Multiplex methods for detecting different analytes and different subgroups / variants of analytes in a sample

Abstract

The technology provided herein relates to multiplexed methods and kits for detecting different analytes and different subgroups / variants of analytes in a sample, as well as in vitro methods and optical multiplexed systems for screening, identifying, and / or testing substances and / or drugs and for disease diagnosis.

Description

[Technical Field]

[0001] The technology provided herein relates to multiplex methods and kits for detecting different analytes and different subgroups / variants of analytes in a sample in parallel by sequential signal encoding of the analytes, as well as in vitro methods and optical multiplex systems for screening, identifying and / or testing substances and / or drugs and for diagnosing diseases. [Background technology]

[0002] The analysis and detection of small amounts of analytes in biological and non-biological samples has become common routine in clinical and analytical environments. Many analytical methods have been established for this purpose. Some of these use coding techniques that assign a specific readable code to a specific first analyte that differs from the code assigned to a specific second analyte.

[0003] One prior art in this field is the so-called "single-molecule fluorescence in situ hybridization" (smFISH), which was developed primarily 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, mRNAs of interest are detected via specific, directly labeled probe sets. After one round of hybridization and detection, a set of mRNA-specific probes is eluted from the mRNA, and over several rounds, the same set of probes with different (or the same) fluorescent labels is used in subsequent rounds of hybridization and imaging to generate a gene-specific color-coded scheme. This technique requires several differently tagged probe sets for each transcript and requires denaturing these probe sets after every detection round.

[0004] Further developments of this technology will not use directly labeled probe sets. Instead, the oligonucleotides in the probe set provide nucleic acid sequences that act as initiators for hybridization chain reaction (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.

[0005] Another technique, called "multiplexed error robust fluorescence in situ hybridization" (merFISH), was 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 from a total of 16 sequence elements. Following hybridization of the specific probe sets to the mRNA of interest, a so-called readout hybridization is performed. In each readout hybridization, one of the 16 fluorescently labeled oligonucleotides complementary to one of the sequence elements is hybridized. All readout oligonucleotides have the same fluorescent color. After imaging, the fluorescent signal is destroyed by irradiation, and another round of readout hybridization is performed without a denaturation step. As a result, a binary code is generated for each mRNA species. Only a single hybridization round is used for binding of a specific probe set to the mRNA of interest, followed by 16 rounds of hybridization of readout oligonucleotides labeled with a single fluorescent color, resulting in a unique signal signature of four signals over the 16 rounds.

[0006] Further developments of this technique will improve throughput by using two different fluorescent colors, eliminating the signal through disulfide cleavage between the readout oligonucleotide and the fluorescent label, and using 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. USA. 113(39), pp. 11046-11051.

[0007] A technique called "intron seqFISH" is described in 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 consecutive hybridizations. In each of these consecutive hybridizations, three readout probes, each labeled with a different fluorophore, are hybridized to corresponding elements of the mRNA-specific probe set. After imaging, the readout probes are stripped with 55% formamide buffer, followed by the next hybridization. After five rounds of color-coding, each with four consecutive hybridizations, the color coding is complete.

[0008] EP 0611828 discloses the use of a bridging element to mobilize 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 mobilizes a bridging nucleic acid molecule. This bridging nucleic acid ultimately mobilizes a signal-generating nucleic acid. This document also describes the use of a bridging element with multiple binding sites for signal-generating elements, such as branched DNA, for signal amplification.

[0009] 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 are hybridized to this sequence element. These preamplifier oligonucleotides contain multiple binding sites for amplifier oligonucleotides that are hybridized in subsequent steps. These amplifier oligonucleotides provide multiple sequence elements for labeled oligonucleotides. In this way, a branched oligonucleotide tree is constructed, leading to signal amplification.

[0010] A further development of this method, called "RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues," by Wang et al. (2012), uses a different design of mRNA-specific probes, J. Mol. Diagn. 14(1), pp. 22-29. Here, two mRNA-specific oligonucleotides must hybridize in close proximity to provide a sequence that can recruit a preamplifier oligonucleotide. This increases the specificity of the method by reducing the number of false-positive signals.

[0011] Choi et al. (2010), Programmable in situ amplification for multiplexed imaging of mRNA expression, Nat. Biotechnol. 28(11), p.1208-1212, discloses a method known as "HCR-hybridization chain reaction." The mRNA of interest is detected through 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, hybridization chain reaction is based on metastable oligonucleotide hairpins that self-assemble into polymers after the first hairpin is opened via the initiator sequence.

[0012] Further developments of this technology, similar to the RNAscope technology, use so-called split initiator probes that must hybridize in close proximity to form initiator sequences for 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).

[0013] 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.

[0014] EP 2992115 B1 describes a method of sequential single molecule hybridization, providing a technique for detecting and / or quantifying nucleic acids in cells, tissues, organs or organisms through sequential barcoding.

[0015] However, the methods known in the art have many drawbacks: in particular, they are inflexible, expensive, complicated, time-consuming, and frequently provide inaccurate results. In particular, the codability of existing methods is low and they do not meet the requirements of modern molecular biology and medicine.

[0016] Against this background, the objective underlying the present disclosure is to provide a method by means of which the drawbacks of the prior art methods may be reduced or even avoided. Summary of the Invention

[0017] The present disclosure relates to a novel multiplex method for the parallel detection of different analytes and different subgroups / variants of analytes in a sample by sequential signal encoding of said analytes and variants.

[0018] In particular, the present disclosure relates to a multiplex method for detecting different analytes and different subgroups / variants of analytes in a sample, the method comprising: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes to encode at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and if the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; contacting the sample with at least two different sets of analyte-specific probes for at least one analyte and variants thereof; The analyte-specific probes contained in these different sets interact with the same analyte, but specifically with different substructures of the same analyte; wherein the analyte-specific probes of the first set of analyte-specific probes interact with a substructure contained in all variations of the analyte; a second set of analyte-specific probes (subgroup-specific probes) interacting with substructures contained only in specific variants of the analyte; the analyte-specific probes of the first set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); the analyte-specific probes of the second set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); the analyte-specific probe identifier elements (T) of the first set of analyte-specific probes and the analyte-specific probe identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes are different for binding of different decoding oligonucleotides and / or non-signal decoding oligonucleotides; (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide is (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; one set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in a first connect element (t); (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) contained in the decoding oligonucleotide; (bb) a signal element; (D) detecting a signal produced by the signal element; (E) selectively removing decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining specific binding of the analyte-specific probes to the analytes to be encoded; (F) performing at least three (3) additional cycles comprising steps B) through E) to generate a coding scheme with a code word for each analyte; (G) performing at least one (1) additional cycle comprising steps B) through E) to identify subgroup-specific probes, wherein the cycle may stop at step (D); Includes.

[0019] Furthermore, the present disclosure relates to the use of improved decoding oligonucleotides to improve the efficiency of encoding schemes. So-called "multi-decoders" allow the recruitment of more than one signal oligonucleotide, thus allowing the generation of new signal types by utilizing two or more different signal-oligonucleotide combinations without reducing the brightness of the signal.

[0020] In a further aspect, embodiments of the present disclosure relate in particular to a multiplex method for detecting different analytes in a sample by sequential signal encoding of said analytes, the method comprising: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes to encode at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and if the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; Things and; (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide is (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; a set of decoding oligonucleotides for each analyte differs from another set of decoding oligonucleotides for a different analyte in the first connect element (t); Things and; (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide; (bb) a signal element; and (D) detecting a signal produced by the signal element; (E) selectively removing decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining specific binding of the analyte-specific probes to the analytes to be encoded; (F) performing at least three (3) additional cycles comprising steps B) through E) to generate a coding scheme with code words for each analyte, in particular the last cycle may stop at step (D); Includes.

[0021] In a further aspect, embodiments of the present disclosure include: (A) at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and when the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; and at least twenty (20) different sets of analyte-specific probes; (B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; a set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in the identifier connector element (t); at least one set of decoding oligonucleotides per analyte; (C) a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide; (bb) a signal element, one set of signal oligonucleotides; The present invention relates to a kit for multi-analyte encoding, comprising:

[0022] In a further aspect, embodiments of the present disclosure relate to an in vitro method for the diagnosis of a disease selected from the group comprising cancer, a neurological disease, a cardiovascular disease, an inflammatory disease, an autoimmune disease, a disease caused by a viral or bacterial infection, a skin disease, a musculoskeletal disease, a dental disease, and a prenatal disease, comprising the use of a multiplex method according to the present disclosure.

[0023] In a further aspect, embodiments of the present disclosure provide an in vitro method for the diagnosis of a disease in a plant selected from the group comprising a disease caused by biotic stress, preferably of infectious and / or parasitic origin, or a disease caused by biotic stress, preferably caused by nutritional disorders and / or an unfavorable environment, comprising the use of a multiplex method according to the present disclosure.

[0024] In a further aspect, some embodiments of the present disclosure comprise at least: at least one reaction vessel for containing a kit or part of a kit according to the present disclosure; a detection unit including a microscope, in particular a fluorescence microscope; -Camera and a liquid handling device; The present disclosure relates to an optical multiplexing system suitable for the method according to the present disclosure, comprising:

[0025] In a further aspect, some embodiments of the disclosure relate to a kit for multiplexed analyte encoding, comprising: (A) at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and when the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; and at least twenty (20) different sets of analyte-specific probes; (B) at least one set of decoding oligonucleotides per analyte, wherein for each analyte, in each set of decoding oligonucleotides, each decoding oligonucleotide is (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) containing a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, one set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in the identifier connect element (t); at least one set of decoding oligonucleotides per analyte; (C) a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) contained in the decoding oligonucleotide; (bb) a signal element, a set of signal oligonucleotides; Including, The kit comprises at least two different sets of analyte-specific probes for the analyte; Analyte-specific probes are included in these different sets that interact with the same analyte, but specifically interact with different substructures of the same analyte; wherein the analyte-specific probes of the first set of analyte-specific probes interact with substructures contained in all variations of the analyte; Analyte-specific probes of a second set of analyte-specific probes (subgroup-specific probes) interact with substructures contained only in specific variants of the analyte; the analyte-specific probes of the first set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); the analyte-specific probes of the second set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); The identifier element (T) of the analyte-specific probes of the first set of analyte-specific probes and the identifier element (T) of the analyte-specific probes of the second set of analyte-specific probes are different.

[0026] Additionally, some embodiments include: (A) optionally, at least twenty (20) different sets of analyte-specific probes for encoding at least twenty different analytes, each set of analyte-specific probes interacting with a different analyte, and if the analytes are nucleic acids, each set of analyte-specific probes including at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe being: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; at least twenty (20) different sets of analyte-specific probes; (B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; a set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in the identifier connector element (t); at least one set of decoding oligonucleotides per analyte; (C) a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide; (bb) a signal element; a set of signal oligonucleotides comprising: The present invention relates to a kit for multi-analyte encoding, comprising:

[0027] In a further aspect, some embodiments comprise: (a) contacting a test sample containing a sample with a substance and / or drug; (b) detecting different analytes in a sample by sequential signal encoding of said analytes in a method according to the present disclosure; In accordance with the present invention, there is provided an in vitro method for screening, identifying and / or testing substances and / or drugs, comprising:

[0028] In a further aspect, embodiments of the present disclosure extend the multiplex method for detecting different analytes (described in the first aspect) by targeting subgroups of targets in a sample. When performed as described, sequential signal encoding of one set of probes and at least one additional set of probes are added to distinguish target subgroups.

[0029] Decoding (multiple rounds) of the primary analyte is performed as described (A-I in embodiment 1). Additional signals are generated and analyzed in combination with the primary analyte encoding to identify subgroups of said analytes. The method comprises: (A1) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and where the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; (A2) contacting at least one analyte subgroup with a set of at least five (5) subgroup-specific probes that differ in the nucleotide sequence of their identifier elements (T) from the analyte-specific probes of another set of analyte-specific probes; (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide is (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; one set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in a first connect element (t); (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) contained in the decoding oligonucleotide; (bb) a signal element; and (G) detecting a signal produced by the signal element; (H) selectively removing decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining specific binding of the analyte-specific probes to the analytes to be encoded; (I) performing at least three (3) additional cycles comprising steps B) through E) to generate a coding scheme with a code word for each analyte; (J) performing at least one (1) additional cycle comprising steps B) to E) to identify the subgroup-specific probes contacted in step A2), wherein the final cycle may terminate at step (D); Includes.

[0030] According to the present disclosure, a unique tag (identifier) ​​is used for each target (e.g., the mRNA of one single gene) or for groups of targets, which can be formed to indicate a particular identity, process, biological function, or disease (e.g., cell type, inflammation, signal processing, cancer).

