A method for simultaneously or sequential detecting, counting, localizing genome sites and / or extra-genomic nucleic acid elements

Abstract

New methods and kits are disclosed for simultaneously or sequential determining the number and localization of nuclear genome sites as well as extra-genomic nucleic elements (such as mRNA). This is essential for unravelling the complexities of the genome and understanding its functional organization, which has wide-ranging implications for fields such as genetics, genomics, medicine, and evolutionary biology.

Description

[0001] A METHOD FOR SIMULTANEOUSLY OR SEQUENTIAL DETECTING, COUNTING LOCALIZING GENOME SITES AND / OR EXTRA-GENOMIC NUCLEIC ACID

[0002] ELEMENTS

[0003] FIELD OF THE DISCLOSURE

[0004] Determining the number and localization of nuclear genome sites is essential for unravelling the complexities of the genome and understanding its functional organization, which has wide-ranging implications for fields such as genetics, genomics, medicine, and evolutionary biology.

[0005] Determining the number and localization of genomic sites helps to understand how genetic information is organized within cells. This knowledge provides insights into the spatial arrangement of genes, regulatory elements, and other functional regions of the genome. It can reveal patterns of gene clustering, chromatin structure, and interactions between different genomic regions.

[0006] Identifying the precise location of genes within the genome is crucial for studying their functions and understanding their roles in various biological processes. By knowing the positions of genes on chromosomes, researchers can associate them with specific traits or diseases and investigate how they are regulated.

[0007] Many genetic disorders are caused by mutations or variations in specific genes. By pinpointing the exact genomic location of disease-associated genes, scientists can better understand the underlying genetic mechanisms and develop diagnostic tests or targeted therapies. Additionally, studying the spatial organization of the genome can reveal how chromosomal rearrangements or structural variations contribute to diseases like cancer.

[0008] Comparing the organization of genomes across different species provides also valuable insights into evolutionary relationships and processes. By identifying conserved regions or gene clusters in related species, scientists can infer evolutionary changes and gain a deeper understanding of how genomes have evolved over time.

[0009] Advances in genomic technologies, such as high-resolution imaging techniques and nextgeneration sequencing, have made it increasingly feasible to study the organization of the genome at finer scales. By characterizing the number and localization of genomic sites, researchers can refine genome assembly methods, improve gene annotation, and develop more accurate models of genome architecture.

[0010] The number and localization of nuclear genome sites can be determined by several methods:

[0011] 1) Determination of the karyotype by analysis of metaphase chromosomes (e.g. classical cytogenetic analysis of amniotic fluid)

[0012] 2) Chromosome painting (combination of cytogenetic analysis of metaphase chromosomes and FISH)

[0013] 3) FISH (counting of individual chromosomes by probes binding to DNA)

[0014] 4) Real-time PCR

[0015] 5) Microarray

[0016] 6) Next generation sequencing methods

[0017] However, each of these methods also do have limitations and disadvantages.

[0018] For example, metaphase chromosome analysis provides a limited resolution for detecting smaller chromosomal abnormalities, such as sub-microscopic deletions or duplications, which may be relevant in certain genetic disorders. Metaphase chromosome analysis also typically require cell culture to obtain dividing cells, which can be time-consuming and may introduce artefacts or cell culture-related abnormalities. Further, metaphase chromosome analysis may miss certain chromosomal abnormalities if the abnormal cells are not present in the sample analyzed or if the abnormality is present in a mosaic form (only in a subset of cells).

[0019] Chromosome painting allows visualization of whole chromosomes or large chromosomal regions, but it may not provide detailed information about specific genes or smaller genomic rearrangements. Thus, chromosome painting is often used in combination with other techniques, such as FISH, to provide additional information. This adds complexity to the analysis and requires multiple experiments.

[0020] FISH is typically used to analyze specific chromosomal regions or individual chromosomes, making it less suitable for genome-wide analysis or screening for multiple abnormalities simultaneously or sequential. However, FISH may not detect smaller structural variants or copy number variations beyond the resolution of the probes used and interpreting FISH results can be complex, and falsepositive or false-negative results can occur due to technical issues, probe limitations, or sample characteristics.

[0021] Real-time PCR is typically used to quantify the amount of specific DNA sequences or detect specific mutations. It provides limited information about the overall genome structure or larger-scale genomic rearrangements. However, real-time PCR requires prior knowledge of the specific target sequence or mutation to be analyzed. It may not be suitable for detecting novel or unknown genetic variants. Further, real-time PCR is a relatively low-throughput method, and analyzing multiple targets or a large number of samples can be time-consuming and labor-intensive.

[0022] Microarrays have a limited resolution for detecting small structural variants or copy number variations, especially compared to newer techniques like next-generation sequencing. Microarrays have fixed probe sets, and their ability to detect genetic variants depends on the specific probes present on the array. They may not cover the entire genome or certain regions of interest adequately.

[0023] The analysis of next-generation sequencing data can be computationally demanding and requires specialized bioinformatics expertise. Handling and interpreting large datasets can be challenging. As such, next-generation sequencing methods can be relatively expensive compared to other techniques, especially for large-scale or whole-genome sequencing projects. Further, nextgeneration sequencing is not error-free, and false-positive or false-negative results can occur due to sequencing errors, library preparation artifacts, or data analysis limitations. Additionally, nextgeneration sequencing can generate a vast amount of genetic information, including incidental findings or information unrelated to the initial purpose of the analysis. Handling and interpreting such information raise ethical and privacy concerns.

[0024] The subject-matter of the present application addresses these issues and overcomes some of the before-mentioned limitations. BACKGROUND

[0025] The review article of Cekan (2004) "Methods to find out the expression of activated genes", Reprod Biol Endocrinol. 2004 Sep 23;2:68. doi: 10.1186 / 1477-7827-2-68.1 describes methods of identifying genes that have been activated by inner or outer impulses. The activation and subsequent expression of a gene can be detected by its transcription into a corresponding messenger ribonucleic acid (mRNA). Principles of the methods for identification of individual activated genes, as well as groups of activated genes are described, the former methods being mostly based on subtractive hybridization and serial analysis of gene expression (SAGE), the latter on microarrays.

