an "analyte" excludes DNA.

[0025] A "sample" as referred to herein is a composition in liquid or solid form suspected of containing the analyte to be encoded.

[0026] As used herein, "oligonucleotide" refers to a short nucleic acid molecule such as DNA, PNA, LNA, or RNA. The length of an oligonucleotide ranges from 4 to 200 nucleotides (nt), preferably 6 to 80 nucleotides, more preferably 8 to 60 nucleotides, more preferably 10 to 50 nucleotides, and more preferably 12 to 35 nucleotides, depending on the number of consecutive sequence elements. A nucleic acid molecule can be fully or partially single-stranded. An oligonucleotide can be linear or contain a hairpin or loop structure. An oligonucleotide can contain modifications such as biotin, a labeling moiety, a blocking moiety, or other modifications.

[0027] An "analyte-specific probe" consists of at least two elements: a so-called binding element (S) that specifically interacts with one of the analytes, and a so-called identifier element (T) that contains a "unique identifier sequence." The binding element (S) can be a nucleic acid, such as a hybridization sequence or an aptamer, or a peptide structure, such as an antibody. The "unique identifier sequence" contained in the analyte-specific probe is unique in sequence compared to other unique identifiers. "Unique" in this context means that it specifically identifies only one analyte, such as cyclin A, cyclin D, or cyclin E, or alternatively, it specifically identifies only a group of analytes, regardless of whether the group of analytes includes a gene family. Thus, an analyte or group of analytes coded by this unique identifier can be distinguished from all other analytes or groups of analytes coded based on the unique identifier sequence of the identifier element (T). Or, in other words, there is only one "unique identifier sequence" for a particular analyte or group of analytes, but not more than one, i.e., not even two. Due to the uniqueness of the unique identifier sequence, the identifier element (T) hybridizes to exactly one type of decoding oligonucleotide. The length of the unique identifier sequence ranges from 8 to 60 nt, preferably 12 to 40 nt, and more preferably 14 to 20 nt, depending on the number of analytes to be encoded in parallel and the required stability of the interaction. The unique identifier may be a sequence element of an analyte-specific probe, attached directly or via a linker, covalent bond, or high-affinity binding mode, such as an antibody-antigen interaction, streptavidin-biotin interaction, etc. The term "analyte-specific probe" is understood to include multiple probes whose binding elements (S) may be different, such that each probe binds to the same analyte but to different portions of it, e.g., different (e.g., adjacent) or overlapping portions of the nucleotide sequence contained in the encoded nucleic acid molecule. However, each of the multiple probes contains the same identifier element (T).

[0028] A "decoding oligonucleotide" consists of at least two sequence elements. One sequence element capable of specifically binding to a unique identifier sequence is called the "first connector element" (t), and the second sequence element that specifically binds to a signal oligonucleotide is called the "translator element" (c). The length of the sequence elements ranges from 8 to 60 nt, preferably 12 to 40 nt, and more preferably 14 to 20 nt, depending on the number of analytes to be coded in parallel, the required stability of the interaction, and the number of different signal oligonucleotides used. The lengths of the two sequence elements may or may not be the same.

[0029] As used herein, a "signal oligonucleotide" comprises two elements: a so-called "second connector element" (C) having a nucleotide sequence capable of specifically hybridizing to at least a portion of the nucleotide sequence of the translator element (c) of a decoding oligonucleotide, and a "signal element" that provides a detectable signal. This element can actively generate a detectable signal or provide such a signal via manipulation, e.g., fluorescence excitation. Typical signal elements are, for example, enzymes, fluorophores, radioactive elements, or dyes that catalyze a detectable reaction.

[0030] A "set" refers to a plurality of moieties or objects, e.g., analyte-specific probes or decoding oligonucleotides, whether the members are identical or different from one another. In one embodiment of the present invention, a single set refers to a plurality of oligonucleotides.

[0031] An "analyte-specific probe set" refers to multiple moieties or entities, e.g., analyte-specific probes that are different from each other and bind to separate regions of an analyte. A single analyte-specific probe set is further characterized by the same unique identifier.

[0032] A "decoding oligonucleotide set" refers to multiple decoding oligonucleotides specific to a particular unique identifier required to achieve encoding, regardless of the length of the codeword. Each and every decoding oligonucleotide included in a "decoding oligonucleotide set" binds to the same unique identifier element (T) of an analyte-specific probe.

[0033] "Essentially complementary" when referring to two nucleotide sequences means that both sequences can specifically hybridize with each other under stringent conditions, thereby forming a hybrid nucleic acid molecule having sense and antisense strands linked to each other via hydrogen bonds (Watson-Crick base pairs). "Essentially complementary" includes not only perfect base pairing along the entire strand, i.e., perfectly complementary sequences, but also imperfectly complementary sequences, which still have the ability to hybridize with each other under stringent conditions. It is well-recognized among experts that "essentially complementary" sequences have at least 88% sequence identity with a completely or perfectly complementary sequence.

[0034] Next, "percent sequence identity" or "percent identity" means that the sequence being compared ("comparison sequence") is compared to the described or claimed sequence ("reference sequence") after alignment of the sequence. The percent identity is then determined according to the following formula: percent identity = 100[1 - (C / R)] where C is the number of differences between the reference and comparison sequences over the length of the alignment between the reference and comparison sequences; (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference; and (iiii) the alignment must start at position 1 of the aligned sequence; R is the number of bases or amino acids in the reference sequence over the length of the alignment with the comparison sequence, with any gaps made in the reference sequence also counted as bases or amino acids.

[0035] If there is an alignment between a comparison sequence and a reference sequence in which the percent identity calculated above is about or greater than the specified minimum percent identity, then the comparison sequence has a specified minimum percent identity to the reference sequence, even though there may be alignments in which the percent identity calculated herein above is less than the specified percent identity.

[0036] In an "incubation" step as understood herein, each moiety or entity, such as a probe or oligonucleotide, is brought into contact with each other under conditions known to those skilled in the art that allow a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions, etc. Thus, such a step may preferably be carried out in a liquid environment, such as a buffer system known in the art.

[0037] The "removal" step according to the present invention may involve washing the moiety or object to be removed, such as a probe or oligonucleotide, under specific conditions, e.g., pH, temperature, salt conditions, etc., as known in the art.