[0031] Surprisingly, the methods and kits of the present disclosure lead to reduced complexity: many different probes with different binding sequences share the same unique tag (one per target). These tags have reduced sequence complexity (vs. one per target) and also have certain predetermined properties (e.g., thermodynamic stability).

[0032] Advantages of the methods and kits according to the present disclosure include: a) Maximum flexibility in the process for determining tag identity, e.g., use of more or fewer signals and / or rounds, number of fluorophores, variation in total number of signals per tag → fewer targets (e.g., 20) can be identified with high confidence in fewer rounds (e.g., 4) than in the case of a larger number of targets (e.g., 100, which require 8 rounds for the same level of confidence), even if the exact same unique tag is used in both cases. b) All unique tags are used (reused) in many successive rounds of hybridization, and all primary probes contribute (are informative about their identity) in every round of identification. c) All tags share the same predetermined property (e.g., thermodynamic stability that allows selective denaturation). d) In some advantageous embodiments, the unique tag is designed as follows: - Non-cross hybridization (compatibility) between all oligonucleotides (probe, decoder, readout) in this step, so that all tag sequences can be used together. - Non-crossover hybridization between connector elements (bridges) of different unique tags. The hybridization stability of the unique tag should be in a narrow range: as stable as possible (fast hybridization, i.e., short cycle times), but significantly different (in this case less stable) from the primary probe (due to differential denaturation without removing the primary probe).

[0033] Thus, this description particularly relates to the use of a set of labeled and unlabeled nucleic acid sequences for the specific quantitative and / or spatial detection of different analytes in parallel via specific hybridization. This technique allows for the discrimination of different analytes beyond the available differential detection signals. This discrimination is realized through sequential signal encoding of the analytes, achieved through several cycles of specific hybridization, signal detection, and selective elution of the hybridized nucleic acid sequences. In contrast to other state-of-the-art methods, the oligonucleotides providing the detectable signal do not interact directly with sample-specific nucleic acid sequences, but are mediated by so-called "decoding oligonucleotides." This mechanism decouples the dependency between analyte-specific oligonucleotides and signal oligonucleotides. The use of decoding oligonucleotides allows for much greater flexibility while dramatically reducing the number of different signal oligonucleotides required, which in turn increases the coding capacity achieved in a given number of detection rounds. The use of decoding oligonucleotides leads to sequential signal encoding techniques that are, for example, more flexible, inexpensive, simple, fast, and / or accurate than other methods.

[0034] Examples for the use of kits and methods according to the present disclosure that include subgroup-specific probes are as follows: 1.) Fusion-transcript detection in cancer research Gene fusion events generating chimeric proteins are responsible for several cancer types, accounting for approximately 20% of all tumors (Mitelman 2007). Detection of RNA fusions has facilitated the molecular characterization and diagnosis of various tumors (reviewed by Neckles 2020). Recent approved molecules targeting oncogenic fusion transcripts for degradation suggest that they represent promising therapeutic targets. However, the inter- and intratumoral variability of oncogenic fusion transcripts needs to be understood in more detail, ideally at the cellular level or even subcellular degradation. ·Mitelman, F., Johansson, B., & Mertens, F. (2007): The impact of translocations and gene fusions on cancer causation. Nature Reviews. Cancer, 7(4), 233-245. ·Neckles, C, Sundara Rajan, S, Caplen, NJ. (2020):Fusion transcripts:Unexploited vulnerabilities in cancer? WIREs RNA;11:e1562.https: / / doi.org / 10.1002 / wrna.1562 2.) RNA subgroup (alternative splicing) RNA splicing is a fundamental process in gene expression, and alternative splicing plays an important role in transcriptome complexity, cell type differentiation, and biological development. Detection of spliced ​​products is important because aberrant splicing can lead to many diseases, including cancer and neurodegeneration. Variability in splicing between individual cells is primarily responsible for heterogeneity in gene expression. Investigating RNA splicing variants at the single-cell level helps decipher regulatory circuits and classify and understand cell types and subtypes (Walks 2011). Single-molecule FISH (smFISH) has previously been applied to detect RNA splicing variants. Vargas (2011) demonstrated that unspliced ​​pre-mRNA, spliced ​​introns, and spliced ​​mRNA can be simultaneously detected in a single cell, but this does not allow for multiplexing. ·T.Maniatis, B.Tasic.(2002):Alternative pre-mRNA splicing and proteome expansion in metazoans.Nature,418,pp.236-243 ·Z.Waks,AMKlein,PASilver.(2011):Cell-to-cell variability of alternative RNA splicing.Mol.Syst.Biol.,7,p.506 ·DYVargas, K. Shah, M. Batish, M. Levandoski, S. Sinha, SA Marras, P. Schedl, S. Tyagi. (2011): Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell, 147, pp. 1054-1065 3.) Viral transcript length Many viral genomes possess multiple promoters that can lead to mRNA species of various lengths, some of which share several segments in common. Detecting the correct length and composition is crucial for understanding the current phase of viral infection. For example, the HBV genome serves as a template for the synthesis of multiple genomic and subgenomic viral mRNA transcripts: four viral promoters, Core, Pre S1, Pre S2, and X, and two enhancers, Enhancer I and Enhancer II, regulate HBV transcription (Zheng 2004). Quantitation of each of these subgenomic mRNA transcripts is important for understanding the phase of infection and replication status. ·Zheng, Y., Li, J. & Ou, J. (2004): Regulation of Hepatitis B Virus Core Promoter by Transcription Factors HNF1 and HNF4 and the Viral X Protein Regulation of Hepatitis B Virus Core Promoter by Transcription Factors HNF1 and HNF4 and the Viral X Protein.78,6908-6914.

[0035] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to the specific components of the method steps described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the singular and / or plural unless the context clearly indicates otherwise. For clarity, when a parameter range bounded by numerical values ​​is provided, it is further understood that the range is deemed to include those limits. [Brief explanation of the drawings]

[0036] [Figure 1] The embodiment, wherein the analyte is a nucleic acid and the probe set comprises oligonucleotides that specifically bind to the analyte, the probes comprising unique identifier sequences that allow hybridization of decoding oligonucleotides. [Figure 2] The embodiment in which the analyte is a protein and the probe set comprises proteins (here antibodies) that specifically bind to the analyte, the probes comprising unique identifier sequences that allow hybridization of decoding oligonucleotides. [Figure 3] 1 is a flowchart of a method according to the present disclosure. [Figure 4] Alternative options for the application of decoding and signal oligonucleotides. [Figure 5] Example for signal encoding of three different nucleic acid sequences with two different signal types and three detection rounds; in this example, the encoding scheme includes error detection. [Figure 6] Number of generated codewords versus number of detection cycles (logarithmic scale). [Figure 7] Calculated total efficiency of the five-round coding scheme based on the efficiency of the single stages. [Figure 8] Comparison of relative transcript abundance between different experiments [Figure 9]Correlation of relative transcript abundance between different experiments. [Figure 10] Comparison of intercellular distribution of signals. [Figure 11] Comparison of the subcellular distribution of the signal. [Figure 12] Distribution patterns of different cell cycle-dependent transcripts. [Figure 13] Detection of multiple targets using 8 rounds of coding with two labels (A and B) and without label (-). Targets 1, 2, 3, 4, 5, 20 and n are represented. Rounds 1, 2, 3 and 8 of the coding scheme are represented. [Figure 14] Detection of multiple targets can be achieved by a coding scheme using detectable markers. The termination scheme can also include a "0" as a marker, meaning that no transcript is detected at a specific location. As a result, a coding scheme can be represented by the following constructs using only two gene-specific probes: 1) with detectable label F: detectable during imaging; 2) with detectable label F and quencher Q: undetectable during imaging; 3) with quencher Q: undetectable during imaging; 4) without label F: undetectable during imaging; 5) without signaling oligonucleotide: undetectable during imaging; 6) with decoder oligonucleotide that cannot recruit signaling oligonucleotide; 7) without decoder oligonucleotide: undetectable during imaging. [Figure 15]Detection of distinct target subgroups using additional subgroup-specific probe sets (Round 0). This procedure involves contacting an analyte-specific probe set at the common (shared) portion and further contacting the subgroup-specific probe set with a subgroup-determining (exclusive) portion (see Round 1; description, A2). Detection of analytes using probe sets bound to the shared portion (Rounds 1-4) using the decoding scheme also described. Round 5: In at least one specialized round, only subgroup-specific probe sets are detected. The presence of the exclusive portion of the target is then combined with the results from previous rounds, making it possible to distinguish between subgroup 1' within group 1 and subgroup 2' within group 2. [Figure 16] Possible structures of multi-decoders. Numbers indicate examples. (A) is a unique identifier sequence, (a) is the corresponding sequence of the decoding oligonucleotide or multi-decoder, and (c1) to (c3) are different sequence elements that specifically bind to different signal oligonucleotides. Examples 2 to 5 show different versions of multi-decoders. The order of the different sequence elements as well as the number of signal oligonucleotide binding elements are not fixed. Example 1 shows a normal decoding oligonucleotide, since there is only one signal oligonucleotide binding element (c1). [Figure 17] Example for signal encoding three different nucleic acid sequences by using a multi-decoder and two different signal oligonucleotides and three detection rounds to generate three different signal types. In this example, the encoding scheme includes error detection and correction. [Figure 18]Number of codewords generated versus number of detection cycles (logarithmic scale). The number of codewords for merFISH does not increase exponentially with the number of detection cycles, but becomes less efficient with each additional round. In contrast, the number of codewords for intronSeqFISH, the disclosed method without multiple decoders, and the method with multiple decoders increases exponentially. The slope of the curve for the disclosed method with multiple decoders is significantly higher than the prior invention, with over 20,000,000 times more codewords available after 20 rounds of detection. DETAILED DESCRIPTION OF THE INVENTION

[0037] Disclosed herein are novel multiplex methods and kits for detecting different analytes and different subgroups / variants of analytes in a sample.

[0038] This disclosure describes the use of sets of labeled and unlabeled nucleic acid sequences for the specific quantitative and / or spatial detection of different analytes in parallel via specific hybridization. This technique allows for the discrimination of more different analytes than the available differential detection signals. Discrimination can be achieved through sequential signal encoding of the analytes, achieved by several cycles of specific hybridization, signal detection, and selective elution of the hybridized nucleic acid sequences.

[0039] In contrast to other state-of-the-art methods, the oligonucleotides that provide the detectable signal do not directly interact with the sample-specific nucleic acid sequence, but are mediated by so-called "decoding oligonucleotides." This mechanism separates the dependency between the analyte-specific oligonucleotide and the signal oligonucleotide. The use of decoding oligonucleotides allows for much greater flexibility while dramatically reducing the number of different signal oligonucleotides required, which in turn increases the encoding capacity achieved in a given number of detection rounds.

[0040] The use of decoding oligonucleotides leads to sequential signal encoding techniques that are more flexible, cheaper, simpler, faster and / or more accurate than other methods.

[0041] A.Definition According to the present disclosure, an "analyte" is an entity that is specifically detected as being present or absent in a sample and, if present, encodes it. It can be any type of entity, including a protein, polypeptide, protein, or nucleic acid molecule (e.g., RNA, PNA, or DNA) of interest. An analyte provides at least one site for specific binding with an analyte-specific probe. The term "analyte" may be substituted herein for "target." According to the present disclosure, an "analyte" includes a complex of interest, such as at least two individual nucleic acid, protein, or peptide molecules. In one embodiment of the present disclosure, an "analyte" excludes a chromosome. In another embodiment of the present disclosure, an "analyte" excludes DNA. According to the present disclosure, the term "analyte" can include a group of different variants / embodiments of the same analyte, such as splicing variants of the analyte, variants containing different introns and / or exons, and sequences containing UTRs and / or sequences with different lengths. In particular, the basic sequence and variants having at least 50%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity.

[0042] In some embodiments, the analyte may be a "coding sequence," "encoded sequence," "structural nucleotide sequence," or "structural nucleic acid molecule," which refers to a nucleotide sequence that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of a coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding sequences may include, but are not limited to, genomic DNA, cDNA, ESTs, and recombinant nucleotide sequences.

[0043] A "sample," as used herein, refers to a composition in liquid or solid form suspected of containing the analyte to be encoded. In particular, the sample is a biological sample, preferably containing biological tissue, more preferably containing biological cells and / or extracts and / or cell parts. For example, the cells are prokaryotic or eukaryotic cells, particularly mammalian cells, especially human cells. In some embodiments, the biological tissue, biological cells, extracts and / or cell parts are fixed. In particular, the analyte is fixed in a permeabilized sample, such as a cell-containing sample.