[0026] The article by Lin et al., (2019) "Detection of copy number variants with chromosomal microarray in 10 377 pregnancies at a single laboratory", Acta Obstet Gynecol Scand. 2020;99:775-782, DOI: 10.1111 / aogs.13886, describes the analysis of 10377 pregnancy cases by chromosomal microarray (CMA). In this study, prenatal SNP-array analysis was used to detect clinically significant CNVs in fetuses with abnormal ultrasound or pregnancies of advanced maternal age.

[0027] The article by Ma et al., (2015) "Quantitative Analysis of Copy Number Variants Based on RealTime LightCycler PCR", Curr Protoc Hum Genet; 80: 7.21.1-7.21.8. doi:10.1002 / 0471142905. hg0721s80; describes PCR which is visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples.

[0028] The review-article by Ried et al., 1998 "Chromosome painting: a useful art", Human Molecular Genetics, 1998, Vol. 7, No. 10 Review Article describes the technology of "chromosome painting" by hybridization of fluorescently labeled chromosome-specific, composite probe pools to cytological preparations.

[0029] The article Xie et al., (2022) "Enhancer transcription detected in the nascent transcriptomic landscape of bread wheat", Genome Biology, 23:109 https: / / doi.org / 10.1186 / sl3059-022-02675-l describes methods for nascent RNA sequencing combined with epigenome profiling. Against this background, it is an object underlying the present disclosure to provide a method by means of which the disadvantages of the prior art methods can be reduced or even overcome.

[0030] In an article from Kishi et al, (2018) "SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissue", bioRxiv, doi: https: / / doi.org / 10.1101 / 401810 a method was disclosed utilizing a multiplexed amplification of FISH signals in a single cell gene expression assay. However, the authors had to use inter alia single-stranded DNA primers extended by catalytic hairpins in order to achieve effective amplification of probe-signal. The target nucleic acid sequences had to be significant different for detection.

[0031] Thus, there was a need to find new highly multiplexed methods for detecting highly similar nucleic acid sequences both in genomic as well as extra-genomic nucleic acid target sequences (such as mRNA).

[0032] SUMMARY OF THE DISCLOSURE

[0033] In a first aspect the disclosure pertains to a new method for simultaneously or sequential detecting, counting, localizing at least two nucleic acid elements selected from nuclear genome sites and / or non-genomic nucleic acid elements, such as mRNA, that is defined by an indirect detection process that includes a coding system.

[0034] In one embodiment the disclosure pertains to a method wherein the at least two nucleic acid elements mentioned before are detected by in s / tu-hybridization and comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the detected nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein. wherein, optionally, the method uses a detection and / or coding process comprising: multiple rounds of detection to cover a coding of sites detected simultaneously or sequential; performed in situ; resulting in spatially resolved images where copies of a certain transcribed genome element can be counted and localized; detection of genome elements simultaneously or sequential because of the coding system.

[0035] In one embodiment the method comprises the general steps of:

[0036] 1. Selecting a transcribed genome element of interest;

[0037] 2. Designing probes that are specific and complementary to nucleic acid molecules, in particular to RNA molecules but that preferably exclude exon areas;

[0038] 3. Providing and preparing a sample;

[0039] 4. Immobilizing the tissue section on a holder;

[0040] 5. Hybridizing specific probes to the tissue section;

[0041] 6. Building up a coding process by repeating round of switching the detectable label;

[0042] 7. Imaging the tissue section after colorization in each repeating round;

[0043] 8. Performing a computer aided decoding process of all images that were taken; and / or

[0044] 9) Counting specific signals and / or measure the intensity.

[0045] In a second aspect the disclosure pertains to a method for an in s / tu-hybridization multiplex reaction characterized by comprising the steps of: a.) adding an analyte-specific probe oligonucleotide comprising a binding element which is essentially complementary to an analyte nucleic acid element to be detected, wherein the nucleic acid element comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein; and comprising an identifier element (T) comprising a nucleotide sequence which is unique to the analyte nucleotide sequence; and b.) adding a decoding oligonucleotide comprising an identifier element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique set identifier sequence of the identifier element (T) of the corresponding binding element, and a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; and / or c.) adding a signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and a signal element facilitating a signal which is specific for the polymorphic analyte; and wherein the monitoring probe is added before, during and / or after adding the decoding oligonucleotide in step b).

[0046] In a third aspect the disclosure pertains to a kit, comprising a.) an analyte-specific probe oligonucleotide comprising a binding element which is essentially complementary to an analyte nucleic acid element to be detected, wherein the nucleic acid element comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein;, and comprising an identifier element (T) comprising a nucleotide sequence which is unique to the analyte nucleotide sequence or otherwise distinguishes the analyte from one or more additional analytes in an analyte detection set; and b.) a decoding oligonucleotide comprising an identifier element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique set identifier sequence of the identifier element (T) of the corresponding binding element, and a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; and / or c.) a signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and a signal element facilitating a signal which is specific for the polymorphic analyte.

[0047] In a fourth aspect the disclosure pertains to an in vitro method for diagnosis of a disease such as selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases and prenatal diseases comprising the use of the multiplex method disclosed hereinunder.

[0048] In a fifth aspect the disclosure pertains to an optical multiplexing system suitable for the method disclosed hereinunder, comprising at least: one reaction vessel for containing the kits or part of the kits as disclosed hereinunder; a detection unit comprising a microscope, in particular a fluorescence microscope a camera a liquid handling device.