[0038] It will be appreciated that in one embodiment of the method according to the present invention, multiple analytes can be coded in parallel. This requires the use of different sets of analyte-specific probes in step (1). The analyte-specific probes of a particular set are different from the analyte-specific probes of another set. This means that the analyte-specific probes of set 1 bind to analyte 1, the analyte-specific probes of set 2 bind to analyte 2, the analyte-specific probes of set 3 bind to analyte 3, etc. In this embodiment, the use of different sets of decoding oligonucleotides is also required in step (4). The decoding oligonucleotides of a particular set are different from the decoding oligonucleotides of another set. This means that the decoding oligonucleotides of set 1 bind to the analyte-specific probes of set 1 of the analyte-specific probes, the decoding oligonucleotides of set 2 bind to the analyte-specific probes of set 2 of the analyte-specific probes, the decoding oligonucleotides of set 3 bind to the analyte-specific probes of set 3 of the analyte-specific probes, etc. In this embodiment, where multiple analytes are encoded in parallel, different sets of analyte-specific probes may be provided in step (1) as premixes of different sets of analyte-specific probes, and / or different sets of decoding oligonucleotides may be provided in step (4) as premixes of different sets of decoding oligonucleotides. Each mixture may be contained in a single vial. Alternatively, different sets of analyte-specific probes and / or different sets of decoding oligonucleotides may be provided separately in steps (1) and / or (4).

[0039] A "kit" is a combination of individual elements useful for carrying out the uses and / or methods of the present invention, the elements being optimized for use together in the method. The kit may also include additional reagents, chemicals, buffers, reaction vials, etc. that may be useful for carrying out the methods according to the present invention. Such kits integrate all essential elements needed to carry out the methods according to the present invention, thus minimizing the risk of error. Thus, such kits also allow semi-skilled laboratory staff to carry out the methods according to the present invention.

[0040] The features, properties, advantages and embodiments identified herein apply to the methods, uses and kits according to the invention even if not specifically indicated therein.

[0041] In one embodiment of the present invention, the sample is a biological sample, preferably comprises biological tissue, more preferably comprises biological cells.Biological samples can be derived from organs, organoids, cell cultures, stem cells, cell suspensions, primary cells, viral, bacterial or fungal infected samples, eukaryotic or prokaryotic samples, smears, disease samples, tissue sections.

[0042] The method is particularly suitable for encoding, identifying, detecting, counting or quantifying analytes or single analyte molecules in a biological sample, i.e., a sample containing nucleic acids or proteins as the analytes of interest. It will be understood that the biological sample may be in the form as it is in its natural environment (i.e., liquid, semi-liquid, solid, etc.), or may be treated as, for example, a dry film on the surface of a device which may be re-liquefied before the method is performed.

[0043] In another embodiment of the present invention, prior to step (2), the biological tissue and / or biological cells are fixed.

[0044] This method has the advantage that the analytes to be encoded, e.g., nucleic acids or proteins, are immobilized and cannot escape, thereby preparing them for better detection or encoding by the method according to the invention. Fixation of the sample can be carried out, for example, with formalin, ethanol, methanol, or other components known to those skilled in the art.

[0045] In still further embodiments, within the set of analyte-specific probes, each analyte-specific probe comprises a binding element (S1, S2, S3, S4, S5) that specifically interacts with a different substructure of one of the encoded analytes.

[0046] By this means, the signal strength obtained at the end of the method or cycle, respectively, is increased, making the method even more robust and reliable. It is understood that the individual probes of a set bind to the same analyte, but their binding positions or binding sites in or on the analyte are different. Thus, the binding elements S1, S2, S3, S4, S5, etc. of the first, second, third, fourth, fifth, etc. analyte-specific probes bind to different positions, which may or may not overlap.

[0047] In another embodiment of the method according to the invention, the method further comprises the following additional steps: (9) removing unbound signal oligonucleotides from the sample; (10) detecting a signal.

[0048] By this means, the method is further developed to the extent that the encoded analyte can be detected by any means adapted to visualize the signal element. Examples of detectable physical characteristics include, for example, light, chemical reaction, molecular weight, radioactivity, etc.

[0049] In a further embodiment of the method according to the invention, the method further comprises the following additional steps: (11) A step is performed to selectively remove the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probe to the encoded analyte.

[0050] This procedure establishes the need for another round of binding of additional decoding oligonucleotides to the same analyte-specific probe, thus ultimately resulting in a code or encoding scheme containing two or more signals. This step is achieved by applying conditions and factors known to those skilled in the art, such as pH, temperature, salt conditions, oligonucleotide concentration, polymer, etc.

[0051] In another embodiment of the present invention, a method comprises the following steps: (12) Repeating steps (4) through (11) at least once [(42) through (112)] to generate a coding scheme consisting of at least two signals.

[0052] By this means, codes of two or more signals are set, i.e., two [(42)-(112)], three [(43)-(113)], four [(44)-(114)], five [(45)-(115)], etc. [(4 n )-(11 n ) rounds, signals are coded as 2, 3, 4, 5, etc., where "n" is an integer representing the number of rounds. The coding capacity of the method according to the invention increases depending on the nature of the analyte and the needs of the operator.

[0053] In one embodiment of the present invention, the coding scheme is predetermined and assigned to the analyte being coded.

[0054] This approach allows for precise experimental setup by providing the proper sequential order of decoding and signal oligonucleotides used, and therefore allows for the correct assignment of specific analytes to their respective coding schemes.

[0055] The decoding oligonucleotide used in the repeat steps (42)-(112) may contain a translator element (c2) that is identical to the translator element (c1) of the decoding oligonucleotide used in the previous steps (4)-(11). In another embodiment of the invention, a decoding oligonucleotide is used in the repeat steps (42)-(112) that contains a translator element (c2) that is different from the translator element (c1) of the decoding oligonucleotide used in the previous steps (4)-(11).

[0056] The decryption elements are generated in rounds, i.e., second round (42)-(112) with translator element c2, third round (43)-(113) with translator element c3, fourth round (44)-(114) with translator element c4, fifth round (45)-(115) with translator element c5, and "n" round (46) with translator element cn. n )~(11 n ), where "n" is an integer representing the number of rounds.

[0057] The signal oligonucleotide used in the repeat steps (42)-(112) may contain a signal element that is identical to the signal element of the decoding oligonucleotide used in the previous steps (4)-(11). In a further embodiment of the invention, a signal oligonucleotide is used in the repeat steps (42)-(112) that contains a signal element that is different from the signal element of the decoding oligonucleotide used in the previous steps (4)-(11).

[0058] By this means, the same or a different signal can be provided in each round, resulting in a coding scheme characterized by a signal sequence consisting of multiple different signals. This means allows for the creation of a unique code or codeword that is different from all other codewords in the coding scheme.

[0059] In another embodiment of the invention, the binding element (S) of the analyte-specific probe comprises a nucleic acid comprising a nucleotide sequence that allows specific binding to, preferably specific hybridization to, the encoded analyte.

[0060] This procedure creates the conditions for encoding nucleic acid analytes, such as specific DNA molecules, e.g., genomic DNA, nuclear DNA, mitochondrial DNA, viral DNA, bacterial DNA, extracellular or intracellular DNA, or specific mRNA molecules, e.g., hnRNA, miRNA, viral RNA, bacterial RNA, extracellular or intracellular RNA.