[0044] As used in this disclosure, "cell," "cell line," and "cell culture" can be used interchangeably, and all such designations include progeny. Thus, the words "transformants" or "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.

[0045] A "coding scheme" may refer to a set of code words associated with the analytes to be detected. Each code word refers to one of the analytes and can be distinguished from all other code words. A code word according to the present invention is a sequence of signatures provided by the detection cycles of the method. A signature within a code word is a detectable signal or the absence of a signal. A code word does not need to include all the different signals used in the method. The number of signatures in a code word is defined by the number of detection cycles.

[0046] 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 nt, more preferably 8 to 60 nt, more preferably 10 to 50 nt, and more preferably 12 to 35 nt, depending on the number of consecutive sequence elements. The nucleic acid molecule can be completely or partially single-stranded. The oligonucleotide can be linear or can contain a hairpin or loop structure. The oligonucleotide can contain modifications such as biotin, a labeling moiety, a blocking moiety, or other modifications.

[0047] 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.

[0048] In particular, in some embodiments, the binding element (S) comprises an affinity portion from an affinity agent, or a portion which is an affinity agent in its entirety, selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptidic allergen, a recombinant allergen, an allergen-idiotypic antibody, an autoimmune-inducing structure, a tissue rejection-inducing structure, an immunoglobulin constant region and derivatives, mutants or combinations thereof. In further advantageous embodiments, the antibody fragment is a Fab, scFv; a single domain or fragment thereof, a bis-scFv, Fab2, Fab3, a minibody, a maxibody, a diabody, a triabody, a tetrabody or a tandub, in particular a single-chain variable fragment (scFv).

[0049] A "unique identifier sequence," when included by an analyte-specific probe, is unique in its 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, that it specifically identifies only one group of analytes, independent of whether the group of analytes comprises a gene family. Thus, the analyte or group of analytes to be encoded by this unique identifier can be distinguished from all other analytes or groups of analytes to be encoded 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 multiple, 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, 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 can be a sequence element of an analyte-specific probe, linked directly or by a linker, covalent bond or high-affinity binding scheme, e.g., antibody-antigen interaction, streptavidin-biotin interaction, etc. The term "analyte-specific probe" should be understood to include multiple probes that may differ in their binding element (S), such that each probe binds to the same analyte, but possibly to different portions thereof, e.g., different (e.g., adjacent) or overlapping sections of the nucleotide sequence comprised by the nucleic acid molecule to be encoded. However, each of the multiple probes contains the same identifier element (T).

[0050] A "decoding oligonucleotide" consists of at least two sequence elements: one sequence element capable of specifically binding to a unique identifier sequence, called the "identifier connector element" (t) or "first connector element" (t), and a second sequence element that specifically binds to a signal oligonucleotide, called the "translator element" (c). The length of the sequence elements ranges from 8 to 60 nt, preferably 12 to 40 nt, more preferably 14 to 20 nt, depending on the number of analytes to be encoded in parallel, the required stability of the interaction, and the number of different signal oligonucleotides used. The length of the two sequence elements may or may not be the same.

[0051] In some advantageous embodiments, the decoding oligonucleotide in the kits and / or methods of the present disclosure can be a "multi-decoder." A "multi-decoder" is a decoding oligonucleotide consisting of at least three sequence elements. One sequence element (identifier connector element (t)) can specifically bind to a unique identifier sequence (identifier element (T)), and at least two other sequence elements (translator elements (c)) specifically bind to different signal oligonucleotides (each of these sequence elements specifically binds to a signal oligonucleotide that is different from all other signal oligonucleotides recruited by other elements of the multi-decoder). 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 detected in parallel, the required stability, and the number of different signal oligonucleotides used. The lengths of the sequence elements may or may not be the same.

[0052] Thus, in some advantageous embodiments, the decoding oligonucleotide comprises: an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; and - at least two translator elements (c), each containing a nucleotide sequence that allows specific hybridization of a different signal oligonucleotide; A multi-decoder including:

[0053] Thus, the first translator element binds to a different signal oligonucleotide as the second translator element, in particular the signal oligonucleotides differ in the type of signal element, e.g., fluorophore, contained therein.

[0054] A "signal oligonucleotide," as used herein, comprises at least two elements: a so-called "translator connector element" (C) or "second connector element" (C) having a nucleotide sequence capable of specifically hybridizing to at least a section 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 either actively generate a detectable signal or provide such a signal through operation, e.g., fluorescence excitation. Typical signal elements are, for example, enzymes that catalyze a detectable reaction, fluorophores, radioactive elements, or dyes.

[0055] A "set" refers to a plurality of moieties or entities, such as analyte-specific probes or decoding oligonucleotides, whether the individual members of this plurality are identical or different from one another. In an analyte-specific probe set, the analyte-specific probes are identical in their identifier element (T), but may contain different binding elements (S) that interact specifically with the same analyte, but with different substructures of the same analyte that are to be encoded.

[0056] "Selective denaturation" can be a process that eliminates bound decoding and signal oligonucleotides with maximum efficiency while allowing the target-specific probe to remain hybridized with maximum efficiency. The total efficiency of these two combined events can be at least 0.22 for 2 detection cycles, 0.37 for 3 detection cycles, 0.47 for 4 detection cycles, 0.55 for 5 detection cycles, 0.61 for 6 detection cycles, 0.65 for 7 detection cycles, 0.69 for 8 detection cycles, 0.72 for 9 detection cycles, and 0.74 for 10 detection cycles, 0.76 for 11 detection cycles, and 0.78 for 12 detection cycles.

[0057] In one embodiment of the present disclosure, a single set refers to a plurality of oligonucleotides.

[0058] "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 probe set specific for a single analyte is further characterized by at least the same unique identifier.

[0059] A "subgroup-specific probe set" may contain the same features as an "analyte-specific probe set," but differs in the encoding and decoding information. A "subgroup-specific probe set" is used only to add information (mainly presence / absence) to the code already encoded by the "analyte-specific probe set."

[0060] Analyte "subgroups" or "variants," as they are also called, refer to embodiments of an analyte, where the variants contain common or "shared" elements (e.g., identical nucleic acid sequences) contained in all embodiments (or variants) of the same analyte, and at least one additional element that distinguishes the analyte (target)-subgroups / variants from each other and / or from the base analyte.

[0061] In some embodiments, at least one analyte subgroup / variant with at least five (5) sets is contacted with a subgroup-specific probe that differs in the nucleotide sequence of the identifier element (T) from the analyte-specific probes of another set of analyte-specific probes.

[0062] A "decoding oligonucleotide set" refers to multiple decoding oligonucleotides specific to a particular unique identifier required to achieve encoding independent 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.

[0063] In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides can be converted into a "code word." For example, the code words can be "101" and "110" for the analytes, with a value of 1 representing binding and a value of 0 representing no binding. The code words can also have longer lengths in other embodiments (see FIG. 13). The code words can be directly related to the specific unique identifier sequence of the analyte-specific probe. Thus, different analyte-specific probes can match a particular code word, which can then be used to identify different analytes of the analyte-specific probes based on the binding pattern of the decoding oligonucleotides. However, if there is no apparent binding, the code word is "000" in this example.

[0064] In some embodiments, the values ​​in each code word may also be assigned in different ways. For example, a value of 0 may represent binding, while a value of 1 represents no binding. Similarly, a value of 1 may represent binding of the secondary nucleic acid probe to one type of signaling entity, while a value of 0 may represent binding of the secondary nucleic acid probe to another type of distinguishable signaling entity. These signaling entities may be distinguished, for example, via different fluorescent colors. In some cases, the values ​​in a code word need not be limited to 0 and 1. Values ​​may also be drawn from a larger alphabet, such as a system of triplets (e.g., 0, 1, and 2) or quadruplets (e.g., 0, 1, 2, and 3). Each different value may be represented, for example, by a different distinguishable signaling entity, including (in some cases) one value that may be represented by the absence of signal.

[0065] The code words for each analyte can be assigned sequentially or randomly. For example, a first analyte can be assigned 101, while a second nucleic acid target can be assigned 110. Furthermore, in some embodiments, code words can be assigned using an error detection or correction system, such as a Hamming system, a Golay code, or an extended Hamming system (or a SECDED system, i.e., single error correction, double error detection). Generally speaking, such systems can be used to identify where an error occurred, and in some cases, such systems can also be used to correct the error and determine what the correct code word should be. For example, a code word such as 001 can be detected as invalid and corrected using such a system relative to 101 if, for example, 001 has not been previously assigned to a different target sequence. A variety of different error correction codes can be used, many of which have been previously developed for use in the computer industry; however, such error correction systems have not generally been used in biological systems. Further examples of such error correction codes are discussed in more detail below.

[0066] "Essentially complementary," when referring to two nucleotide sequences, means that both sequences can specifically hybridize to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule in which the sense and antisense strands are linked to each other via hydrogen bonds (Watson and Crick base pairing). "Essentially complementary" encompasses not only perfect base pairing throughout the entire strand, i.e., perfect complementary sequences, but also complementary sequences that are not perfect but still have the ability to hybridize to each other under stringent conditions. It is well-recognized among experts that "essentially complementary" sequences have at least 88% sequence identity to a completely or perfectly complementary sequence.

[0067] "Percent sequence identity" or "percent identity" similarly refers to the alignment of a described or claimed sequence ("reference sequence") with the sequence to be compared ("comparison sequence"), followed by comparison of the sequence to the claimed or described sequence. 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 sequence and the comparison sequence over the length of the alignment between the reference sequence and the comparison sequence; (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 begin 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 generated in the reference sequence also counted as bases or amino acids.

[0068] If there is an alignment between the comparison sequence and the reference sequence in which the percent identity as calculated above is about equal to or greater than the specified minimum percent identity, then the comparison sequence has the specified minimum percent identity to the reference sequence, even though there may be an alignment in which the percent identity as calculated herein above is less than the specified percent identity.

[0069] In an "incubation" step as understood herein, the respective moieties or entities, such as probes or oligonucleotides, are brought into contact with each other under conditions well known to those skilled in the art that allow for a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions, etc. Such a step may therefore preferably be carried out in a liquid environment, such as a buffer system well known in the art.

[0070] The "removing" step according to the present disclosure may involve washing away the moiety or object to be removed, such as a probe or oligonucleotide, under certain conditions, such as pH, temperature, salt conditions, etc., as known in the art.

[0071] It is understood that in embodiments of the methods according to the present disclosure, multiple analytes can be encoded 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. This embodiment also requires the use of different sets of decoding oligonucleotides in the methods according to the present disclosure.

[0072] The decoding oligonucleotides of a particular set are different from the decoding oligonucleotides of another set, meaning that the decoding oligonucleotides of set 1 bind to the analyte-specific probes of said set 1 of analyte-specific probes, the decoding oligonucleotides of set 2 bind to the analyte-specific probes of said set 2 of analyte-specific probes, the decoding oligonucleotides of set 3 bind to the analyte-specific probes of said set 3 of analyte-specific probes, etc.

[0073] In this embodiment, if multiple analytes are to be encoded in parallel, different sets of analyte-specific probes can be provided as premixes of different sets of analyte-specific probes and / or different sets of decoding oligonucleotides can be provided as premixes of different sets of decoding oligonucleotides. Each mixture can be contained in a single vial. Alternatively, different sets of analyte-specific probes and / or different sets of decoding oligonucleotides can be provided in separate stages.

[0074] A "kit" is a combination of individual elements useful for carrying out the uses and / or methods of the present disclosure, the elements being optimized for use together in the method. The kit may also contain additional reagents, chemicals, buffers, reaction vials, etc. that may be useful for carrying out the methods according to the present disclosure. Such kits integrate all essential elements needed to carry out the methods according to the present disclosure, thus minimizing the risk of error. Thus, such kits also enable semi-skilled laboratory staff to carry out the methods according to the present disclosure.

[0075] The terms "quencher" or "quencher dye" or "quencher molecule" refer to a dye or equivalent molecule, such as the nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which can reduce the fluorescence of a fluorescent reporter dye or donor dye. A quencher dye can be a fluorescent dye or a non-fluorescent dye. When a quencher is a fluorescent dye, its fluorescence wavelength is generally substantially different from that of the reporter dye, and quencher fluorescence is typically not monitored during an assay. Some embodiments of the present disclosure disclose signal oligonucleotides comprising a quencher and / or a quencher combined with a signal element (see FIG. 14), such that the signal oligonucleotide is not detectable during imaging.

[0076] In one embodiment of the present disclosure, sample is biological sample, preferably comprises biological tissue, more preferably comprises biological cell.Biological sample can be derived from organ, organoid, cell culture, stem cell, cell suspension, primary cell, virus, bacteria or fungus infected sample, eukaryotic or prokaryotic sample, smear, disease sample, tissue section.