[0049] In a sixth aspect the disclosure pertains to an in vitro method for screening, identifying and / or testing a substance and / or drug comprising:

[0050] (a) contacting a test sample comprising a sample with a substance and / or drug

[0051] (b) detecting different analytes in a sample by sequential signal-encoding of said analytes with a method as disclosed hereinunder.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Fig. 1: Embodiment where the analyte is a nucleic acid and the probe set comprises oligonucleotides specifically binding to the analyte. The probes comprise a unique identifier sequence allowing hybridization of decoding oligonucleotides.

[0054] Fig. 2: Embodiment where the analyte is a nucleic acid comprising non-coding nucleic acid elements (e.g., introns) (black) and coding nucleic acid sequences (e.g., exons) (grey) and the probe set comprises oligonucleotides specifically binding to the nucleic acid elements comprising non- coding nucleic acid elements. The probes comprise a unique identifier sequence allowing hybridization of decoding oligonucleotides.

[0055] Fig. 3: Flowchart of the method according to the disclosure.

[0056] Fig. 4: Alternative options for the application of decoding and signal oligonucleotides.

[0057] Fig. 5: Example for signal encoding of three different nucleic acid sequences by two different signal types and three detection rounds; in this example, the encoding scheme includes error detection.

[0058] Fig. 6: Number of generated code words (logarithmic scale) against number of detection cycles.

[0059] Fig. 7: Calculated total efficiency of a 5-round encoding scheme based on single step efficiencies.

[0060] Fig. 8: Comparison of relative transcript abundances between different experiments.

[0061] Fig. 9: Correlation of relative transcript abundances between different experiments.

[0062] Fig. 10: Comparison of intercellular distribution of signals.

[0063] Fig. 11: Comparison of intracellular distribution of signals.

[0064] Fig. 12: Distribution pattern of different cell cycle dependent transcripts.

[0065] Fig. 13: Detection of multiple targets using a 8 round code with 2 labels (A and B) and no label (-). The targets 1, 2, 3, 4, 5, 20 , and n are represented. The rounds 1, 2, 3, and 8 of the coding scheme are represented. Herein, the blank is part of the code.

[0066] Fig. 14: Detection of multiple targets can be performed by an encoding scheme using a detectable marker. The ending scheme may comprise also the „0" as a marker. That means that at a specific position the transcript is not detected. Consequently, the encoding scheme may be represented by the following constructs using only two gene specific probes:

[0067] 1) With detectable label F: detectable during imaging 2) With detectable label F and quencher Q: not detectable during imaging

[0068] 3) With quencher Q: not detectable during imaging

[0069] 4) Without label F: not detectable during imaging

[0070] 5) Without signaling oligonucleotide: not detectable during imaging

[0071] 6) With a decoder oligonucleotide that cannot recruit a signaling oligonucleotide

[0072] 7) Without decoder oligonucleotide: not detectable during imaging

[0073] Fig. 15: Possible structures of a multi-decoder. The numbers depict the examples. (A) is the unique identifier sequence, (a) is the corresponding sequence of the decoding oligonucleotide or multi-decoder and (cl) 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 is not fixed. Example 1 shows a normal decoding oligonucleotide since there is only one signal oligonucleotide binding element (cl).

[0074] Fig. 16: Example for signal encoding of three different nucleic acid sequences by using multi-decoders and two different signal oligonucleotides creating three different signal types and three detection rounds. In this example, the encoding scheme includes error detection and correction.

[0075] Fig. 17: Number of generated code words (logarithmic scale) against number of detection cycles. The number of code words for merFISH does not exponentially increase with the number of detection cycles but gets less effective with each added round. In contrast, the number of code words for intronSeqFISH, the method of the present disclosure without using multi-decoders, the method with multi-decoders increases exponentially. The slope of the curve for the method using multi-decoders is much higher than that of the prior invention, leading to more than 20000000 times more code words usable after 20 rounds of detection.

[0076] Fig 18 Typical intron-exon structure of DNA, primarily transcribed RNA, matured RNA with respect to the position of the probe binding sites.

[0077] Fig 19 Structure of the genomic element in the transcription complex and the detection of the signal. Fig 20 - Fluorescence image of the analytic probes binding to genomic elements within cells. Standard Molecular Cartography was performed on human liver samples using defined probes for specific markers. Note that 1, 2 and 3 different signals in the area shown here can be recognized depending on the genomic situation. Also, cells without any signal are recognized.

[0078] DETAILED DESCRIPTION OF THE DISCLOSURE

[0079] Disclosed herein are methods and kits for specifically detecting, counting, and localizing nuclear genome sites, wherein the detection method is defined by an indirect multiplex detection process that includes a coding system.

[0080] The present disclosure aims for the detection of genomic regions which preferably are not encoding for the final gene-product. As such in particular intron-regions or other regions which are not included in mature proceesed mRNA molecules are preferred as detection targets, although also partially promotor and / or exon-regions may be part of the detected sequence.

[0081] The detection of intron or other regions not included in mature processed mRNA molecules opens a number of advantages of the present disclosure:

[0082] • It has been found that intron regions are abundant in the human genome, occupying a significant portion of the total DNA sequence. By targeting introns, a larger pool of potential targets for genomic detection is made available compared to the limited number of exons, which constitute a smaller fraction of the genome.

[0083] • Further, it has been found that intron regions tend to have higher levels of genetic variation compared to exons. By targeting introns, a broader range of genetic variants, including single nucleotide polymorphisms (SNPs), insertions, deletions, and structural variants can be targeted. This can be particularly useful for population studies, genetic diversity analyses, and identifying disease-associated variants.

[0084] • Additionally, it has been found that intron regions generally exhibit lower sequence conservation compared to exons. This lower conservation increases the specificity of detection assays by reducing the likelihood of cross-reactivity or non-specific binding to highly similar sequences in related genes.