[0061] In an alternative embodiment of the invention, the binding element (S) of the analyte-specific probe comprises an amino acid sequence that allows specific binding to the encoded analyte, preferably the binding element is an antibody.

[0062] This procedure creates the conditions for encoding a nucleic acid analyte, such as an mRNA, for example an mRNA that encodes a particular protein.

[0063] In another embodiment, the analyte to be encoded or detected is a nucleic acid, preferably DNA or RNA, more preferably mRNA, and / or alternatively the analyte to be decoded is a peptide or protein.

[0064] By this means, the present invention is adapted to the detection of those types of analytes that are of most importance in clinical routine or in the focus of biological problems.

[0065] It is to be understood that the features mentioned above and below can be used not only in the combination indicated in each case, but also in other combinations or in separate ways without departing from the object of the invention.

[0066] The present invention will now be further described by embodiments that provide further features, characteristics and advantages of the present invention. The embodiments are purely exemplary in nature and are not intended to limit the scope or spirit of the present invention. Features mentioned in specific embodiments are general features of the present invention that are not only applicable to the specific embodiment, but also applicable in a separated manner in the context of any embodiment of the present invention. [Brief explanation of the drawings]

[0067] The present invention will now be described and explained in more detail by reference to the following non-limiting examples and drawings. [Figure 1] 1 illustrates an embodiment in which the analyte is a nucleic acid and the probe set comprises oligonucleotides that specifically bind to the analyte. The probes contain unique identifier sequences that allow hybridization of decoding oligonucleotides. [Figure 2] 2 illustrates an embodiment in which the analyte is a protein and the probe set comprises a protein (here, an antibody) that specifically binds to the analyte. The probe comprises a unique identifier sequence that allows hybridization of a decoding oligonucleotide. [Figure 3] FIG. 3 is a flow chart of the method according to the present invention. [Figure 4] Figure 4 shows alternative options for applying decoding and signal oligonucleotides. [Figure 5] 5 shows an example of signal encoding of three different nucleic acid sequences with two different signal types and three rounds of detection. In this example, the encoding scheme includes error detection. [Figure 6] Figure 6 shows the number of generated codewords (logarithmic scale) versus the number of detection cycles. [Figure 7] Figure 7 shows the calculated total efficiency of the five-round encoding scheme based on the efficiency of the single steps. [Figure 8] Figure 8. Comparison of relative transcript abundance between different experiments. [Figure 9] Figure 9. Correlation of relative transcript abundance between different experiments. [Figure 10] Figure 10 shows a comparison of the intercellular distribution of the signal. [Figure 11] Figure 11 shows a comparison of the intracellular distribution of signals. [Figure 12] FIG. 12. Distribution patterns of different cell cycle-dependent transcripts. DETAILED DESCRIPTION OF THE INVENTION

[0068] example 1. Introduction The methods disclosed herein are used to specifically detect many different analytes in parallel. The technique allows for the differentiation of a larger number of analytes than the number of distinct signals available. The process preferably involves at least two successive rounds of specific binding, signal detection, and selective denaturation (if a subsequent round is required), ultimately generating a signal code. To separate the dependency between analyte-specific binding and the oligonucleotides that provide the detectable signal, so-called "decoding" oligonucleotides are introduced. The decoding oligonucleotides transcribe the information of the analyte-specific probe set into signal oligonucleotides.

[0069] In a first application variant, the analyte or target is a nucleic acid, e.g., DNA or RNA, and the probe set comprises oligonucleotides that are partially or completely complementary to the entire sequence or a subsequence of the nucleic acid sequence to be detected (Figure 1). A nucleic acid sequence-specific oligonucleotide probe set comprising an analyte-specific probe (1) comprising a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, and an identifier element (T) that comprises a nucleotide sequence unique to the set of analyte-specific probes (unique identifier sequence).

[0070] In a second application variant, the analyte or target is a protein and the probe set comprises one or more proteins, for example antibodies (Figure 2): a protein-specific probe set comprising an analyte-specific probe (1) comprising a binding element (T) and an identifier element (T), such as a (hyper)variable region of an antibody, which interacts specifically with the target protein to be detected;

[0071] In a third application variant, at least one analyte is a nucleic acid and at least a second analyte is a protein, and at least a first probe set binds to a nucleic acid sequence and at least a second probe set specifically binds to a protein analyte. Other combinations are possible.

[0072] 2. General Method According to the Invention To better understand the workflow, the following workflow is limited to the first application variant. A skilled person can easily adapt the exemplary workflow to other applications. The method steps are shown in the flowchart of FIG. 3.

[0073] Step 1: Application of analyte or target-specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides with sequences complementary to the target nucleic acid. In this example, a probe set of five different probes is shown, each containing a sequence element complementary to a distinct subsequence of the target nucleic acid sequence (S1-S5). In this example, the regions do not overlap. Each of the oligonucleotides targeting the same nucleic acid sequence each contains an identifier element or unique identifier sequence (T).

[0074] Step 2: Hybridization of the probe set. The probe set hybridizes to the target nucleic acid sequence under conditions that allow specific hybridization. After incubation, the probes hybridize to their corresponding target sequences, providing the identifier element (T) for the next step.

[0075] Step 3: Removal of unbound probe After hybridization, unbound oligonucleotides are removed, for example, by a washing step.

[0076] Step 4: Application of decoding oligonucleotides. A decoding oligonucleotide consisting of at least two sequence elements (t) and (c) is applied. Sequence element (t) is complementary to the unique identifier sequence (T), while sequence element (c) provides a region for subsequent hybridization of a signal oligonucleotide (translator element).

[0077] Step 5: Hybridization of the Decoding Oligonucleotides. The decoding oligonucleotides hybridize to the unique identifier sequence of the probe (T) via their complementary first sequence element (t). After incubation, the decoding oligonucleotides provide the translator sequence element (c) for the subsequent hybridization step.

[0078] Step 6: Removal of excess decoding oligonucleotides After hybridization, unbound decoding oligonucleotides are removed, for example, by a washing step.

[0079] Step 7: Applying a signal oligonucleotide. A signal oligonucleotide is applied. The signal oligonucleotide comprises at least one second connector element (C) that is essentially complementary to the translator sequence element (c) and at least one signal element that provides a detectable signal (F).

[0080] Step 8: Hybridization of the signal oligonucleotide. The signal oligonucleotide hybridizes to the translator element (c) of the decoding oligonucleotide through the complementary sequence connector element (C). After incubation, the signal oligonucleotides hybridize to their corresponding decoding oligonucleotides, providing a detectable signal (F).

[0081] Step 9: Removal of excess signal oligonucleotides After hybridization, unbound signal oligonucleotides are removed, for example, by a washing step.