[0077] The present methods are particularly qualified for encoding, identifying, detecting, counting, or quantifying analytes or single analyte molecules in biological samples, i.e., samples containing nucleic acids or proteins as said analytes, etc. It is understood that the biological sample may be in the form as it is in its native environment (i.e., liquid, semi-liquid, solid, etc.) or in the form as it is processed, for example, as a dry film on a device surface that can be re-liquefied before the present methods are performed.

[0078] In another embodiment of the present disclosure, the biological tissue and / or biological cells are fixed prior to step (2). For example, in some embodiments, cells and / or tissues are fixed prior to introducing the probe to preserve the location of analytes, such as nucleic acids, within the cells. Techniques for fixing cells are known to those skilled in the art. By way of non-limiting example, cells may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, and acetic acid. In one embodiment, cells may be fixed using Hepes-glutamic acid buffer-mediated organic solvent (HOPE).

[0079] This approach has the advantage that the analyte to be encoded, e.g., a nucleic acid or protein, is immobilized and cannot escape, thereby preparing the analyte for better detection or encoding by the methods of the present disclosure.

[0080] In yet a further embodiment 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 analytes to be encoded.

[0081] By this means, the method becomes even more robust and reliable, as the signal strength obtained at the end of the method or cycle, respectively, is enhanced. It is understood that the individual probes of a set, while binding to the same analyte, differ in their binding position or site at or on the analyte. 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 or at different positions, which may or may not overlap.

[0082] In an advantageous embodiment, the present disclosure relates to a kit for multi-analyte encoding, comprising: (A) at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and if the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes, and optionally also at least one subgroup-specific probe set, which specifically interact with different substructures of the same analyte, to distinguish targets (analytes / variants) with shared and exclusive portions of the analyte, and each analyte-specific probe (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; at least twenty (20) different sets of analyte-specific probes; (B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; one set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in the identifier connector element (t); at least one set of decoding oligonucleotides per analyte; (C) a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide; (bb) a signal element; a set of signal oligonucleotides comprising: Includes.

[0083] A multiplex method or assay, according to the present disclosure, allows for the simultaneous measurement of multiple analytes, which can be used to determine the presence or absence of multiple predetermined (known) analytes, such as nucleic acid target sequences, in a sample. An analyte may be "predetermined" in that its sequence is known to design a probe that binds to that target.

[0084] In some advantageous embodiments, in accordance with the present disclosure, at least 20, particularly at least 25, particularly at least 30 different analytes are detected and / or quantified in parallel in a sample. There may be, for example, at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable analyte-specific probes applied to the sample, e.g., simultaneously or sequentially.

[0085] In some advantageous embodiments, multiplex twenty (20) or more different sets of analyte-specific probes to encode at least 20 different analytes or more are required, particularly more than 50, more than 100, or more than 200. In the multiplexed methods of the present disclosure, particularly at least 20 different analyte groups (e.g., mRNA molecules) or tags are targeted.

[0086] In an advantageous embodiment, the kit comprises at least two different sets of analyte-specific probes for each analyte, The analyte-specific probes contained in these different sets interact with the same analyte, but specifically with different substructures of the same analyte, the analyte-specific probes of the first set of analyte-specific probes interact with substructures contained in all variations of the analyte; the analyte-specific probes of the second set of analyte-specific probes interact with a substructure contained only in a specific variant of the analyte (subgroup-specific probe set); the analyte-specific probes of the first set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); the analyte-specific probes of the second set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); The identifier elements (T) of the analyte-specific probes of the first set of analyte-specific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes differ for binding to different decoding oligonucleotides and / or non-signal decoding oligonucleotides.

[0087] In some advantageous embodiments, at least four rounds are performed to retrieve information about the identity of the analyte, and multiplexed readings increase the accuracy of identification and avoid false positives. Unique tags can be identified by a variety of techniques, including, for example, hybridization with directly or indirectly labeled probes, or by sequencing (by synthesis, ligation). In particular, the identity of the tag can be coded by one single signal (binary code), two or more signals, which can be fluorescent labels (e.g., linked to oligonucleotides).

[0088] In some advantageous embodiments, according to the present disclosure, the kit does not include a set of analyte-specific probes as defined under item A).

[0089] Preferably, when the analytes in the kit or method according to the present disclosure are nucleic acids, each set of analyte-specific probes comprises at least five (5) analyte-specific probes, particularly at least ten (10), particularly at least fifteen (15), particularly at least twenty (20) analyte-specific probes that specifically interact with different substructures of the same analyte. Nucleic acid analytes include specific DNA molecules, such as genomic DNA, nuclear DNA, mitochondrial DNA, viral DNA, bacterial DNA, extracellular or intracellular DNA, etc., and specific mRNA molecules, such as hnRNA, miRNA, viral RNA, bacterial RNA, extracellular or intracellular RNA, etc.

[0090] Preferably, when the analyte in the kit or method according to the present disclosure is a peptide, polypeptide or protein, each set of analyte-specific probes comprises at least two (2) analyte-specific probes, particularly at least three (3) analyte-specific probes, in particular at least four (4) analyte-specific probes that specifically interact with different substructures of the same analyte.

[0091] In some advantageous embodiments, according to the present disclosure, the kit comprises at least two different sets of signal oligonucleotides, the signal oligonucleotides in each set comprising different signal elements and comprising different connector elements (C).

[0092] In particular, the kit may comprise at least two different sets of decoding oligonucleotides per analyte, the decoding oligonucleotides contained in these sets comprising the same identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets per analyte differ in their translator elements (c) comprising nucleotide sequences that allow specific hybridization of the signal oligonucleotides.

[0093] In some embodiments, the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides contained in these sets comprise the same identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets for at least one analyte differ in their translator element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0094] In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte containing different translator elements (c) corresponds to the number of different sets of signal oligonucleotides containing different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interact with the same identifier element (T) that is unique for a particular analyte. In particular, all sets of decoding oligonucleotides for different analytes may contain the same type of translator element (c).

[0095] In another aspect, the present disclosure is generally directed to a method comprising exposing a sample to a plurality of analyte-specific probes; determining, for each of the analyte-specific probes, the binding of the analyte-specific probes in the sample; generating a code word based on the binding of the analyte-specific probe, a decoding oligonucleotide, and a signal oligonucleotide; and matching the code word to a valid code word for at least a portion of the code word. In certain embodiments, this pattern of binding or hybridization of the analyte-specific probe, the decoding oligonucleotide, and the signal oligonucleotide can be converted into a "code word." For example, the code words can be "101" and "110" for a first analyte and a second analyte, respectively, where a value of 1 represents binding and a value of 0 represents no binding of the decoding oligonucleotide and / or binding of the signal oligonucleotide with no signal element and / or an inactivated signal element. Thus, the analyte in the detection round / cycle is not detectable during imaging.

[0096] To generate such zeros (0) in the code words for each analyte, the kit includes: (D) at least one set of non-signal decoding oligonucleotides for binding to the specific identifier element (T) of the analyte-specific probe; wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T); Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0097] To generate such zeros (0) in the code words for each analyte, the kit includes: (D) at least one set of non-signal decoding oligonucleotides for binding to the specific identifier element (T) of the analyte-specific probe; wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T), Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence, and a translator element that does not interact / bind with the signal oligonucleotide due to an unstable binding sequence and / or because the translator element shorts (c), which comprises a nucleotide sequence that enables specific hybridization of the signal oligonucleotide.

[0098] In some advantageous embodiments, the kit comprises: (D) at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of the analyte-specific probes each set of non-signal decoding oligonucleotides interacts with a different identifier element (T); Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0099] In some advantageous embodiments, the different sets of non-signal-decoding oligonucleotides may be included in a premix of the different sets of non-signal-decoding oligonucleotides or may be present separately.

[0100] Furthermore, in some advantageous embodiments, the kit comprises: (E) Set of non-signal oligonucleotides and each non-signal oligonucleotide may comprise: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the translator element (c); (bb) A quencher (Q), a signal element and a quencher (Q), or no signal element.

[0101] In some advantageous embodiments, the kit comprises: (E) At least two sets of non-signal oligonucleotides and each non-signal oligonucleotide comprises (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the translator element (c); (bb) A quencher (Q), a signal element and a quencher (Q), or no signal element.

[0102] In some advantageous embodiments, the different sets of non-signal oligonucleotides can be included in a premix of the different sets of non-signal oligonucleotides or can be present separately.

[0103] Furthermore, in some embodiments, the decoding oligonucleotides in a particular set of decoding oligonucleotides interact with the same identifier element (T) that is unique for a particular analyte.

[0104] In some advantageous embodiments, the different sets of decoding oligonucleotides may be included in a premix of different sets of decoding oligonucleotides or may be present separately. In some advantageous embodiments, the different sets of analyte-specific probes may be included in a premix of different sets of analyte-specific probes or may be present separately. In some advantageous embodiments, the different sets of signal oligonucleotides may be included in a premix of different sets of signal oligonucleotides or may be present separately.

[0105] In some advantageous embodiments, a mixture of decoding oligonucleotides and / or multi-decoders that specifically hybridize to the unique identifier sequences of the probe sets is provided. In some embodiments, the decoding oligonucleotides contain at least two sequence elements: a first element complementary to the unique identifier sequence of the corresponding probe set and a second sequence element (translator element) that provides a sequence for specific hybridization of a signal oligonucleotide, where the translator element determines the type of signal generated by the decoding oligonucleotide. In some embodiments, a multi-decoder is used that contains at least three sequence elements: a first element complementary to the unique identifier sequence of the corresponding probe set and at least a further sequence element (translator element) that provides a sequence for specific hybridization of at least two different signal oligonucleotides. The translator element determines the type of signal generated by the multi-decoder. Different possible structures of the multi-decoder can be seen in Figure 17. Because the multi-decoder recruits a complete signal oligonucleotide per translator element, the intensity of the signal in each channel is no lower than the intensity of the signal associated with the decoding oligonucleotide.

[0106] The use of multiple decoders further increases the efficiency of the encoding scheme. Figure 17 shows a possible encoding scheme using multiple decoders based on the same conditions as used for the example with a decoding oligonucleotide having two sequence elements. It can be clearly seen that an encoding scheme based on multiple decoders can produce a higher Hamming distance using the same number of rounds and the same number of different signal oligonucleotides used in the example of Figure 5.

[0107] As mentioned above, the analyte to be encoded can be a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA, a peptide, a polypeptide, a protein and / or mixtures thereof.

[0108] In some advantageous embodiments, the binding element (S) comprises an amino acid sequence that allows specific binding to the analyte to be encoded. The binding element (S) may comprise an affinity portion from an affinity substance, or a portion that is an affinity substance in its entirety, selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions, and combinations thereof.

[0109] In some advantageous embodiments, the binding element (S) may comprise or is an antibody or antibody fragment selected from the group consisting of a Fab, scFv; single domain or fragment thereof, bis-scFv, F(ab)2, F(ab)3, minibody, diabody, triabody, tetrabody and tandab.

[0110] The present disclosure particularly relates to a multiplex method for detecting different analytes in a sample by sequential signal encoding of the analytes, comprising the following steps: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes to encode at least twenty (20) different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and where the analytes are nucleic acids, each set of analyte-specific probes includes at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, and each analyte-specific probe is (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs from an analyte-specific probe of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) that is unique to said analyte; (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide is (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; a set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in a first connect element (t); (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide; (bb) a signal element; and (D) detecting a signal produced by the signal element; (E) selectively removing decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining specific binding of the analyte-specific probes to the analytes to be encoded; (F) performing at least three (3) additional cycles comprising steps B) through E) to generate a coding scheme with a code word for each analyte, in particular the last cycle may stop at step (D); Includes:

[0111] As mentioned above, the method according to the present disclosure involves selectively removing decoding oligonucleotides and signal oligonucleotides from a sample, thereby essentially maintaining specific binding of the analyte-specific probe to the analyte to be encoded. In particular, all steps are performed sequentially. However, some steps, in particular contacting steps A) to C), especially B) and C), can be performed simultaneously.

[0112] This establishes the need for another round / cycle of binding of additional decoding oligonucleotides to the same analyte-specific probe, thus ultimately resulting in a code or encoding scheme containing multiple signals. This step is achieved by applying conditions and factors well known to those skilled in the art, such as pH, temperature, salt conditions, oligonucleotide concentration, polymer, etc.