[0085] • Further, it has been found that many regulatory elements, such as enhancers, silencers, and promoters, are located within intron regions. Targeting introns allows identifying and characterizing these regulatory elements, providing insights into gene regulation and expression patterns. • Further, it has been found that intron regions are intimately involved in the splicing process, which leads to the generation of different mRNA isoforms through alternative splicing. By targeting introns, alternative splicing events and their potential functional consequences can be investigated.

[0086] • Further, it has been found that intron regions generally experience less selective pressure compared to exons, as they are not directly translated into protein sequences. This reduced functional constraint allows for more sequence variation and flexibility, making introns valuable targets for evolutionary and comparative genomics studies.

[0087] • Last, but not least, it has been found that intron regions can give rise to non-coding RNA molecules, such as long non-coding RNAs (IncRNAs). Targeting introns enables the study of these non-coding RNA transcripts and their potential roles in various biological processes.

[0088] Overall, targeting intron regions provides a broader perspective on genetic variation, gene regulation, alternative splicing, and non-coding RNA molecules. By focusing on introns, the disclosed subjectmatter can unlock a wealth of information about genomic structure, function, and diversity.

[0089] Thus, in a first aspect the disclosure pertains to a new method for detecting, counting, localizing nuclear genome sites that is defined by an indirect detection process that includes a coding system.

[0090] In one embodiment the disclosure pertains to a method for an in s / tu-hybridization of a nucleic acid element, wherein the nucleic acid element comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein;

[0091] Wherein, optionally, the method uses a detection and / or coding process comprising: multiple rounds of detection to cover a coding of sites detected simultaneously or sequential ; performed in situ; resulting in spatially resolved images where copies of a certain transcribed genome element can be counted and localized; detection of genome elements simultaneously or sequential because of the coding system.

[0092] In one embodiment the method comprises the general steps of:

[0093] 1. Selecting a transcribed genome element of interest;

[0094] 2. Designing probes that are specific and complementary to RNA molecules but that preferably exclude exon areas;

[0095] 3. Providing and preparing a sample;

[0096] 4. Immobilizing the tissue section on a holder;

[0097] 5. Hybridizing specific probes to the tissue section;

[0098] 6. Building up a coding process by repeating round of switching the detectable label;

[0099] 7. Imaging the tissue section after colorization in each repeating round;

[0100] 8. Performing a computer aided decoding process of all images that were taken; and / or

[0101] 9) Counting specific signals and / or measure the intensity.

[0102] For example, in one embodiment a method for spatially detecting at least one target analyte in at least one cell from a tissue sample is used comprising:

[0103] (1) contacting at least one genome element as defined herein in at least one cell in a tissue sample with at least one probe comprising a genome element binding domain and an identifier oligonucleotide, wherein the identifier oligonucleotide comprises a unique nucleic acid sequence which identifies the genome element bound to the genome element binding domain;

[0104] (2) providing a force to a location of the tissue sample sufficient to release the identifier oligonucleotide;

[0105] (3) collecting the released identifier oligonucleotide;

[0106] (4) hybridizing to the released identifier oligonucleotide a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe comprises: a nucleic acid complementary to a portion of the identifier oligonucleotide, a nucleic acid sequence comprising a unique molecular identifier, a first amplification primer binding site, and wherein the second nucleic acid probe comprises: a nucleic acid complementary to a portion of the identifier oligonucleotide, and a second amplification primer binding site, and wherein the first and the second nucleic acid probes hybridize to the identifier oligonucleotide such that the first and the second nucleic acid probes are adjacent but not overlapping; (5) ligating the hybridized first and second nucleic acid probes together;

[0107] (6) amplifying the ligation product produced in step (5); and

[0108] (7) identifying the released identifier oligonucleotide by sequencing the amplified products produced in step (6), thereby spatially detecting the at least one target analyte in the at least one cell in a tissue sample.

[0109] In one embodiment, the method relates to an RNA imaging-based technique (utilizing single molecule FISH or smFISH). In such an embodiment fluorescently tagged oligo probes label at least one genome element as defined herein, and by directly counting the number of fluorescent RNA targets, each gene's expression profile is determined. This detection can be expanded by adding combinatorial labeling, sequential rounds of imaging and error robust barcoding to greatly increase the multiplexing capacity and enable spatially resolved, single-cell gene expression profiling.

[0110] In such embodiments a combinatorial barcoding scheme is implemented where each targeted gene in the (custom designed) gene panel is assigned a unique binary barcode. Sequential rounds of imaging are used to generate the barcode, a sequence of zeros and ones. This combinatorial barcoding scheme dramatically increases multiplexing capacity. Additionally, this barcoding system is further designed with error robustness. If there is a readout error, the system can assign the readout to the nearest correct barcode. With such a spatial profiling method transcripts can be spatially localized with nanometer-scale resolution, mapping gene expression across whole tissues and discovering the complex arrangement of cell types and states.

[0111] In one embodiment the method comprises the steps of:

[0112] (i) contacting a biological sample comprising a first genome element as defined herein with a first agent, wherein the first agent comprises a first binding species that specifically binds to the genome element as defined herein, and a first oligonucleotide conjugated to the binding species;

[0113] (ii) contacting the biological sample with a second agent, wherein the second agent comprises a first reactive species and a second oligonucleotide conjugated to the first reactive species, to hybridize at least a portion of the second oligonucleotide to at least a portion of the first oligonucleotide;

[0114] (iii) contacting the biological sample with a first labeling species, wherein the first labeling species reacts with the first reactive species to deposit the first labeling species or a derivative thereof in the biological sample; (iv) removing the second agent from the biological sample following deposition of the first labeling species or the derivative thereof;

[0115] (v) contacting the biological sample with a third agent, wherein the third agent comprises a second binding species that specifically binds to a second target analyte in the biological sample, and a third oligonucleotide conjugated to the second binding species;

[0116] (vi) contacting the biological sample with a fourth agent, wherein the fourth agent comprises a second reactive species and a fourth oligonucleotide conjugated to the second reactive species, to hybridize at least a portion of the fourth oligonucleotide to at least a portion of the third oligonucleotide; and

[0117] (vii) contacting the biological sample with a second labeling species, wherein the second labeling species reacts with the second reactive species to deposit the second labeling species or a derivative thereof in the biological sample.