[0082] Step 10: Signal detection The signal provided by the signal oligonucleotide is detected.

[0083] In the final detection round, the following steps (steps 11 and 12) are not required.

[0084] Step 11: Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotide is dissolved. Destabilization can be achieved through different mechanisms well known to those skilled in the art, for example, increased temperature, denaturing agents, etc. The target or analyte-specific probe is not affected by this step.

[0085] Step 12: Removal of denatured decoding oligonucleotides. The denatured decoding oligonucleotides and signal oligonucleotides are removed (e.g., by a washing step), leaving the specific probe sets with free unique identifier sequences available for reuse in the next round of hybridization and detection (steps 4-10). This detection cycle (steps 4-12) is repeated "n" times until the planned encoding scheme is completed.

[0086] Note that in each round of detection, the type of signal provided by a particular unique identifier is controlled by the use of a particular decoding oligonucleotide. As a result, the sequence of the decoding oligonucleotide applied in the detection cycle transcribes the binding specificity of the probe set into a unique signal sequence.

[0087] 3. Alternative Options for Applying Decoding and Signal Oligonucleotides The steps of decoding oligonucleotide hybridization (steps 4-6) and signal oligonucleotide hybridization (steps 7-9) can also be combined in two alternative ways, as shown in FIG.

[0088] Option 1: Simultaneous Hybridization. Instead of steps 4-9 of Figure 3, specific hybridization of decoding oligonucleotides and signal oligonucleotides can also be performed simultaneously, which, after removal of excess decoding and signal oligonucleotides, can result in the same result as shown in step 9 of Figure 3.

[0089] Option 2: Pre-incubation. In addition to option 1 in Figure 3, the decoding and signal oligonucleotides can be pre-incubated in separate reactions before being applied to the target nucleic acid with the specific probe set already bound.

[0090] 4. Example of signal encoding of three different nucleic acid sequences with two different signal types and three detection rounds Figure 3 shows a general concept of the generation and detection of specific signals mediated by decoding oligonucleotides. The general concept of encoding that can be achieved by this procedure is not shown. To illustrate the use of the process shown in Figure 3 for generating an encoding scheme, Figure 5 shows a general example of a multiple round encoding experiment using three different nucleic acid sequences. In this example, the encoding scheme includes error detection.

[0091] Step 1: Target Nucleic Acids. In this example, three different target nucleic acids (A), (B), and (C) need to be detected and distinguished by using only two different types of signals. Before starting the experiment, a specific encoding scheme is set up. In this example, the three different nucleic acid sequences are encoded by three rounds of detection using two different signals (1) and (2) and a resulting Hamming distance of 2 to allow for error detection. The planned code words are as follows: Sequence A: (1)-(2)-(2), Sequence B: (1)-(1)-(1), Sequence C: (2)-(1)-(2).

[0092] Step 2: Hybridization of Probe Sets. For each target nucleic acid, a unique probe set is applied and hybridizes specifically to the nucleic acid sequence of interest. Each probe set provides a unique identifier sequence (T1), (T2), or (T3). In this way, each different target nucleic acid is uniquely labeled. In this example, sequence (T) is labeled with (T1), sequence (B) with (T2), and sequence (C) with (T3). The diagram summarizes steps 1-3 of Figure 3.

[0093] Step 3: Hybridization of Decoding Oligonucleotides. For each unique identifier present, a specific decoding oligonucleotide is applied so that it specifically hybridizes to the corresponding unique identifier sequence by its first sequence element (here, (t1) to (T1), (t2) to (T2), (t3) to (T3)). Each decoding oligonucleotide provides a translator element that defines the signal generated after hybridization of the signal oligonucleotide. Here, nucleic acid sequences (A) and (B) are labeled with translator element (c1), and sequence (C) is labeled with translator element (c2). The diagram summarizes steps 4 to 6 of Figure 3.

[0094] Step 4: Hybridization of signal oligonucleotides. For each type of translator element, a signal oligonucleotide with a specific signal (2) that is distinguishable from the signals of other signal oligonucleotides is applied. This signal oligonucleotide can specifically hybridize to the corresponding translator element. The diagram summarizes steps 7 to 9 in Figure 3.

[0095] Step 5: Signal detection of the coding scheme. Different signals are detected. Note that in this example, nucleic acid sequence (C) can be distinguished from other sequences by the unique signal (2) it provides, while sequences (A) and (B) provide the same type of signal (1) and cannot be distinguished after the first detection cycle. This is due to the fact that the number of different nucleic acid sequences detected exceeds the number of different signals available. The diagram corresponds to step 10 in Figure 3.

[0096] Step 6: Selective Denaturation. The decoding (and signal) oligonucleotides of all nucleic acid sequences to be detected are selectively denatured and removed, as described in steps 11 and 12 of Figure 3. The unique identifier sequences of the different probe sets can then be used for the next round of hybridization and detection.

[0097] Step 7: Second Round Detection. The next round of hybridization and detection is performed as described in steps 3-5. Note that in this new round, the mixture of different decoding oligonucleotides is changed. For example, the decoding oligonucleotides for nucleic acid sequence (A) used in the first round consist of sequence elements (t1) and (c1), while the new decoding oligonucleotides consist of sequence elements (t1) and (c2). Note that all three sequences can now be clearly distinguished due to their unique combination with the signals from the first and second rounds.

[0098] Step 8: Third round of detection. Again, a new combination of decoding oligonucleotides is used, resulting in a new signal combination. After signal detection, the resulting code words of the three different nucleic acid sequences are not only unique and distinguishable, but also contain a Hamming distance of 2 from other code words. Due to the Hamming distance, errors in signal detection (signal exchange) will not result in a valid code word and can therefore be detected. In this way, three different nucleic acids can be distinguished with two different signals in three detection rounds, making error detection possible.

[0099] 5. Advantages over conventional technologies Coding Strategy One particular advantage of the method according to the invention compared to state of the art methods is the use of decoding oligonucleotides which break the dependency between the target-specific probe and the signal oligonucleotide.

[0100] When two different molecular tags are used without separating the target-specific probes and signal generation, two different signals can be generated for only a specific target. Each of these molecular tags can be used only once. Multiple readouts of the same molecular tag do not increase information about the target. To create a coding scheme, either the target-specific probe set must be changed after each round (SeqFISH) or multiple molecular tags must be present in the same probe set (merFISH, intron-SeqFISH, etc.).