[0113] In another embodiment of the present disclosure, the method may comprise repeating steps (B) to (E) at least three times to generate a coding scheme, by which codes of four signals are determined for four cycles / rounds performed by the user, where "n" is an integer representing the number of rounds. The coding capacity of the method according to the present disclosure is increased herein depending on the nature of the analyte and the needs of the operator. In one embodiment of the present disclosure, the coding scheme is predetermined and assigned to the analyte to be coded.

[0114] However, this approach allows for precise experimental setup by providing the appropriate sequential order of the decoding and signal oligonucleotides used, thus enabling accurate assignment of specific analytes to each encoding scheme. The decoding oligonucleotide used in the iterative steps (B)-(D2) can contain a translator element (c2) that is identical to the translator element (c1) of the decoding oligonucleotide used in the previous steps (B)-(E). In another embodiment of the present disclosure, a decoding oligonucleotide is used in the iterative steps (B)-(E) that contains a translator element (c2) that is different from the translator element (c1) of the decoding oligonucleotide used in the previous steps (B)-(E). It is understood that the decoding element may or may not change from round to round, i.e., in the second round (B)-(E) it contains translator element c2, in the third round (B)-(E) it contains translator element c3, in the fourth round (B)-(E) it contains translator element c4, etc., where "n" is an integer representing the number of rounds.

[0115] The signal oligonucleotide used in each iteration step (B)-(E) can contain a signal element that is identical to the signal element of the decoding oligonucleotide used in the previous step (B)-(E). In further embodiments of the present disclosure, a signal oligonucleotide is used in each iteration step (B)-(E) that contains a signal element that is different from the signal element of the decoding oligonucleotide used in the previous step (B)-(E). In some embodiments, a non-signal oligonucleotide and / or a non-signal decoding oligonucleotide for each analyte is used, resulting in a value of 0 in the code word for this cycle / position. In some embodiments, in each iteration cycle, a decoding oligonucleotide for each analyte does not contact the sample, also resulting in a value of 0 in the code word for this cycle / position.

[0116] By this means, the same or a different signal is provided in each round, resulting in a coding scheme characterized by a signal sequence consisting of many different signals. This means allows the generation of a unique code or code word that is different from all other code words in the coding scheme. In another embodiment of the present disclosure, 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 analyte to be coded.

[0117] In another embodiment of the present disclosure, subgroups (variants) of the same type of analyte may be detected by performing step G) to detect the presence or absence of exclusive elements.

[0118] In an advantageous embodiment, the sample is contacted with at least two different sets of analyte-specific probes per analyte; The analyte-specific probes contained in these different sets interact with the same analyte, but specifically with different substructures of the same analyte, the analyte-specific probes of the first set of analyte-specific probes interact with substructures contained in all variations of the analyte; Analyte-specific probes of a second set of analyte-specific probes (subgroup-specific probe set) interact with substructures contained only in specific variants of the analyte; the analyte-specific probes of the first set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); the analyte-specific probes of the second set of analyte-specific probes contain the same identifier element (T) that contains a nucleotide sequence that is unique to the analyte to be encoded (the unique identifier sequence); The identifier elements (T) of the analyte-specific probes of the first set of analyte-specific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes differ with respect to binding to different decoding oligonucleotides and / or non-signal decoding oligonucleotides.

[0119] In some advantageous embodiments, all steps are automated, particularly steps B) through F) using a robotic system and / or optical multiplexing system according to the present disclosure. In some examples, these steps may be performed in a fluidic system.

[0120] As described above, the method according to the present disclosure generates a coding scheme with a code word for each analyte. Thus, each analyte can be associated with a specific code word, the code word including several positions, each position corresponding to one cycle, generating multiple distinct coding schemes with multiple code words. In particular, the coding schemes can be predetermined and assigned to the analytes to be coded.

[0121] In some advantageous embodiments, the code word obtained for each analyte in the cycle performed comprises a detected signal and additionally at least one element corresponding to no detected signal, such as 0, 1 or 0, 1, 2, etc. (see also Figures 13 and 14). In particular, when using a no-signal probe according to Figure 14, numbers 2-4, or a no-signal decoding oligonucleotide as shown in Figure 14, number 15, or when no decoding oligonucleotide is contacted with the corresponding identifier sequence contained in the analyte-specific probe that interacts with the corresponding analyte in the sample in one cycle, no signal is detected for at least one analyte in at least one cycle. In this cycle, the position has the value zero (0).

[0122] In some advantageous embodiments, for at least one individual analyte, the code word position is zero (0). In particular, code word zero (0) is generated without using a decoding oligonucleotide having an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte. As mentioned above, in some embodiments, if the code word position is zero (0) for at least one individual analyte in this cycle, then the corresponding decoding oligonucleotide having an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte is not used.

[0123] Furthermore, in some advantageous embodiments, the sample is contacted with at least two different sets of signal oligonucleotides, the signal oligonucleotides in each set comprising a different signal element and comprising a different connector element (C).

[0124] In a more particular embodiment, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte; the decoding oligonucleotides contained in these different sets contain the same identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; The different sets of decoding oligonucleotides for each analyte differ in the translator element (c), which contains a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0125] In a more particular embodiment, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte; the decoding oligonucleotides contained in these different sets contain the same identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; The different sets of decoding oligonucleotides for each analyte differ in a translator element (c) that contains a nucleotide sequence that allows specific hybridization of the signal oligonucleotide; Only one set of decoding oligonucleotides per analyte is used per cycle, and / or different sets of decoding oligonucleotides are used in different cycles in combination with a corresponding set of signal oligonucleotides in the same cycle, in particular Sets of decoding oligonucleotides and / or non-signal decoding oligonucleotides are reserved for the optional detection of subgroups of analytes.

[0126] In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte containing different translator elements (c) corresponds to the number of different sets of signal oligonucleotides containing different connector elements (C). All sets of decoding oligonucleotides for different analytes may contain the same type of translator element (c).

[0127] In some advantageous embodiments of the method according to the present disclosure, the sample is contacted with at least one set of non-signal decoding oligonucleotides for binding to specific identifier elements (T) of the analyte-specific probe, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T), and each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence that enables specific hybridization of the signal oligonucleotide.

[0128] As described above, the sample may be contacted with at least two (2) different sets of non-signal-decoding oligonucleotides for binding to at least two different identifier elements (T) of the analyte-specific probe, each set of non-signal-decoding oligonucleotides interacting with a different identifier element (T), each non-signal-decoding oligonucleotide comprising an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of a unique identifier sequence, and no translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide.

[0129] In some advantageous embodiments of the methods according to the present disclosure, the different sets of non-signal-decoding oligonucleotides may be included in a premix of the different sets of non-signal-decoding oligonucleotides or may be present separately.

[0130] Furthermore, in some advantageous embodiments of the methods according to the present disclosure, the sample is contacted with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the translator element (c); (bb) A quencher (Q), a signal element and a quencher (Q), or no signal element.

[0131] In a further embodiment, the sample is and contacting the nucleic acid sequence with at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the translator element (c); (bb) a quencher (Q), a signal element and a quencher (Q), or no signal element.

[0132] As noted above, the different sets of non-signal oligonucleotides can be included in a premix of the different sets of non-signal oligonucleotides or can be present separately.

[0133] In a further embodiment, the decoding oligonucleotides in a particular set of decoding oligonucleotides interact with the same identifier element (T) that is unique for a particular analyte.

[0134] As noted above, different sets of decoding oligonucleotides may be included in a premix of different sets of decoding oligonucleotides or may be present separately, different sets of analyte-specific probes may be included in a premix of different sets of analyte-specific probes or may be present separately, and different sets of signal oligonucleotides may be included in a premix of different sets of signal oligonucleotides or may be present separately.

[0135] In some advantageous embodiments of the method according to the present disclosure, the binding element (S) comprises a nucleic acid comprising a nucleotide sequence that allows specific binding to, preferably specific hybridization to, the analyte to be encoded.

[0136] In some advantageous embodiments of the method according to the present disclosure, after step A) and before step B), unbound analyte-specific probes may be removed, in particular by washing, and after further step B) and before step C), unbound decoding oligonucleotides may be removed, in particular by further washing, and after step C) and before step D), unbound signal oligonucleotides may be removed, in particular by washing.

[0137] In some advantageous embodiments of the methods according to the present disclosure, analyte-specific probes may be incubated with the sample, thereby allowing specific binding of the analyte-specific probe to the analyte to be encoded; decoding oligonucleotides may be incubated with the sample, thereby allowing specific hybridization of the decoding oligonucleotide to the identifier element (T) of each analyte-specific probe; and signal oligonucleotides may be incubated with the sample, thereby allowing specific hybridization of the signal oligonucleotide to the translator element (T) of each decoding oligonucleotide.

[0138] As mentioned above, the analyte to be encoded can be a nucleic acid, preferably DNA, PNA, RNA, particularly mRNA, a peptide, a polypeptide, a protein, or a combination thereof. Accordingly, the binding element (S) can comprise an amino acid sequence that allows specific binding to the analyte to be encoded. Examples of binding elements (S) include affinity moieties from affinity substances or their entirety selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenesis or pathogenicity factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions, and combinations thereof. In particular, the binding element (S) can be an antibody or antibody fragment selected from the group consisting of Fab, scFv; single domains or fragments thereof, bis-scFv, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and tandabs.

[0139] By this means, the method is further extended to the extent that the coded analyte can be detected by any means adapted to visualize the signal element. Examples of detectable physical properties include, for example, light, chemical reaction, molecular weight, radioactivity, etc.

[0140] In some advantageous embodiments, the signal generated by the signal element, and thus in particular the binding of the signal oligonucleotide to the decoding oligonucleotide that interacts with the corresponding analyte probe that binds to the individual analyte, is (a) imaging at least a portion of the sample; and / or (b) using optical imaging techniques; and / or (c) using fluorescent imaging techniques; and / or (d) multicolor fluorescence imaging techniques; and / or (e) Super-resolution fluorescence imaging technology is determined by

[0141] The kits and methods according to the present disclosure may be ideally used for in vitro methods for the diagnosis of diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases.

[0142] Furthermore, the kits and methods according to the present disclosure may also be ideally used for in vitro methods for the diagnosis of diseases in plants selected from the group comprising diseases caused by biotic stress, preferably of infectious and / or parasitic origin, or diseases caused by abiotic stress, preferably caused by nutritional disorders and / or unfavorable environment.

[0143] Furthermore, the kits and methods according to the present disclosure may also be ideally used for in vitro methods for screening, identifying and / or testing substances and / or drugs, including: (a) contacting a test sample containing a sample with a substance and / or drug; (b) detecting different analytes in a sample by sequential signal encoding of said analytes in a method according to the present disclosure; Includes.

[0144] An optical multiplexing system suitable for the method according to the present disclosure comprises at least: a reaction vessel for containing a kit or part of a kit according to the present disclosure; - with a detection unit including a microscope, in particular a fluorescence microscope; - With camera; a liquid handling device; Includes.

[0145] In some embodiments, the optical multiplexing system may further include heating and cooling equipment and / or a robotic system.

[0146] Some examples of suitable construction techniques or materials that may be adapted for use in connection with the present disclosure may be found, for example, in commonly assigned U.S. Pat. No. 6,734,401, entitled "ENHANCED SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS" (Bedingham et al.) and U.S. Patent Application Publication No. 2002 / 0064885, entitled "SAMPLE PROCESSING DEVICES." Other usable device configurations are described, for example, in U.S. Provisional Application No. 60 / 214,508, filed June 28, 2000, entitled "THERMAL PROCESSING DEVICES AND METHODS"; U.S. Provisional Application No. 60 / 214,642, filed June 28, 2000, entitled "SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS"; U.S. Provisional Application No. 60 / 237,072, filed October 2, 2000, entitled "SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS"; and U.S. Provisional Application No. 60 / 260,063, filed January 6, 2001, entitled "SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS." No. 60 / 284,637, filed April 18, 2001, entitled "ENHANCED SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS"; and U.S. Patent Application Publication No. 2002 / 0048533, entitled "SAMPLE PROCESSING DEVICES AND CARRIERS." Other possible device constructions can be found, for example, in U.S. Patent No. 6,627,159, entitled "CENTRIFUGAL FILLING OF SAMPLE PROCESSING DEVICES" (Bedingham et al.).

[0147] An optical multiplexing system according to the present disclosure may include multiple process chambers (e.g., reaction vessels), each for holding an individual sample, and one or more sets of probes, such as a set of analyte-specific probes, a set of decoding / non-signal oligonucleotides, and / or a set of signal / non-signal oligonucleotides. For example, the process chambers may be in a rotatable disk or in a movable well plate, such as a 96-well plate; motors for rotating the disk or moving the well plate may be included, and in particular the motors may be part of a robotic system.