[0118] In one embodiment the present disclosure describes the usage of a set of labeled and unlabeled nucleic acid sequences for specific quantitative and / or spatial detection of different analytes in parallel via specific hybridization. The technology allows the discrimination of more different analytes than different detection signals are available. The discrimination may be realized via sequential signalcoding of the analytes achieved by several cycles of specific hybridization, detection of signals and selective elution of the hybridized nucleic acid sequences.

[0119] In contrast to other state-of-the-art methods, the oligonucleotides providing the detectable signal are not directly interacting with sample-specific nucleic acid sequences but are mediated by so called "decoding-oligonucleotides". This mechanism decouples the dependency between the analytespecific oligonucleotides and the signal oligonucleotides. The use of decoding-oligonucleotides allows a much higher flexibility while dramatically decreasing the number of different signal oligonucleotides needed which in turn increases the coding capacity achieved with a certain number of detection rounds.

[0120] In one embodiment a set of first probes are encompassed, wherein the probes are intron-specific (that is, probes which comprise a binding element which is more than 90%, optionally more than 95%, optionally more than 99%, or optionally to 100% complementary to an intronic or other noncoding sequence or other target sequence not included in a mature mRNA molecule) and comprise further at least an identifier oligonucleotide, which allows the indirect readout by decoding-oligonucleotides as described hereinunder.

[0121] In some cases these first probes are used in isolation. Alternately, in some cases these probes are used in combination with a second set of probes probes that anneal to or are specific to (such as probes which comprise a binding element which is more than 90%, optionally more than 95%, optionally more than 99%, or optionally to 100% complementary to) an exonic segment or a segment otherwise retained in a mature mRNA molecule, such as a 3' untranslated region or 3' untranslated region.

[0122] Similarly, some methods comprise probing a sample using a first set of probes and a second set of probes, and detecting the probes using, for example, the sequential detection approaches disclosed herein. The sequential detection may be effected sequentially, with the first probe set being detected first and the second probed set detected subsequently. Alternately, the first probe set and the second probe set may be detected concurrently, through interleaved or alternating decoding.

[0123] Some such methods optionally further comprise overlaying or deleting a signal associated with the first probe set or the second probe set, so as to identify signal associated with expressed gene products such as transcripts or mature mRNA distinct from the signal arising from binding to the corresponding portion of the genomic DNA encoding the mature mRNA.

[0124] Thus, systems, compositions and methods herein allow the specific detection and subcellular localization of genomic loci encoding particular transcripts, such as by assaying using a first probe set that targets noncoding DNA or DNA from a locus that does not template RNA included in a final processed mRNA, such as intron templating DNA. Similarly, the systems, methods and compositions herein allow the specific detection and subcellular localization of transcripts such as mature mRNA molecules, and distinguishing their signals from those of probes bound to the loci that encode them.

[0125] The utilization of decoding-oligonucleotides leads to a sequential signal-coding technology that is more flexible, cheaper, simpler, faster and / or more accurate than other methods.

[0126] DEFINITIONS

[0127] The term "sample" herein may be any biological sample. The sample may comprise eukaryotic or procaryotic cells. The sample may derive from any kind of animal (including Homo, rat, mouse, mammalia, birds, fish, insects, worms), plant, or fungi. The sample can be selected from any organ, any tissue, any kind of culture, any specimen taken for diagnostic purposes (e.g. smear, liquid biopsy, tissue biopsy etc.). The sample may comprise DNA and / or RNA. Samples may taken from alive or dead organisms. Cells may not be complete and samples may contain only partial cells. The sample may be frozen, fixed or embedded.

[0128] The term "nucleus" as used herein is an enclosed environment containing the genomic information. The nucleus may be described also by the term „nuclear region" or „nuclear area". Because of the preparation of a sample, the nucleus may contain only a part of the genome. The term "cell" as used herein is the smallest unit of life and comprises a number (for example, at least one, at least two, at least 5, at least 10, at least 20) genome elements that can be differentiated. In the background of the invention, the cell may be dead or alive, is eukaryotic or prokaryotic. Because of the preparation of a sample, the cell may not be complete and contain only a part of the cell.

[0129] The term "genome" as used herein is a complete identity or a part of this. It can be DNA or RNA.

[0130] The term "exon" as used herein is a part of a genome element that is not excised from primarily transcribed RNA and thus will be present in the final functional RNA molecule. Exonic sequence elements can be translated or untranslated regions (3'UTR or 5'-UTR). In contrast, non-exonic sequences (the term "introns" as used herein) are at least partially excised from functional RNA molecules. In one embodiment the disclosure pertains to a method for an in s / tu-hybridization wherein an genome element (e.g. intron) is detected that is excised by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50% from primarily transcribed RNA and, thus, less of 95%, less of 90%, less of 85%, less of 80%, less of 75%, less of 70%, less of 65%, less of 60%, less of 55%, less of 50% will be present in the final functional (i.e., translated) RNA molecule

[0131] The term "transcription" is used herein for a process during which one strand of the genome sequence of the genome element is copied into a complementary RNA (i.e., mRNA) strand. These single-stranded copies are independent molecules (not covalently connected to another molecule) but may be connected for a certain time to the genome element by a non-covalently binding (e.g., by hydrogen bounding). The connection can be stabilized by a fixation method (e.g., methanol, formalin etc.)