[0101] Following the method according to the invention, different signals are achieved by using the same unique identifier (molecular tag) and decoding oligonucleotides reusing a small number of different, mostly cost-intensive, signal oligonucleotides, which offers several advantages in contrast to other methods. (1) The encoding scheme is not defined by the target-specific probe set, as in all other prior art methods. Here, the encoding scheme is transcribed by the decoding oligonucleotide. This allows for much greater flexibility in the number of rounds and freedom in selecting the codeword signals. Looking at prior art methods (e.g., merFISH or intron-SeqFISH), the encoding scheme (number, type, and sequence of detectable signals) for every target sequence is predefined by the presence of different tag sequences on the specific probe sets (4 out of 16 different tags per probe set for merFISH, and 5 out of 60 different tags for intron-SeqFISH). To generate a sufficient number of different tags per probe set, the methods use fairly complex oligonucleotide designs, with several tags present on a single target-specific oligonucleotide. To change the encoding scheme for a particular target nucleic acid, the specific probe set must be replaced. The method according to the present invention describes the use of a single unique tag sequence (unique identifier) ​​per analyte, which can be reused in each detection round to generate new information. The encoding scheme is defined by the order of the decoding oligonucleotides used in the detection round. Thus, the coding scheme is not predefined by specific probes (or unique tag sequences), but can be adjusted to different needs during an experiment, by simply changing the decoding oligonucleotides used in a detection round or by adding additional detection rounds. (2) The number of different signal oligonucleotides must match the number of different tag sequences in prior art methods (16 for merFISH and 60 for intron-SeqFISH). Using the method according to the present invention, the number of different signal oligonucleotides matches the number of different signals used. This means that the number of signal oligonucleotides remains constant for the methods described herein and does not exceed the number of different signals, but increases with the complexity of the encoding scheme in prior art methods (more detection rounds require more different signal oligonucleotides). As a result, the methods described herein result in very low complexity (interaction of signal oligonucleotides with the environment or with each other) and dramatically reduce the cost of the assay, since the major cost factor is the signal oligonucleotides. (3) In prior art methods, the number of distinct signals generated by a target-specific probe set is limited by the number of distinct tag sequences that the probe set can provide. Because each additional tag sequence increases the total size of the target-specific probe, there is a limit to the number of distinct tags that a single probe can provide. This limit is imposed by the size-dependent increase in several issues (unintended inter- and intramolecular interactions, cost, diffusion rate, stability, errors during synthesis, etc.). Furthermore, there is a limit to the total number of target-specific probes that can be applied to a particular analyte. In the case of nucleic acids, this limit is imposed by the length of the target sequence and the proportion of suitable binding sites. These factors severely limit the number of distinct signals that a probe set can provide (four signals for merFISH and five signals for intron-SeqFISH). This limit substantially affects the number of distinct code words that can be generated in a given number of detection rounds. In the present approach, only one tag is required, which can be freely reused in all detection rounds. This allows for low oligonucleotide complexity / length while simultaneously achieving the maximum possible coding efficiency (number of colors, ラウンドの数) resulting in a significant difference in the encoding capacity of our method compared to other methods, as shown in Figures 1 and 5. This results in our approach requiring far fewer detection rounds to generate the same amount of information. Fewer detection rounds translates to lower costs, shorter experimental time, lower complexity, higher stability and success rates, less data to be collected and analyzed, and more accurate results.

[0102] Coding Capabilities All three methods compared in Table 1 below use specific probe sets that do not degenerate between different detection rounds. In the case of intron-SeqFISH, four detection rounds are required to generate the pseudocolor of one encoding round, and therefore data are given only for rounds 4, 8, 12, 16, and 20. The merFISH method uses a fixed number of four signals, and therefore data begins with the fewest possible rounds. After eight detection rounds, our method exceeds the maximum encoding capacity reached with 20 rounds of merFISH (indicated by one asterisk), and after 12 rounds of detection, it exceeds the maximum encoding capacity of intronFISH (indicated by two asterisks). The method according to the present invention envisages the use of three different signals (as in intron-SeqFISH). [Table 1]

[0103] As shown in Figure 6, the number of code words in merFISH does not increase exponentially with the number of detection cycles, but rather decreases with each additional round. In contrast, the number of code words in intron-SeqFISH in the method of the present invention does increase exponentially. The slope of the curve for the proposed method is much higher than that of intron-SeqFISH, resulting in over 10,000 times more usable code words after 20 rounds of detection.

[0104] Note that maximum efficiency of this encoding capacity is also achieved in the case of seqFISH, where specific probes are denatured for each detection round and a new set of probes is specifically hybridized to the target sequence for each detection round. However, this method has a major disadvantage over techniques that use only one specific hybridization for their encoding scheme (all other methods). (1) For efficient denaturation of specific probes, rather harsh conditions (high temperature, high concentration of denaturant, long incubation time) must be used, which makes the possibility of loss or damage of the analyte much higher. (2) For each round of detection, a unique probe set must be used for every target nucleic acid sequence. Therefore, the number of specific probes required for an experiment is proportional to the number of different signals required for the coding scheme. This dramatically increases the complexity and cost of the assay. (3) Because the hybridization efficiency of all target nucleic acid molecules is subject to several stochastic effects, the variation in signal intensity between different detection rounds is much higher than that of methods using only one specific hybridization event, reducing the proportion of complete coding. (4) The time required for specific hybridization is much longer than that for hybridization of signal or decoding oligonucleotides (as can be seen in the methods sections of the Intron-SeqFISH, merFISH, and seqFISH publications), which dramatically increases the time required to complete an experiment.

[0105] For these reasons, all other methods use a single specific hybridization event and accept the major drawback of lower code complexity, and therefore the need for more detection rounds and higher oligonucleotide design complexity.

[0106] The method according to the invention combines the advantages of seqFISH (mainly the complete flexibility in terms of the encoding scheme) with all the advantages of methods using only one specific hybridization event, while eliminating the major problems of such methods.

[0107] Note that the large number of code words generated after 20 rounds can also be used to introduce a higher Hamming distance (difference) between different code words, allowing for error detection of 1, 2, or even more errors, and even error correction. Thus, even very high coding power is still practically adequate.

[0108] 6. Selective denaturation, oligonucleotide assembly, and reuse of unique identifiers are surprisingly efficient. The key element of the method according to the present invention is the sequential process of decoding oligonucleotide binding, signal oligonucleotide binding, signal detection, and selective denaturation. To generate the encoding scheme, this process must be repeated several times (depending on the length of the codeword). Because the same unique identifier is reused in every detection cycle, all events from the first detection cycle to the last detection cycle are interdependent. Furthermore, selective denaturation depends on two different events: the decoding oligonucleotide must be dissolved from the unique identifier with the highest efficiency, while the specific probe must remain hybridized with the highest efficiency.

[0109] This allows the efficiency E of the overall encoding process to be described by the following equation:

number

[0110] Based on this formula, the efficiency of each single step can be estimated for a given total efficiency of the method. The calculation is based on the assumption that each process has the same efficiency. The total efficiency represents the fraction of all signals present that can be successfully decoded.