[0148] The optical multiplexing system according to the present disclosure may further include at least one or more optical modules, and in particular, the system includes a housing having at least one or more positions adapted to receive the optical modules, each of the plurality of optical modules being removable from its position in the housing.

[0149] In some advantageous embodiments, an optical multiplexing system according to the present disclosure may include a detector and, in particular, an optical fiber bundle coupled to the multiple optical modules for carrying fluorescent light from the multiple optical modules to the detector.

[0150] In particular, the optical modules include optical channels, each optical module having a light source selected for excitation of a different one of the dyes and a lens for capturing the emitted fluorescent light, and the optical modules are optically configured to interrogate the fluorescent dyes at different wavelengths.

[0151] In further embodiments, the system may include a microfluidic cartridge (also referred to herein as a microfluidic device) having at least one flow-through. The optical multiplexing system includes a fluorescent imaging system. Additional features of the system may be a temperature measurement and / or control system. In some embodiments, the system includes a pressure measurement and control system for applying a variable pneumatic pressure, e.g., to the microfluidic cartridge. The optical multiplexing system may include a storage device, e.g., a well plate, for holding multiple reagents. Additionally, the optical multiplexing system may include a liquid handling system, particularly including at least one robotic pipettor, e.g., for aspirating, mixing, and dispensing reagent mixtures, e.g., into the microfluidic cartridge and / or into reaction vessels. Additionally, the system may include means for data storage, processing, and output; and a system controller, particularly for coordinating the various devices and functions.

[0152] In some embodiments, methods according to the present disclosure involve encoding a nucleic acid analyte, such as an mRNA, for example an mRNA that encodes a particular protein.

[0153] In some advantageous embodiments, the methods described herein are used for the specific detection of many different analytes in parallel. This technique makes it possible to distinguish between a greater number of analytes than the number of different signals available. The process involves at least four 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 a detectable signal, so-called "decoding" oligonucleotides are introduced. The decoding oligonucleotides transcribe the information of the analyte-specific probe set to the signal oligonucleotides.

[0154] In certain embodiments, the method comprises the steps of: 1. providing one or more analyte-specific probe sets, where the analyte-specific probe set is composed of one or more different probes, each differing in a binding moiety that specifically interacts with the analyte, and where all probes of a single probe set are tethered to a sequence element (unique identifier) ​​that is unique for the single probe set, allowing specific hybridization of decoding oligonucleotides; 2. specific binding of the probe sets to their target binding sites of the analyte; 3. removing unbound probes (e.g., by a wash step); and 4. providing a mixture of decoding oligonucleotides that specifically hybridize to the unique identifier sequences of the probe sets, where the decoding oligonucleotides have at least two sequence elements, a first element complementary to the unique identifier sequence of the corresponding probe set and a sequence for specific hybridization of a signal oligonucleotide. 4. Specific hybridization of the decoding oligonucleotide to the unique identifier sequence provided by the bound probe set, the translator element determining the type of signal mobilized to the decoding oligonucleotide; 5. Specific hybridization of the decoding oligonucleotide to the unique identifier sequence provided by the bound probe set; 6. Removal of unbound decoding oligonucleotides (e.g., by a washing step); 7. Providing a mixture of signal oligonucleotides consisting of a nucleic acid sequence that specifically hybridizes to the translator element of one of the decoding oligonucleotides used in the previous hybridization step with a detectable signal; 8. Specific hybridization of the signal oligonucleotide; 9. Removal of unbound signal oligonucleotide; 10. Detection of the signal; 11. Selective release of the decoding oligonucleotide and the signal oligonucleotide, while binding of the specific probe set to the analyte is largely or completely unaffected; and 12.and removing (e.g., by a washing step) the released decoding and signal oligonucleotides while leaving the binding of the specific probe sets to the analytes largely or completely unaffected. Steps 4 through 12 are repeated at least three times until a sufficient number of signals are detected to generate a coding scheme for each different analyte of interest.

[0155] It is to be understood that the aforementioned characteristics and those to be described hereinafter may not only be used in the combinations shown in the individual cases, but may also be used in other combinations or in isolated form without departing from the scope of the present disclosure.

[0156] The present disclosure will now be further described by embodiments that provide further characteristics, features, and advantages of the present disclosure. The embodiments are purely exemplary in nature and do not limit the scope or area of ​​the present disclosure. The characteristics described in specific embodiments are general characteristics of the present disclosure that are not only applicable to the specific embodiment, but also applicable in isolation in the context of any embodiment of the present disclosure.

[0157] The method disclosed herein is used for the specific detection of many different analytes in parallel. This technique allows for the discrimination of a greater number of analytes than the number of different 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.

[0158] Methods and Examples In a variant application, 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 comprises an analyte-specific probe (1) that comprises 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 identifier sequence) unique to said set of analyte-specific probes.

[0159] In an advantageous embodiment of the present disclosure, the analyte / target is a nucleic acid, e.g., RNA, and two probe sets (Figure 15) containing oligonucleotides that are partially or completely complementary to unique regions of a single nucleic acid sequence target. Each nucleic acid sequence-specific oligonucleotide probe set (1 and 1', 2 and 2') contains two sets of analyte-specific probes, both containing a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, each with a different identifier element (T) containing a nucleotide sequence (unique identifier sequence) that is unique to the set of analyte-specific probes. The two sets are used to decode the analyte (1, 2) and detect the presence / absence of an exclusive element that distinguishes between analyte subgroups (1' and 2').

[0160] In a further application variant, the analyte or target is a protein and the probe set comprises one or more proteins, e.g., antibodies (Figure 2). A protein-specific probe set comprises 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 specifically interacts with the target protein to be detected.

[0161] In a further 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.

[0162] A general embodiment of the method may be as follows: Step 1: Application of at least 20 analyte- or target-specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides having 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 respective 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 contains an identifier element or unique identifier sequence (T). Step 2: Hybridization of the probe set. The probe set is hybridized to the target nucleic acid sequence under conditions that allow for specific hybridization. After incubation, the probes are hybridized to their corresponding target sequences, providing the identifier element (T) for the next step. Step 3: Removal of unbound probe After hybridization, unbound oligonucleotides are removed, for example, by a washing step. 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). Step 5: Hybridization of the Decoding Oligonucleotide. The unique identifier sequence of the probe (T) and the decoding oligonucleotide are hybridized via their complementary first sequence element (t). After incubation, the decoding oligonucleotide provides the translator sequence element (c) for the subsequent hybridization step. Step 6: Removal of excess decoding oligonucleotides After hybridization, unbound decoding oligonucleotides are removed, for example, by a washing step. 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). Step 8: Hybridization of the signal oligonucleotide. The signal oligonucleotide is hybridized to the translator element (c) of the decoding oligonucleotide via the complementary sequence connector element (C). After incubation, the signal oligonucleotides hybridize to their corresponding decoding oligonucleotides, providing a detectable signal (F). Step 9: Removal of excess signal oligonucleotides After hybridization, unbound signal oligonucleotides are removed, for example, by a washing step. Step 10: Signal detection The signal provided by the signal oligonucleotide is detected.

[0163] The following steps (steps 11 and 12) are not necessary for the final detection round.

[0164] Step 11: Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotide is abolished. Destabilization can be achieved through different mechanisms well known to the skilled artisan, such as increased temperature, denaturing agents, etc. The target or analyte-specific probe is not affected by this step. Step 12: Removal of Modified Decoding Oligonucleotides. The modified decoding and signal oligonucleotides are removed (e.g., by a washing step), leaving 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 at least four times until the planned encoding scheme is completed.

[0165] In some advantageous embodiments, in step 13, further cycles of steps 4 to 10 are performed to read out subgroup / variant-specific signals.

[0166] Another embodiment of the general method of the present disclosure using multiple decoders may be as follows (FIG. 16): Step 1: Target Nucleic Acids: In this example, three different target nucleic acids (A), (B) and (C) must be detected and distinguished by using only two different types of signal oligonucleotides. Before the experiment begins, a specific encoding scheme is established. In this example, the three different nucleic acid sequences are encoded by three different signal types (1), (2) and (1 / 2) and three rounds of detection with a resulting Hamming distance of 3 to allow for error detection. The planned code words are: Sequence A: (1)-(1)-(2) Arrangement B: (2)-(2)-(1 / 2) Sequence C: (1 / 2)-(1 / 2)-(1). Step 2: Hybridization of probe sets: For each target nucleic acid, its own probe set is applied and specifically hybridizes to the corresponding 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 (A) is labeled with (T1), sequence (B) with (T2), and sequence (C) with (T3). The illustration in Figure 16 summarizes steps 1-3 of Figure 3. Step 3: Hybridization of Decoding Oligonucleotides and Multidecoders: For each unique identifier present, a specific decoding oligonucleotide or multidecoder is applied, specifically hybridizing by its first sequence element to the corresponding unique identifier sequence (here (t1) to (T1), (t2) to (T2), and (t3) to (T3)). Each decoding oligonucleotide or multidecoder provides one translator or two translator elements that determine the signal generated after hybridization of the signal oligonucleotide. Here, nucleic acid sequence (A) is labeled with (c1), (B) is labeled with (c2), and (C) is labeled with both translator elements (c1) and (c2), resulting in signal (1 / 2). The illustration in Figure 16 summarizes steps 4-6 of Figure 3. Step 4: Hybridization of signal oligonucleotides: For each type of translator element, a signal oligonucleotide is applied that has a specific signal that is distinguishable from the signals of other signal oligonucleotides. This signal oligonucleotide can specifically hybridize to the corresponding translator element. The illustration in Figure 16 summarizes steps 7-9 in Figure 3. Step 5: Signal detection for encoding scheme: Different signals are detected. Note that in this example, nucleic acids (A), (B), and (C) can already be distinguished after the first round of detection. This contrasts with step 5 in Figure 5, which illustrates the additional signal types (1 / 2) that can be realized due to the multiple decoders. Although the nucleic acid sequences can already be distinguished, additional rounds contribute to the planned Hamming distance of 3. The illustration in Figure 16 corresponds to step 10 in Figure 3. Step 6: Selective Denaturation: The decoding (and signal) oligonucleotides and / or multidecoders 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. Step 7: Second round of detection: A next round of hybridization and detection is performed as described in steps 3-5. Note that in this new round, different decoding oligonucleotides and the mixture of multi-decoders are used. 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 multi-decoder for round 2 consists of sequence elements (t1), (c1), and (c2). Note that after two rounds, a Hamming distance of 2 is already given, which is the final result for the example in Figure 3 after three rounds. Step 8: Third round of detection: Again, new combinations of decoding oligonucleotides and / or multiple decoders are used, leading to new signal combinations. After signal detection, the resulting code words for the three different nucleic acid sequences are not only unique and therefore distinguishable, but also comprise a Hamming distance of 3 to the other code words. Due to the Hamming distance, an error in the detection of a signal (signal exchange) would result in no valid code word being generated, in contrast to the encoding scheme of Figure 3, and therefore can be detected and, due to the Hamming distance of 3, can also be corrected. In this way, three different nucleic acids can be distinguished in three detection rounds using two different signals, allowing error detection and correction.

[0167] Furthermore, in every round of detection, the type of signal provided by a particular unique identifier is controlled by the use of a specific 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.

[0168] 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.

[0169] Option 1: Simultaneous Hybridization. Instead of steps 4-9 of Figure 3, specific hybridization of decoding and signal oligonucleotides can also be performed simultaneously, resulting in the same result as shown in step 9 of Figure 3 after removal of excess decoding and signal oligonucleotides.

[0170] 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 its specific probe set already bound.

[0171] 1. Example for 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. It does not show a general concept of the encoding that can be achieved by this procedure. To illustrate the use of the process shown in Figure 3 for the generation of an encoding scheme, Figure 5 shows a general example for a multiple-round encoding experiment using three different nucleic acid sequences. In this example, the encoding scheme includes error detection.