[0132] The term "translation" is used herein to describe the process by which the genetic information carried by messenger RNA (mRNA) is used to synthesize a specific protein. This process occurs within the ribosomes, cellular structures responsible for protein synthesis. Translation is a crucial step in gene expression, where the sequence of nucleotides in the mRNA is translated into a sequence of amino acids to form a functional protein. The mRNA molecule serves as a template during translation, and it contains a series of codons. Each codon is a three-nucleotide sequence that corresponds to a specific amino acid. There are 20 different amino acids used to construct proteins, and the genetic code dictates which codon codes for which amino acid. For instance, the codon AUG codes for the amino acid methionine and also serves as the start codon, initiating the process of translation.

[0133] During translation, ribosomes move along the mRNA in a process involving three key steps:

[0134] 1. Initiation: The ribosome binds to the mRNA at the start codon (AUG) with the help of initiation factors. This marks the beginning of translation. 2. Elongation: The ribosome moves along the mRNA in a 5' to 3' direction, reading each codon one by one. Each codon is matched with its corresponding tRNA (transfer RNA) molecule carrying the appropriate amino acid. The ribosome catalyzes the formation of peptide bonds between adjacent amino acids, forming a growing polypeptide chain.

[0135] 3. Termination: The process continues until a stop codon (UAA, UAG, or UGA) is encountered on the mRNA. At this point, a release factor binds to the ribosome, causing the ribosome to detach from the mRNA. The newly synthesized polypeptide is then released, and the ribosome disassembles, ready for another round of translation.

[0136] Once translation is complete, the newly formed polypeptide chain folds into its specific three- dimensional shape to become a functional protein, which can play a crucial role in various cellular processes and functions.

[0137] The term "genome element" is used as any kind of genomic nucleic acid that can be transcribed. The genome elements can comprise complete entities, or parts of them.

[0138] The term "target sequence" or "nucleic acid element" is defined herein as a nucleic acid region within the genome and / or a non-genomic nucleic acid to be detected, such as mRNA. In one embodiment "target sequence" or "nucleic acid element" may not be transcribed. In another embodiment it may be transcribed but preferably is not translated into a protein. „Preferably" means that 50-100% of the target sequence is not translated, more preferably 70-100% is not translated, most preferably more than 85% is not translated. Thus, in one embodiment the "nucleic acid element" comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein.

[0139] In this disclosure "simultaneously detecting" means that all probes detecting the analyte may be added in one detection round. For example, genomic as well as mRNA may be detected in one detection round. In this disclosure "sequential detecting" means that the probes detecting the analyte may be added sequentially, i.e., probe set 1 for detecting analyte 1 is added first, then, optionally after a washing step, probe set 2 for detecting analyte 2 is added second, etc. For example, first, genomic nucleic acid elements may be detected in round 1, then followed by the detection of nucleic acid elements of mRNA in round 2, optionally, with a washing step in between round 1 and round 2. In some embodiments both "simultaneously detecting" as well as "sequential detecting" may be performed, e.g., detecting simultaneously analyte 1 and 2 with a first and second set of probes, the, optionally performing a washing step, and then detecting simultaneously analyte 3 and 4 with a third and fourth set of probes.

[0140] The term "probe" is defined herein a nucleic acid that can bind complementary to the target nucleic acid. The probe comprises a target recognition sequence and a detector element. The target recognition sequence can be a single continuous sequence or can be comprised of two or more sequences that are separated from each other by a non-functional or functional sequence. A functional sequence for example can contain a detector element. Other functional elements are conceivable. The length and base composition of a probe can vary and is designed for a certain application and reaction condition. The number of probes to detect the target nucleic acid can vary and is adjusted to the application. If multiple probes are used to detect the target nucleic acid, the probes vary in the target recognition sequence. In this case, the probes contain the same or different detector elements. The probe itself is directly or indirectly detectable using the detector element: a. A directly detectable probe is a probe with a detector element that can be measured directly without further reactions (e.g., the detector element is a fluorophore, another dye, a nanoparticle, a mass-tag, or a similar feature that can be detected directly); b. An indirectly detectable probe is a probe with a detector element that can be measured only if a second, third, fourth, or even higher ordered reaction is used to detect the probe.

[0141] The term "detector element" as used herein can be a single continuous sequence or can be comprised of two or more sequences that are separated from each other by a non-functional or functional sequence. A functional sequence for example can contain a target recognition sequence. Other functional elements are conceivable: a. The detector element is an enzyme to perform a reaction at the localization of probe binding. b. The detector element is one or more nucleic acid tails that can be used to hybridize oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly. c. The detector element is one or more tails that can be used to hybridize oligonucleotide which can be used to hybridize another oligonucleotide with a label (e.g. fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly. d. The detector element is characterized by one or more tails that can be used as a primer for a nucleic acid amplification reaction whose product is used for a hybridization of an oligonucleotide with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly. e. The probe is ligated after binding to the target element and forms a circular probe that can be used for hybridization of one or more oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc) that can be detected directly. f. The probe is ligated after binding to the target element and forms a circular probe that can be used for a nucleic acid amplification reaction whose product is used for a hybridization of one or more oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly.

[0142] The term "biological sample" is defined as a material that is derived from an organism and at least contains detectable nucleic acids, cells or parts of cells. These cells may originate from the same or different organs or even organisms.

[0143] The term "tissue" is used herein for any kind of a sample material that is formed by a certain number of cells of the same or different type with a meaningful structural relationship (or the lack thereof), and thus does comprise genome elements. The term "tissue section" is used herein for a thin section of a tissue favorably done by a cryotome or a microtome.

[0144] The term "repeated rounds for switching the labels" is defined herein in one embodiment as a process that labels the target region using the probe as a feature by a certain label followed by a detection process that detects the label. After detection of the label in a certain round, the label is eliminated, and a new label is introduced followed by a next round of detection process.

[0145] The term "label switching" is defined herein as being performed by different processes such as chemical cleavage and rebinding of another label, photobleaching, exchange of oligonucleotides that can bind to the probe, a higher order oligonucleotide or an amplification product.