[0111] The overall efficiency of the method depends on the efficiency of each single step, which is a function of the different factors described by the formula: Under the assumption of evenly distributed efficiency, the overall efficiency can be plotted against the single-step efficiency, as shown in Figure 7. As can be seen, a practically adequate overall efficiency for an encoding scheme with five detection cycles can only be achieved with a single-step efficiency above 90%. For example, to achieve an overall efficiency of 50%, an average efficiency within each single step of 97.8% is required. These calculations are based on the assumption of 100% signal detection and analysis efficiency. Due to the wide DNA melting curves of oligonucleotides with various sequences, the inventors assumed prior to the experiment that selective denaturation would be inefficient for denaturing the decoding oligonucleotides and that the sequence-specific binding probes would not be sufficiently stable. In contrast to this assumption, the inventors found surprising effectiveness of all steps and high stability of the sequence-specific probes during selective denaturation.

[0112] Experimentally, we achieved an overall decoding efficiency of approximately 30% to 65% based on five detection cycles. Calculation of the efficiency of each single step (Bsp, Bde, Bsi, Ede, Ssp) using the above formula revealed an average efficiency of approximately 94.4% to 98%. These high efficiencies are quite surprising and could not be easily predicted by those skilled in the field.

[0113] 7. Experimental Data background This experiment demonstrates the specific detection of 10-50 different mRNA species in parallel with single-molecule resolution. It is based on five detection cycles, three distinct fluorescent signals, a coding scheme with no signal gaps, and a Hamming distance (error detection) of 2. The experiment demonstrates the effectiveness and functionality of the method according to the present invention.

[0114] Oligonucleotides and their sequences All oligonucleotide sequences (target-specific probes, decoding oligonucleotides, signal oligonucleotides) used in the experiments are listed in the Sequence Listing in the Appendix. The signal oligonucleotide R:ST05*O_Atto594 was ordered from bioers.net GmbH. All other oligonucleotides were ordered from Integrated DNA Technologies. Oligonucleotides were dissolved in water. Stock solutions (100 μM) were stored at -20°C.

[0115] Experimental Overview The 50 different target-specific probe sets are divided into five groups. The names of the transcripts detected and the target-specific probe sets are the same (transcript variant names from www.ensemble.org). The term "new" indicates a modified probe design. All oligonucleotide sequences of the probe sets can be found in the sequence listing. The table lists the unique identifier names of the probe sets as well as the names of the decoding oligonucleotides used in the different detection cycles. The resulting codes indicate the sequence of the fluorescent signals generated during the five detection cycles (G(reen) = Alexa Fluor 488, O(range) = Atto 594, Y(ellow) = Alexa Fluor 546). [Table 2]

[0116] Experimental Variations Several variations of the experiment were performed. Experiments 1-4 differ primarily in the number of transcripts detected in parallel. The groups listed as target-specific probe sets are listed in Table 6. Experiments 5-8 are single-round, single-target controls for comparison with the decoded signals. [Table 3] Experiment details A. Cell seeding and culture HeLa cells were grown to near 100% confluence in HeLa cell culture medium containing DMEM (Thermo Fisher Scientific, Catalog No. 31885) containing 10% FCS (Biochrom, Catalog No. S0415), 1% penicillin-streptomycin (Sigma-Aldrich, Catalog No. P0781), and 1% MEM non-essential amino acid solution (Thermo Fisher Scientific, Catalog No. 11140035). After aspirating the cell culture medium, the cells were trypsinized by incubation in trypsin-EDTA solution (Sigma-Aldrich, Catalog No. T3924) for 5 minutes at 37°C, following a washing step with PBS (1,424 g / L NaHPO*2H2O in water, 0,276 g / L NaH2PO*2H2O, 8,19 g / L NaCl, pH 7.4). The cells were then seeded into the wells of a μ-Slide 8 Well ibidiTreat (Ibidi, Cat. No. 80826). The number of cells per well was adjusted to reach approximately 50% confluence after cell attachment. The cells were incubated overnight in 200 μl of HeLa cell culture medium per well. B. Fixation of cells After aspiration of the cell culture medium and two washing steps with 200 μl per well of 37°C-warmed PBS, the cells were fixed with 200 μl of pre-chilled methanol (-20°C, Roth, Cat. No. 0082.1) for 10 min at -20°C. C. Counterstaining with Sudan Black The methanol was aspirated, and 150 μl of 0.2% Sudan Black solution diluted in 70% ethanol was added to each well. The wells were incubated in the dark for 5 minutes at room temperature. After incubation, the cells were washed three times with 400 μl of 70% ethanol per well to remove excess Sudan Black solution. D. Analyte / Target-Specific Probe Hybridization Prior to hybridization, cells were equilibrated with 200 μl of sm wash buffer, which contained 30 mM Na citrate, 300 mM NaCl, pH 7, 10% formamide (Roth, catalog number P040.1), and 5 mM vanadyl ribonucleoside conjugates (NEB, catalog number S1402S). For each target-specific probe set, 1 μl of a 100 μM oligonucleotide stock solution was added to the mixture. The oligonucleotide stock solution contained equimolar amounts of all target-specific oligonucleotides in the corresponding target-specific probe set. The total volume of the mixture was adjusted to 100 μl with water and mixed with 100 μl of 2x concentrated hybridization buffer, which contained 120 mM Na citrate, 1200 mM NaCl, pH 7, 20% formamide, and 20 mM vanadyl ribonucleoside conjugates. The resulting 200 μl hybridization mixture was added to the corresponding wells and incubated for 2 hours at 37° C. The cells were then washed three times with 200 μl per well for 10 minutes at 37° C. with target probe wash buffer, which contained 30 mM Na citrate, 300 mM NaCl, pH 7, 20% formamide, and 5 mM ribonucleoside vanadyl complex. E. Hybridization of Decoding Oligonucleotides Prior to hybridization, cells were equilibrated with 200 μl of sm wash buffer. For each decoding oligonucleotide, 1.5 μl of a 5 μM stock solution was added to the mixture. The total volume of the mixture was adjusted to 75 μl with water and mixed with 75 μl of 2x concentrated hybridization buffer. 150 μl of the resulting decoding oligonucleotide hybridization mixture was added to the corresponding wells and incubated for 45 minutes at room temperature. Cells were then washed three times with 200 μl per well of sm wash buffer for 2 minutes at room temperature. F. Hybridization of signal oligonucleotides Prior to hybridization, cells were equilibrated with 200 μl of SM wash buffer. The signal oligonucleotide hybridization mixture was the same for all rounds in Experiments 1–4, containing 0 or 3 μM of each signal oligonucleotide (see Table A3) in 1x concentrated hybridization buffer. In each round, 150 μl of this solution was added per well and incubated at room temperature for 45 minutes. The procedure was the same as in Experiments 5–8, except that the final concentrations of each signal oligonucleotide were 0 or 15 μM. Cells were then washed three times with 200 μl per well of room-temperature SM wash buffer for 2 minutes. G. Fluorescence and White Light Imaging The cells were washed once with 200 μl of imaging buffer per well at room temperature. For experiments without Trolox (see Table 7, last column), the imaging buffer contained 30 mM Na citrate, 300 mM NaCl, pH 7, and 5 mM ribonucleoside vanadyl complex. For experiments with Trolox, the imaging buffer further contained 10% VectaCell Trolox Antifade Reagent (Vector Laboratories, catalog number CB-1000), resulting in a final Trolox concentration of 10 mM. A Zeiss Axiovert 200M microscope equipped with a 63x immersion oil objective (Zeiss, apochromat) with a numerical aperture of 1.4, a pco.edge 4.2 CMOS camera (PCO AG), and an LED light source (Zeiss, colibri 7) was used to image the regions. The filter sets and LED wavelengths were adjusted to the optimum for the different fluorophores used. The exposure time per image was 1000 ms for Alexa Fluor 546 and Atto 594, and 400 ms for Alexa Fluor 488. For each experiment, three regions were randomly selected for imaging. For each region, a z-stack of 32 images was detected with a z-step size of 350 nm. One white-light image was also taken from each region. In experiments with more than one detection cycle, the region from the first detection round was re-imaged and imaged for each subsequent round. H. Selective denaturation For selective denaturation, each well was incubated with 200 μl of sm wash buffer for 6 min at 42° C. This procedure was repeated six times.