[0172] Step 1: Target Nucleic Acids. In this example, three different target nucleic acids (A), (B), and (C) must be detected and distinguished by using only two different types of signals. Before the experiment begins, a specific encoding scheme is set up. In this example, 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: Sequence A: (1)-(2)-(2); Sequence B: (1)-(1)-(1); Sequence C: (2)-(1)-(2). Step 2: Hybridization of the probe sets. For each target nucleic acid, its own probe set is applied and specifically hybridizes to the corresponding 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 illustration summarizes steps 1-3 in Figure 3. Step 3: Hybridization of Decoding Oligonucleotides. For each unique identifier present, a specific decoding oligonucleotide is applied, specifically hybridizing to the corresponding unique identifier sequence by its first sequence element (here (t1) to (T1), (t2) to (T2), and (t3) to (T3)). Each decoding oligonucleotide provides a translator element that determines 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 illustration summarizes steps 4 to 6 of Figure 3. Step 4: Hybridization of the signal oligonucleotide. For each type of translator element, a signal oligonucleotide is applied that has a specific signal (2) that is distinguishable from the signals of other signal oligonucleotides. This signal oligonucleotide can specifically hybridize with the corresponding translator element. The illustration summarizes steps 7-9 of Figure 3. Step 5: Signal detection for the encoding 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 cycle of detection. This is due to the fact that the number of different nucleic acid sequences to be detected exceeds the number of different signals available. The illustration corresponds to step 10 in Figure 3. 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. Step 7: Second round of detection. A next round of hybridization and detection is performed as described in steps 3-5. In this new round, the mixture of different decoding oligonucleotides is again changed. For example, the decoding oligonucleotides of nucleic acid sequence (A) used in the first round are composed of sequence elements (t1) and (c1), while the new decoding oligonucleotides are composed of sequence elements (t1) and (c2). Note that all three sequences can be clearly distinguished due to the unique combination of first and second round signals. Step 8: Third round of detection. Again, a new combination of decoding oligonucleotides is used, which leads to a new signal combination. After signal detection, the resulting code words for the three different nucleic acid sequences are not only unique and therefore distinguishable, but also contain a Hamming distance of 2 from other code words. Due to the Hamming distance, an error in signal detection (signal exchange) will result in a valid code word not being generated and therefore detectable. In this way, three different nucleic acids can be distinguished in three detection rounds using two different signals, making error detection possible.

[0173] 2. Advantages over prior art Coding Strategies One particular advantage of the method according to the present disclosure compared to state-of-the-art methods is the use of decoding oligonucleotides that break the dependency between the target-specific probe and the signal oligonucleotide.

[0174] If two different molecular tags are used without separating the target-specific probes and signal generation, only two different signals can be generated for a particular 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 generate a coding scheme, either a change in the target-specific probe set after each round is required (SeqFISH) or multiple molecular tags must be present on the same probe set (as in merFISH or intronSeqFISH).

[0175] Following the method according to the present disclosure, different signals are achieved by using different decoding oligonucleotides reusing the same unique identifier (molecular tag) and a small number of different, mostly cost-intensive, signal oligonucleotides, which leads to several advantages in contrast to other methods. (1) The coding scheme is not determined by the target-specific probe set, as is the case for all other methods of the prior art. Here, the coding scheme is transcribed by the decoding oligonucleotide. This leads to much greater flexibility in the number of rounds and freedom in signal selection for codewords. Looking at prior art methods (e.g., merFISH or intronSeqFISH), the coding scheme (number, type, and sequence of detectable signals) for every target sequence is predetermined by the presence of different tag sequences in specific probe sets (4 out of 16 different tags per probe set for merFISH and 5 out of 60 different tags for intronFISH). To generate a sufficient number of different tags per probe set, the present method uses a fairly complex oligonucleotide design, with several tags present on one target-specific oligonucleotide. To change the coding scheme for a particular target nucleic acid, the specific probe set must be replaced. The method of the present disclosure describes the use of a single unique tag sequence (unique identifier) ​​per analyte, which can be reused in every detection round to generate new information. The coding scheme is determined by the order of the decoding oligonucleotides used in the detection rounds. Therefore, the coding scheme is not predetermined by specific probes (or unique tag sequences), but can be adjusted to different needs even during an experiment. This can be achieved by simply changing the decoding oligonucleotides used in the detection rounds 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 intronSeqFISH). Using the method of the present disclosure, the number of different signal oligonucleotides matches the number of different signals used. Thus, the number of signal oligonucleotides remains constant for the methods described herein and does not exceed the number of different signals, whereas in prior art methods it increases with the complexity of the encoding scheme (more detection rounds require more different signal oligonucleotides). As a result, the method described herein significantly reduces complexity (unintentional interactions of signal oligonucleotides with the environment or with each other) and dramatically reduces the cost of the assay, since the major cost factor is the signal oligonucleotides. (3) In prior art methods, the number of different signals generated by a target-specific probe set is limited by the number of different tag sequences that the probe set can provide. Since each additional tag sequence increases the overall size of the target-specific probe, there is a limit to the number of different tags that a single probe can provide. This limit is imposed by the size-dependent increase of several issues (unintentional 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 lead to a serious limitation in the number of different signals that a probe set can provide (four signals for merFISH and five signals for intronSeqFISH). This limit effectively affects the number of different code words that can be generated by a certain number of detection rounds. In the approach of the present disclosure, only one tag is required, which can be freely reused in all detection rounds. This allows for low oligonucleotide complexity / length and simultaneously the maximum possible encoding efficiency (number of colors). ラウンド数) The enormous difference in encoding power of our method compared to other methods is shown in Figures 1 and 5. Hence, in the disclosed approach, significantly fewer rounds of detection are required to generate the same amount of information. Fewer rounds of detection translate into lower costs, shorter experimental time, lower complexity, improved robustness and success rates, less data to collect and analyze, and more accurate results.

[0176] Codability All three methods compared in Table 1 below use specific probe sets that do not degenerate between different detection rounds. In the case of intronSeqFISH, four detection rounds are required to generate the pseudocolor of one coding round, so data are only given for rounds 4, 8, 12, 16, and 20. The merFISH method uses a fixed number of four signals, so data starts with the minimum possible number of rounds. After eight detection rounds, our method exceeds the maximum coding capacity achieved with 20 merFISH rounds (indicated by one asterisk), and after 12 detection rounds, it exceeds the maximum coding capacity of intronFISH (indicated by two asterisks). In the case of the method according to the present disclosure, the use of three different signals is assumed (as in intronSeqFISH). [Table 1] Table 1: Comparison of coding abilities

[0177] As shown in Figure 6, the number of code words for merFISH does not increase exponentially with the number of detection cycles, but each additional round reduces the effectiveness. In contrast, the number of code words for intronSeqFISH in the method according to the present disclosure increases exponentially. The slope of the curve for the proposed method is significantly higher than intronFISH, leading to over 10,000 times more code words available after 20 detection rounds.

[0178] Note that this maximum efficiency of encoding capacity is also achieved in the case of seqFISH, where specific probes are denatured after every detection round and a new set of probes specifically hybridizes 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, fairly harsh conditions must be used (high temperature, high concentration of denaturing agent, long incubation time), which significantly increases the chance of loss or damage of the analyte. (2) For each round of detection, every target nucleic acid sequence must use its own probe set. Therefore, the number of specific probes required for an experiment corresponds 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 in methods using only one specific hybridization event, resulting in a lower percentage of complete coding. (4) The time required for specific hybridization is significantly longer than for signal or decoding oligonucleotide hybridization (as can be seen in the methods section of the intronSeqFISH, merFISH, and seqFISH publications), dramatically increasing the time required to complete the experiment.

[0179] For these reasons, all other methods use a single specific hybridization event and accept the major downside of lower code complexity and therefore the need for more detection rounds and higher oligonucleotide design complexity.

[0180] The method according to the present disclosure combines all the advantages of methods that use only one specific hybridization event with the advantages of seqFISH (mainly complete flexibility regarding the encoding scheme) while eliminating the major problems of such methods.

[0181] Note that multiple code words generated after 20 rounds can also be used to introduce higher Hamming distances (differences) between different code words, allowing error detection and even error correction of 1, 2 or even more errors. Thus, even very high code powers are actually appropriate.

[0182] For the detection of subgroups / variants (e.g., mRNA splicing variants) within a group of analytes / targets that share a certain portion, two distinct code words can be used. However, signals are generated at the very same physical location, resulting in mixed reads for this naive approach. Using additional rounds to add information to the already established code circumvents this problem.

[0183] As mentioned above, the use of multiple decoders further increases the coding capacity of the coding scheme. Instead of being limited to having exactly the same number of different signal types as different signal oligonucleotides and corresponding translator elements, the use of multiple decoders increases the signal types that can be used to (N x (N + 1)) / 2, where N is the number of different signal oligonucleotides used. For the code used in Table 1 with three different signal oligonucleotides, this means that the following seven different signal types can be used: (S1), (S2), (S3), (S1 / S2), (S1 / S3), (S2 / S3), (S1 / S2 / S3). The effect on coding efficiency can be seen in Table 1b and Figure 18. [Table 2] Table 1b shows the coding capacity of the four methods.

[0184] All four methods compared in Table 1b use specific probe sets that do not degenerate between different detection rounds. For intronSeqFISH, four detection rounds are required to generate the pseudocolor of one encoding round, so data are only given for rounds 4, 8, 12, 16, and 20. The merFISH method uses a fixed number of four signals, so data begins with the minimum possible number of rounds. After four detection rounds, the method using the multi-decoder described herein exceeds the maximum encoding capacity reached with 20 merFISH rounds (indicated by one asterisk), after seven detection rounds it exceeds the maximum encoding capacity of intronFISH (indicated by two asterisks), and after 12 detection rounds it exceeds the maximum encoding capacity of the disclosed method (indicated by three asterisks). The use of three different signal oligonucleotides is envisioned (as in intronSeqFISH).

[0185] 3. The selective denaturation, oligonucleotide assembly and reuse of unique identifiers is surprisingly efficient. The key elements of the disclosed method are the sequential processes 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). Since the same unique identifier is reused in every detection cycle, all events from the first to the last detection cycle are interdependent. Furthermore, selective denaturation depends on two distinct events: the decoding oligonucleotide must be dissolved from the unique identifier with maximum efficiency, while the specific probe must remain hybridized with maximum efficiency.

[0186] Therefore, the efficiency E of the overall coding process can be stated by the following formula: E=B sp x(B de xB si xE de xS sp ) n E = total efficiency B sp = specific probe binding B de = Binding of decoding oligonucleotide B si = Binding of signal oligonucleotide E de = Removal of decoding oligonucleotides S sp = Stability of specific probes during removal process n = number of detection cycles

[0187] 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 here on the assumption that each step has the same efficiency. The total efficiency describes the fraction of the total signal that can be successfully decoded.

[0188] The overall efficiency of the present 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 efficiencies, 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 significantly 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 even based on the assumption of 100% signal detection and analysis efficiency. Due to the broad DNA melting curves of oligonucleotides with various sequences, the inventors assumed prior to the experiment that selective denaturation would function less efficiently than denaturation of decoding oligonucleotides and that 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 sequence-specific probes during selective denaturation.

[0189] Experimentally, the inventors 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 formula given above revealed an average efficiency of approximately 94.4% to 98%. These high efficiencies are quite surprising and cannot be easily predicted by those skilled in the art.

[0190] 4. Experimental Data background This experiment demonstrates the specific detection of 10-50 different mRNA species in parallel with single molecule resolution, based on five detection cycles, three distinct fluorescent signals, a coding scheme with no signal gaps, and a Hamming distance (error detection) of 2. This experiment tests the feasibility and functionality of the method according to the present disclosure.

[0191] 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. Signal oligonucleotide R:ST05 * O_Atto594 was ordered from biomers.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.

[0192] Experimental Overview The 50 different target-specific probe sets are divided into five groups. The names of the transcripts to be detected and the names of 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 decoding oligonucleotide names used in 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 3] JPEG0007828901000004.jpg71163Table 2: Experimental overview

[0193] Experimental Variants 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 decoding signal. [Table 4] Table 3: Experimental Variations

[0194] Experiment details A. Cell seeding and culture HeLa cells were grown to nearly 100% confluency in HeLa cell culture medium, which contained DMEM (Thermo Fisher, Cat.: 31885) with 10% FCS (Biochrom, Cat.: S0415), 1% penicillin-streptomycin (Sigma-Aldrich, Cat.: P0781), and 1% MEM non-essential amino acid solution (Thermo Fisher, Cat.: 11140035). After aspirating the cell culture medium, the cells were resuspended in PBS (1,424 g / L NaHPO4 in water). * 2H2O, 0.276g / l, NaH2PO4 * After a washing step with 2H2O, 8.19 g / l NaCl, pH 7.4, the cells were trypsinized by incubation with trypsin-EDTA solution (Sigma-Aldrich, Cat.: T3924) for 5 minutes at 37°C. The cells were then seeded into the wells of a μ-Slide 8 Well ibidiTreat (Ibidi, Cat.: 80826). The number of cells per well was adjusted to achieve approximately 50% confluency after cell attachment. The cells were incubated overnight with 200 μl of HeLa cell 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.: 0082.1) for 10 min at -20°C. C. Counterstaining with Sudan Black 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 at room temperature for 5 minutes. 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. The sm-wash buffer contained 30 mM Na citrate, 300 mM NaCl, pH 7, 10% formamide (Roth, Cat.: P040.1), and 5 mM vanadyl ribonucleoside conjugates (NEB, Cat.: 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 solution. The 2x concentrated hybridization buffer 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 / well of target probe wash buffer for 10 minutes at 37° C. The target probe wash buffer 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 at room temperature for 45 minutes. Cells were then washed three times with 200 μl / well of sm-wash buffer at room temperature for 2 minutes. 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 and contained 0.3 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 for 45 minutes at room temperature. The procedure was the same for Experiments 5-8, except that the final concentration of each signal oligonucleotide was 0.15 μM. Cells were then washed three times with sm-wash buffer at room temperature for 2 minutes at 200 μl / well. G. Fluorescence and White Light Imaging Cells were washed once with 200 μl of imaging buffer per well at room temperature. In experiments without Trolox (see Table 7, last column), the imaging buffer contained 30 mM NaCitrate, 300 mM NaCl, pH 7, and 5 mM ribonucleoside vanadyl complex. In experiments with Trolox, the imaging buffer further contained 10% VectaCell Trolox Antifade reagent (Vector Laboratories, Cat.: CB-1000), resulting in a final Trolox concentration of 10 mM. A Zeiss Axiovert 200M microscope equipped with a 63x oil immersion 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 for imaging the regions. Filter sets and LED wavelengths were adjusted for the different optimal conditions of the fluorophores used. The exposure time per image was 1000 ms for Alexa Fluor 546 and Atto 594, and 400 ms for Alexa Fluor 488.