[0146] According to the present disclosure an "analyte" may be genomic and / or extra-genomic (e.g. mRNA) nucleic acid sequences to be specifically and simultaneously or sequential detected as being present or absent in a sample and, in case of its presence, to encode it. The "analyte" according to the present invention is a target recognition sequence which may be characterized by comprising intron sequences, in one embodiment the target sequence may be any sequence which is excised from primarily transcribed RNA and is not present in the final functional RNA molecule, such as an intron sequence. However, in some embodiments, at least partially the target sequence can comprise also sequences which are still present in the final functional RNA molecule, such as up to 2%, up to 5%, up to 10% up to 20%, up to 30%, up to 40%, up to 45% and up to 49% of target sequence can be still present in the final functional RNA molecule. However, in one embodiment up to 80%, up to 90%, up to 95%, up to 99%, up to 100% of the target sequence are not present in the final functional RNA molecule anymore.

[0147] In one embodiment the disclosure pertains to the detection of a nucleic acid element, wherein the nucleic acid element comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a "non-coding nucleic acid element" and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein. The target sequence can be a single continuous sequence or target recognition sequence comprising two or more sequences interspersed by other sequences, which are not detected.

[0148] In one embodiment the "non-coding nucleic acid element" comprises preferably an intron. In further embodiments "non-coding genome element" comprises other non-transcribed and / or non-translated elements, selected from the group of "non-transcribed elements" comprising introns, intergenic regions, repetitive sequences like transposons and retrotransposons, short tandem repeats (STRs), long interspersed nuclear elements (LINEs), silent or nonfunctional genes, regulatory elements such as promoters and enhancers and heterochromatin, as well as combinations thereof; and / or selected from the group of "non-translated elements" selected from the group comprising 5' UTR and 3' UTR ends (i.e., at the 5' UTR such as a start codon, e.g., (AUG), ribosome binding sites, and other upstream open reading frames (uORFs); at the 3' UTR such as mRNA downstream of the stop codon (UAA, UAG, or UGA), microRNA binding sites and polyadenylation signals), introns in the mRNA, various mRNA processing elements (cap structure, poly-A tail, and splice junctions), as well as combinations thereof.

[0149] It is important to note that since the "analyte" is a nucleic acid sequence comprising a "non-coding nucleic acid element" (i.e., nucleic acid sequences which are not to transcribed and / or translated into a peptide or protein) the overall detection quality and even allow the distinction between polymorph genes, such as homologs, splice-variants, etc. is improved. "Improvement" in this respect refers to better specificity, better resolution and / or better differentiation quality. In some embodiments these "analytes" may allow the simultaneous and distinct detection of at least 2, at least 3, at least 4, at least 8, at least 16, at least 32, at least 40, at least 50, at least 100 target nucleic acid sequences. It also allows the differentiation between genes within the genome and transcribed genes, such as found in mRNA. It also allows for the distinction of unprocessed or just partially processed and fully processed mRNA (i.e., mRNA where the intronic sequences are already excised). In one embodiment the "analyte" is present "ab initio" in the sample and not added artificial nucleic acid.

[0150] The analyte provides at least one site for specific binding with analyte-specific probes. Sometimes herein the term "analyte" is replaced by "target". An "analyte" according to the disclosure incudes a complex of subjects, e.g., at least two individual nucleic acid. In an embodiment of the disclosure an "analyte" excludes a chromosome. In another embodiment of the disclosure an "analyte" excludes DNA.

[0151] In some embodiments, an analyte may be a "coding sequence", "encoding 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 the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.

[0152] A "sample" as referred to herein is a composition in liquid or solid form suspected of comprising the analytes to be encoded. In particular, the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells and / or extracts and / or part of cells. For example, the cell is a prokaryotic cell or a eukaryotic cell, in particular a mammalian cell, in particular a human cell. In some embodiments, the biological tissue, biological cells, extracts and / or part of cells are fixed. In particular, the analytes are fixed in a permeabilized sample, such as a cell-containing sample.

[0153] As used in the present 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 progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included.

[0154] An "encoding scheme" may describe a set of code words that are 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 hereby is a sequence of signs provided by the detection cycles of the method. A sign within a code word is a detectable signal or the absence of a signal. A code word does not need to comprise of all different signals used in the method. The number of signs in a code word is defined by the number of detection cycles.

[0155] An "oligonucleotide" as used herein, refers to s short nucleic acid molecule, such as DNA, PNA, LNA or RNA. The length of the oligonucleotides is within the range 4-200 nucleotides (nt), preferably 6-80 nt, more preferably 8-60 nt, more preferably 10-50 nt, more preferably 12 to 35 depending on the number of consecutive sequence elements. The nucleic acid molecule can be fully or partially single-stranded. The oligonucleotides may be linear or may comprise hairpin or loop structures. The oligonucleotides may comprise modifications such as biotin, labeling moieties, blocking moieties, or other modifications.

[0156] In one embodiment the "analytic probe" is a probe with a detector element that can be measured directly without further reactions (e.g., the detector element is a fluorophore, another dye, a nanoparticle, a mass-tag, an enzyme, or a similar feature that can be detected directly).

[0157] In one embodiment the detector element is an enzyme to perform a reaction at the localization of probe binding, or the detector element is one or more nucleic acid tails that can be used to hybridize oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly or the detector element is one or more tails that can be used to hybridize oligonucleotide which can be used to hybridize another oligonucleotide with a label (e.g. fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly or the detector element is characterized by one or more tails that can be used as a primer for a nucleic acid amplification reaction whose product is used for a hybridization of an oligonucleotide with a label (e.g., fluorophore, masstag, enzyme, nanoparticle, etc.) that can be detected directly or the probe is ligated after binding to the target element and forms a circular probe that can be used for hybridization of one or more oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly or the probe is ligated after binding to the target element and forms a circular probe that can be used for a nucleic acid amplification reaction whose product is used for a hybridization of one or more oligonucleotides with a label (e.g., fluorophore, mass-tag, enzyme, nanoparticle, etc.) that can be detected directly. In another embodiment the "analytic probe" is a probe with a detector element that can be measured only if a second, third, fourth, or even higher ordered reaction is used to detect the probe, such as described hereinunder.