[0117] In experiments 1 to 4, steps (E) to (H) were repeated five times. Step (H) was omitted in the fifth detection cycle. I.Analysis A semi-automated analysis of the raw data was performed to distinguish specific fluorescent signals from background using a custom ImageJ plugin. The resulting 3D point clouds of all three fluorescent channels were combined in silico using a custom VBA script. The combined 3D point clouds resulting from five detection cycles were aligned with each other using a VBA script. The resulting alignment revealed the codeword for each unique signal detected. Successfully decoded signals were used for quantitative and spatial analysis of the experiment using a custom VBA script and an ImageJ plugin. result 1. Absolute number of decoded signals The absolute number of successfully decoded signals for all transcripts is listed in Table 4 below for each region in each experiment. In summary, the sum of correct codes indicates the total number of decoded signals assigned to detectable transcripts in the corresponding experiment, and the sum of incorrect codes indicates the total number of undetectable decoded signals in the corresponding experiment. The total number of signals includes successfully and unsuccessfully decoded signals. [Table 4]

[0118] Table 4 shows a very small number of incorrectly decoded signals compared to the number of correctly decoded signals. The absolute values ​​of the decoded signals for a particular transcript are very similar between different regions of an experiment. The percentage of the total number of signals that can be successfully decoded ranges from 27.1% to 64.5%. This percentage depends on the number of transcripts and / or the total number of signals present in each region / experiment.

[0119] conclusion The method according to the present invention generates only a small number of incorrectly assigned code words and can therefore be considered specific. Even when the number of signals per region is very large and the number of transcripts detected in parallel is very large, the percentage of signals that can be successfully decoded is very high. Due to the high percentage of assignable signals and high specificity, this method is practically useful. 2. Comparison of relative transcript abundance between different experiments The overlap of transcripts detected between experiments is used in the analysis, as shown in Figure 8 for both comparisons (A and B). Each bar represents the average abundance of all three regions in the experiment. The standard deviation between these regions is also shown. 3. Correlation of relative transcript abundance between different experiments As can be seen in Figure 9, the mean relative abundance of transcripts in experiment 1 correlates with the abundance of overlapping transcripts in experiments 3, 4, and 2. The correlation and linear regression equations are shown for each correlation. Figure 8 shows low standard deviations, indicating low variation in relative abundance between different regions of a single experiment. The difference in relative abundance between transcripts from different experiments is also very low. This is the case for the comparison of transcripts from group 1 (Figure 8A) detected in experiments 1, 2, and 3. The same is true for the comparison of transcripts from groups 2, 3, and 4, which overlapped between experiments 1, 2, and 4. The very high correlation of these abundances can also be seen in Figure 9. The abundances of transcripts from experiment 1 correlate very well with those from other multi-round experiments. The correlation coefficients are between 0.88 and 0.91, while the slopes of the linear regressions are between 0.97 and 1.05.

[0120] conclusion The relative abundance of transcripts correlates very well not only between different regions of one experiment, but also between different experiments. This can be clearly seen by comparing Figures 3 and 4. The main difference between the experiments is the number of different targets and, therefore, the total number of signals detected. Therefore, the number of transcripts detected and the number and density of signals do not interfere with the method's ability to accurately quantify the number of transcripts. The very good correlation further supports the specificity and robustness of the method, even with a very large number of signals. 4. Comparison of intercellular distribution of signals Figure 10 shows maximum projections of image stacks: A: Region 1 of experiment 7 (single-round single-transcript experiment detecting SPOCK1), B: 2D projection of all selected signals from experiment 1, region 1 assigned to SPOCK1, C: Region 1 of experiment 8 (single-round single-transcript experiment detecting THRAP3), D: 2D projection of all selected signals from experiment 1, region 1 assigned to THRAP3. 5. Comparison of subcellular distribution of signals Maximum projections of the image stacks are shown in Figure 11. Magnified subregions of the corresponding regions are shown: A: Region 1 of experiment 8 (single-round single-transcript experiment detecting THRAP3); B: 2D projection of selected signals from experiment 1, region 1 assigned to THRAP3; C: Region 1 of experiment 5 (single-round single-transcript experiment detecting DDX5); D: 2D projection of all selected signals from experiment 1, region 1 assigned to DDX5. Figure 10 shows the large differences in intercellular distribution between different transcripts. SPOCK1 appears to be highly abundant in some cells but nearly absent in others (Figure 10A). THRAP3 shows a more uniform distribution across all cells in the region (Figure 10C). These spatial distribution patterns can also be clearly observed in the point clouds assigned to the corresponding transcripts from Experiment 1 (Figures 10B and D). Figure 11 shows the significant differences in subcellular distribution between the different transcripts. THRAP3 can be observed primarily at the periphery of the cell (cytoplasm) (Figure 11A), while DDX5 shows higher abundance in the center of the cell (nucleus) (Figure 11C). These subcellular distributions can also be observed in the point clouds from Experiment 1 assigned to THRAP3 and DDX5 (Figures 11B and D).