[0195] In 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. In addition, one white-light image was acquired from that region. In experiments with multiple detection cycles, the region from the first detection round was rediscovered and imaged in all subsequent rounds. H. Selective denaturation For selective denaturation, all wells were incubated with 200 μl of sm-wash buffer for 6 min at 42° C. This procedure was repeated six times. Steps (E) to (H) were repeated five times in experiments 1 to 4. Step (H) was omitted for 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 resulting combined 3D point clouds of five detection cycles were aligned to each other using a VBA script. The resulting alignment revealed a 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.

[0196] result 1. Absolute number of signals decoded The absolute number of successfully decoded signals for all transcripts is listed for each region for each experiment in Table 4 below. In summary, the sum of correct codes indicates the total number of decoded signals assigned to detectable transcripts in the corresponding experiment, while the sum of incorrect codes is the total number of decoded signals that were not detectable in the corresponding experiment. The total number of signals includes successfully decoded signals as well as unsuccessfully decoded signals. [Table 5] JPEG0007828901000007.jpg111165Table 4: Absolute number of decoded signals

[0197] Table 4 shows that there are very few incorrectly interpreted signals compared to the number of correctly interpreted signals. The absolute values ​​for the interpreted signals of a particular transcript are very similar between different regions of an experiment. The percentage of the total number of signals that can be successfully interpreted 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.

[0198] conclusion The method disclosed herein generates only a small number of incorrectly assigned code words and can therefore be considered specific.Even if the number of signals per region is very large and the number of transcripts detected in parallel is very large, the rate of successfully decoded signals is very high.Due to the high rate of assignable signals and high specificity, the method is practically useful.

[0199] Comparison of relative transcript abundance between different experiments The overlap of detected transcripts between experiments is used for analysis, as shown in Figure 8 for both comparisons (A and B). Each bar corresponds to the average abundance of all three regions in the experiment. The standard deviation between these regions is also shown.

[0200] Correlation of relative transcript abundance between different experiments As can be seen in Figure 9, the mean relative abundance of transcripts from experiment 1 correlates with the abundance of overlapping transcripts in experiments 3, 4, and 2. The correlation coefficient as well as the equation for the linear regression are shown for each correlation.

[0201] Figure 8 shows low standard deviations, indicating low variability in relative abundances between different regions of a single experiment. The differences in relative abundances between transcripts from different experiments are also very small. This is the case for the comparison of transcripts from group 1 (Figure 8A), which were 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 0.88–0.91, while the slopes of the linear regressions are 0.97–1.05.

[0202] 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 this method, even with a very large number of signals.

[0203] Comparison of intercellular distribution of signals In Figure 10, maximum projections of image stacks are shown: 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 that are 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 that are assigned to THRAP3.

[0204] Comparison of intracellular distribution of signals In Figure 11, maximum projections of image stacks are shown. 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 that are 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 that are assigned to DDX5.

[0205] Figure 10 shows the large differences in cell-to-cell distribution between different transcripts. SPOCK1 appears 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).

[0206] Figure 11 shows the striking differences in subcellular distribution between the different transcripts. THRAP3 can be observed primarily in the periphery of the cell (cytoplasm) (Figure 11A), whereas 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).

[0207] conclusion Next to the confidence of the quantification, point clouds from multiple-round experiments also show the same intra- and intercellular distribution pattern of transcripts, as clearly demonstrated by direct comparison of point clouds assigned to signals from single-round experiments that detect only one distinctive mRNA species.

[0208] Distribution patterns of different cell cycle-dependent transcripts All images in Figure 12 represent region 1 of experiment 1. In each image, point clouds are shown, which can be assigned to specific transcripts: A: CCNA2, B: CENPE, C: CCNE1, and D: total 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 contribute to 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 predominantly present in the three central cells of region 1. CCNE1 (Figure 12C), also known as cyclin E1, interacts with CDK2 and contributes to 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 distributed very evenly across the other cells. This indicates counterlocalization to the other two transcripts. The corresponding data for the point clouds are derived from point clouds with a very large number of points and a very high point density (Figure 12D provides the findings).

[0209] conclusion The three decoded point clouds of cell cycle-dependent proteins shown in Figure 12 show distribution patterns that can be explained by their corresponding functions. These data strongly suggest that the disclosed method reliably generates biologically relevant data, even when there are few signals per cell (Figure 12C) and when the signal density is very high (Figure 12D).

[0210] Array List In the attached sequence listing, SEQ ID NOs: 1 to 1247 represent the nucleotide sequences of representative target-specific oligonucleotides. The oligonucleotides listed consist of a target-specific binding site (5' end), a spacer / linker sequence (gtaac or tagac), and a unique identifier sequence that is the same for all oligonucleotides in a probe set.

[0211] In the attached sequence listing, SEQ ID NOs: 1248 to 1397 refer to the nucleotide sequences of representative decoding oligonucleotides.

[0212] In the attached sequence listing, SEQ ID NOs: 1398-1400 refer to the nucleotide sequences of representative 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 its 5' end and at its 3' end.

Claims

1. 1. A multiplex method for detecting different analytes and different subgroups / variants of analytes in a sample, comprising: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, each set of analyte-specific probes comprising at least two (2) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs in the nucleotide sequence of said identifier element (T) from an analyte-specific probe of another set of analyte-specific probes; the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and comprise the same nucleotide sequence of said identifier element (T) that is unique to said analyte; contacting the sample with at least two different sets of analyte-specific probes for at least one analyte and variants thereof; The analyte-specific probes contained in these different sets interact with the same analyte, but specifically with different substructures of said same analyte; analyte-specific probes of a first set of analyte-specific probes interact with substructures contained in all variations of the analyte; Analyte-specific probes of a second set of analyte-specific probes (subgroup-specific probes) interact with substructures contained only in specific variants of the analyte; an analyte-specific probe of the first set of analyte-specific probes comprises a first identifier element (T1) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; the analyte-specific probes of the second set of analyte-specific probes comprise a second identifier element (T2) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; a first identifier element (T1) of an analyte-specific probe of a first set of analyte-specific probes and a second identifier element (T2) of an analyte-specific probe of a second set of analyte-specific probes are bound to different decoding oligonucleotides and / or non-signal decoding oligonucleotides; (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including; one set of decoding oligonucleotides for an individual analyte differs in a first connector element (t) from another set of decoding oligonucleotides for a different analyte; (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) contained in the decoding oligonucleotide; (bb) a signal element; and and (D) detecting a signal produced by the signal element; (E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining specific binding of the analyte-specific probe to the analyte to be encoded; (F) performing at least three (3) additional cycles comprising steps B) through E) to generate a coding scheme with a code word for each analyte; (G) performing at least one (1) additional cycle comprising steps B) through E) to identify said subgroup-specific probes; A multiplex method, including:

2. 10. The method of claim 1, comprising additional further cycles comprising steps B) through E) of contacting the sample with the subgroup-specific probe set of claim 1 and identifying variants that interact with the subgroup-specific probes.

3. A method as described in claim 1 or 2, wherein the last cycle of the further cycles comprising steps B) to E) stops at step (D).

4. The method according to any one of claims 1 to 3, wherein all steps are automated, and steps B) to G) are automated by using a robotic system.

5. 5. The method of any one of claims 1 to 4, wherein each analyte is associated with a specific code word, said code word comprising several positions, each position corresponding to one cycle resulting in a plurality of distinct coding schemes with multiple code words.

6. 6. The method of claim 5, wherein for at least one individual analyte, the code word position has a value of zero (0).

7. The method, comprising contacting the sample with at least one set of non-signal decoding oligonucleotides for binding to specific identifier elements (T) of an analyte-specific probe; non-signal decoding oligonucleotides in the same set interact with the same identifier element (T); each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of a particular identifier element (T), and does not comprise a translator element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide; The method according to any one of claims 1 to 6.

8. contacting the sample with at least two (2) different sets of non-signal decode oligonucleotides for binding to at least two different identifier elements (T) of the analyte-specific probes, each set of non-signal decode oligonucleotides interacting with a different identifier element (T); The method of any one of claims 1 to 7, wherein each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence that enables specific hybridization of a signal oligonucleotide.

9. 9. The method of claim 8, wherein the different sets of non-signal-decoding oligonucleotides are contained in a premix of different sets of non-signal-decoding oligonucleotides or are present separately.

10. Contacting the sample with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of said translator element (c); and (bb) comprising a quencher (Q) or a signal element and a quencher (Q), or no signal element; The method according to any one of claims 1 to 8.

11. contacting the sample with at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of said translator element (c); and (bb) comprising a quencher (Q) or a signal element and a quencher (Q), or no signal element; The method according to any one of claims 1 to 10.

12. The method of any one of claims 1 to 11, wherein the decoding oligonucleotides in a particular set of decoding oligonucleotides interact with the same identifier element (T).

13. A multi-analyte kit for carrying out the multiplex method of claim 1, said kit comprising: (A) at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, each set of analyte-specific probes comprising at least two (2) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded; (bb) an identifier element (T) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; Including, an analyte-specific probe of a particular set of analyte-specific probes differs in the nucleotide sequence of said identifier element (T) from an analyte-specific probe of another set of analyte-specific probes; the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and comprise the same nucleotide sequence of said identifier element (T) that is unique to said analyte; the set of analyte-specific probes comprises at least two different sets of analyte-specific probes for at least one analyte and variants thereof; analyte-specific probes of a first set of analyte-specific probes interact with a substructure contained in all variations of the analyte; Analyte-specific probes of a second set of analyte-specific probes (subgroup-specific probes) interact with substructures contained only in specific variants of the analyte; an analyte-specific probe of the first set of analyte-specific probes comprises a first identifier element (T1) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; the analyte-specific probes of the second set of analyte-specific probes comprise a second identifier element (T2) comprising a nucleotide sequence (unique identifier sequence) that is unique to the analyte to be encoded; the first identifier element (T1) and the second identifier element (T2) are different; (B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte, each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including; one set of decoding oligonucleotides for an individual analyte differs from another set of decoding oligonucleotides for a different analyte in the identifier connector element (t); at least one set of decoding oligonucleotides per analyte; (C) a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) contained in the decoding oligonucleotide; (bb) a signal element; and a set of signal oligonucleotides comprising: Includes a kit.

14. 13. An in vitro method for the detection of a marker of a disease selected from the group comprising cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases, diseases caused by biotic stress, preferably of infectious and / or parasitic origin or diseases caused by biotic stress, preferably caused by nutritional disorders and / or an unfavourable environment, comprising detecting the marker of said disease by carrying out in vitro the multiplex method according to any one of claims 1 to 12.

15. An optical multiplexing system for use in a method according to any one of claims 1 to 12 or claim 14, comprising at least: - one reaction vessel containing the kit or part of the kit according to claim 13; a detection unit including a fluorescence microscope; -A camera and - an instrument for handling liquids; An optical multiplex system including:

16. 1. An in vitro method for screening, identifying, and / or testing substances and / or drugs, comprising: (a) contacting a test sample containing a sample with a substance and / or drug; (b) detecting different analytes in a sample by sequential signal encoding of said analytes by the method of any one of claims 1 to 12; An in vitro method comprising:

Images