[0121] conclusion Second, the reliability of the quantification is such that point clouds from multi-round experiments also show the same intra- and intercellular distribution pattern of transcripts, as clearly demonstrated by direct comparison of the signal and assigned point clouds from single-round experiments that detect only one distinctive mRNA species. 6. Distribution Patterns of Different Cell Cycle-dependent Transcripts All images in Figure 12 represent region 1 of experiment 1. Each image shows point clouds assigned to specific transcripts: A: CCNA2, B: CENPE, C: CCNE1, and D: all transcripts. Figure 12 shows transcripts of three distinct cell cycle-dependent proteins. CENPE (Figure 12B), also known as centromere protein E, accumulates during G2 phase and is proposed to be responsible for spindle elongation and chromosome movement. It is absent during interphase. CCNA2 (Figure 12A), also known as cyclin A2, regulates cell cycle progression by interacting with CDK1 during the G2-to-M phase transition. Interestingly, there is clear colocalization of both mRNA species. They are primarily present in the three central cells of region 1. CCNE1 (Figure 12C), also known as cyclin E1, interacts with CDK2 and is involved in the G1-to-S phase transition. Figure 12 clearly shows that the transcript of this gene is absent from the three central cells, but is not distributed very evenly across the other cells, thus indicating a counter-localization to the other two transcripts. The corresponding point cloud data is derived from a point cloud with a very large number of points and a very high point density (Figure 12D gives an impression).

[0122] conclusion The three decoded point clouds of cell cycle-dependent proteins shown in Figure 12 exhibit distribution patterns that can be explained by their corresponding functions. These data strongly suggest that our method reliably generates biologically relevant data even when the number of signals per cell is low (Figure 12C) and the signal density is very high (Figure 12D).

[0123] Sequence Listing In the accompanying sequence listing, SEQ ID NOs: 1-1247 refer to the nucleotide sequences of exemplary target-specific oligonucleotides. The listed oligonucleotides consist of a target-specific binding site (5' end), spacer / linker sequences (gtaac and tagac), and a unique identifier sequence that is the same for all oligonucleotides of a probe set.

[0124] In the attached sequence listing, SEQ ID NOs: 1248 to 1397 refer to the nucleotide sequences of exemplary decoding oligonucleotides.

[0125] In the attached sequence listing, SEQ ID NOs: 1398-1400 refer to the nucleotide sequences of exemplary signal oligonucleotides. For each signal oligonucleotide, the corresponding fluorophore is present twice: one fluorophore is covalently attached to the 5'-end and one fluorophore is covalently attached to the 3'-end. SEQ ID NO: 1398 contains "5Alex488N" at its 5'-end and "3AlexF488N" at its 3'-end. SEQ ID NO: 1399 contains "5Alex546" at its 5'-end and 3Alex546N at its 3'-end. SEQ ID NO: 1400 contains "Atto594" at both its 5'-end and its 3'-end.

Claims

1. 1. A method for sequentially signal-coding analytes in a sample, comprising the steps of: (1) providing a set of analyte-specific probes, each analyte-specific probe comprising: a binding element (S) that interacts specifically with one of the analytes to be encoded, an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) unique to said set of analyte-specific probes; (2) incubating the set of analyte-specific probes with the sample, thereby allowing specific binding of the analyte-specific probes to the analytes to which they are encoded; (3) removing unbound probes from the sample; (4) providing a set of decoding oligonucleotides, each decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is complementary to at least a portion of the unique identifier sequence; - a translator element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide, (5) incubating the set of decoding oligonucleotides with the sample, thereby allowing specific hybridization of the decoding oligonucleotides to the unique identifier sequence; (6) removing unbound decoding oligonucleotides from the sample; (7) providing a set of signal oligonucleotides, each signal oligonucleotide comprising: a second connector element (C) comprising a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the translator element (c); - a signal element; (8) incubating the set of signal oligonucleotides with the sample, thereby allowing specific hybridization of the signal oligonucleotides to the translator element (c), wherein the signal elements do not uniquely identify the analyte; (9) removing unbound signal oligonucleotides from the sample; (10) detecting the signal; Including, (11) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby maintaining the specific binding of the analyte-specific probes to the encoded analytes; Further comprising: (12) Repeat steps (4) to (11) at least once [at least step (4) ２ ) ~ (11 ２ ) repeatedly to generate a coding scheme including at least two signals; Further comprising: Repeated steps (4 ２ ) ~ (11 ２ ) wherein the decoding oligonucleotide comprises a translator element (c2) that is different from the translator element (c1) of the decoding oligonucleotide used in the previous steps (4) to (11) in a nucleotide sequence that allows specific hybridization of a signal oligonucleotide, and comprises a first connector element (t) that is not different from the decoding oligonucleotide used in the previous step, and the signal oligonucleotide comprises a second connector element (C2) that comprises a nucleotide sequence that is complementary to at least a part of the nucleotide sequence of the translator element (c2), method.

2. The method of claim 1 , wherein the sample is a biological sample.

3. 3. The method of claim 2, wherein prior to step (2), the biological sample is fixed.

4. 4. The method of claim 1, wherein within the set of analyte-specific probes, each analyte-specific probe comprises a binding element (S1, S2, S3, S4, S5) that specifically interacts with one different position of the analyte to be coded.

5. The method of claim 1 , wherein the coding scheme is predetermined and assigned to the analyte being coded.

6. The signal oligonucleotide contains a signal element that is different from the signal element of the decoding oligonucleotide used in the previous steps (4) to (11), and the repeat step (4) ２ ) ~ (11 ２ 2. The method of claim 1, wherein

7. The method of any one of claims 1 to 6, wherein the binding element (S) comprises a nucleic acid comprising a nucleotide sequence that allows specific binding to the encoded analyte.

8. The method according to any one of claims 1 to 7, wherein the binding element (S) comprises an amino acid sequence that allows specific binding to the encoded analyte.

9. The method of any one of claims 1 to 8, wherein the analyte to be encoded is a nucleic acid.

10. The method of any one of claims 1 to 9, wherein the analyte to be encoded is a peptide or a protein.

11. 1. A kit for sequentially signal encoding analytes in a sample, comprising: - a set of analyte-specific probes, each analyte-specific probe comprising: a binding element (S) that interacts specifically with one of the analytes to be encoded, an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) unique to said set of analyte-specific probes; a set including and a set of decoding oligonucleotides, each decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is complementary to at least a portion of said unique identifier sequence; a translator element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; a first decoding oligonucleotide comprising: a first connector element (t) comprising a nucleotide sequence that is complementary to at least a portion of said unique identifier sequence; a second translator element (c2) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide different from that of said translator element (c); and a second decoding oligonucleotide comprising: a set including Includes a kit.

12. a first set of signal oligonucleotides, each signal oligonucleotide comprising: a second connector element (C) comprising a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the translator element (c); a first signal element; a second set of signal oligonucleotides, each signal oligonucleotide comprising: a second connector element (C2) comprising a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of said second translator element (c2); a second signal element; The kit of claim 11 further comprising:

Images