[0158] In one embodiment, the "analyte-specific probe" consists of at least two elements, namely the so- called binding element (S) which specifically interacts with one of the analytes, and a so-called identifier element (T) comprising the 'unique identifier sequence'. The binding element (S) may be a nucleic acid such as a hybridization sequence or an aptamer.

[0159] In one embodiment a "probe" consists of at least two elements, namely the so-called binding element (S) which specifically interacts with one of the analytes, and a so-called identifier element (T) comprising the 'unique identifier sequence'.

[0160] The "unique identifier sequence" as comprised by the 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, Cyclin E etc., or, alternatively, it specifically identifies only a group of analytes, independently whether the group of analytes comprises a gene family or not. Therefore, the analyte or a group of analytes to be encoded by this unique identifier can be distinguished from all other analytes or groups of analytes that are 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 a group of analytes, but not more than one, i.e., not even two. Due to the uniqueness of the unique identifier sequence the identifier element (T) hybridizes to exactly one type of decoding oligonucleotides. The length of the unique identifier sequence is within the range 8-60 nt, preferably 12-40 nt, more preferably 14-20 nt, depending on the number of analytes encoded in parallel and the stability of interaction needed. A unique identifier may be a sequence element of the analyte-specific probe, attached directly or by a linker, a covalent bond or high affinity binding modes, e.g., antibody-antigen interaction, streptavidin-biotin interaction etc. It is understood that the term "analyte specific probe" includes a plurality of probes which may differ in their binding elements (S) in a way that each probe binds to the same analyte but possibly to different parts thereof, for instance to different (e.g., neighboring) or overlapping sections of the nucleotide sequence comprised by the nucleic acid molecule to be encoded. However, each of the plurality of the probes comprises the same identifier element (T). A "bipartite labeling probe" comprises a binding sequence capable of hybridizing the analyte and a binding probe sequence capable of binding a detectable signal molecule like a fluorophore or a nucleic acid sequence comprising a fluorophore.

[0161] A "decoding oligonucleotide" or an "adapter" or a / adapter segment" consists of at least two sequence elements. One sequence element that can specifically bind to a unique identifier sequence, referred to as an "identifier connector element "(t) or "first connector element" (t), and a second sequence element specifically binding to a signal oligonucleotide, referred to as "translator element" (c). The length of the sequence elements is within the range 8-60 nt, preferably 12-40 nt, more preferably 14- 20 nt, de-pending on the number of analytes to be encoded in parallel, the stability of interaction needed, and the number of different signal oligonucleotides used. The length of the two sequence elements may or may not be the same.

[0162] In some advantageous embodiments, the decoding oligonucleotide in the kits and / or methods of the present disclosure may be a "multi-decoder". A "multi-decoder" is a decoding oligonucleotide that consists of at least three sequence elements. One sequence element (the 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 different signal oligonucleotides (each of these sequence elements specifically binds a signal oligonucleotide that differs to all other signal oligonucleotides recruited by other elements of the multi-decoder). The length of the sequence elements is within the range 8-60 nt, preferably 12-40 nt, more preferably 14-20 nt, depending on the number of analytes detected in parallel, the stability needed, and the number of different signal oligonucleotides used. The length of the sequence elements may or may not be the same.

[0163] Therefore, in some advantageous embodiments, the decoding oligonucleotide is a multi-decoder comprising: an identifier connector element (t) comprising a nucleotide sequence which 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) comprising each a nucleotide sequence allowing a specific hybridization of a different signal oligonucleotide.

[0164] Therefore, the first translator element binds a different signal oligonucleotide as the second translator element. In particular, the signal oligonucleotides differ in the signal element comprised in the signal oligonucleotide, e.g., in the kind of the fluorophore. A "signal oligonucleotide" or a "reporter" as used herein comprises at least two elements, a so-called "translator connector element" (C) or "second connector element" (C) having a nucleotide sequence specifically hybridizable to at least a section of the nucleotide sequence of the translator element (c) of the decoding oligonucleotide, and a "signal element" which provides a detectable signal. This element can either actively generate a detectable signal or provide such a signal via manipulation, e.g., fluorescent excitation. Typical signal elements are, for example, enzymes that catalyze a detectable reaction, fluorophores, radioactive elements, or dyes. In some embodiments the "signal oligonucleotide" or a "reporter" may bind directly to the analyte.

[0165] A "set" refers to a plurality of moieties or subjects, e.g., analyte-specific probes or decoding oligonucleotides, whether the individual members of said plurality are identical or different from each other. In an analyte specific probe set, the analyte specific probes are identical in the identifier element (T) but may comprise a different binding element (S) for specifically interacting with the same analyte but for specifically interacting with different sub-structures of the same analyte to be encoded.

[0166] "Selective denaturation" may be the process of eliminating bound decoding oligonucleotides and signal oligonucleotides with highest efficiency while at the same time the target specific probes have to stay hybridized with the highest efficiency. The total efficiency of these two combined events may to be at least 0.22 for two detection cycles, 0.37 for three detection cycles, 0.47 for four detection cycles, 0.55 for five detection cycles, 0.61 for six detection cycles, 0.65 for seven detection cycles, 0.69 for eight detection cycles, 0.72 for nine detection cycles and 0.74 for 10 detection cycles, 0.76 for 11 detection cycles and 0.78 for 12 detection cycles.

[0167] In an embodiment of the disclosure a single set refers to a plurality of oligonucleotides.

[0168] An "analyte specific probe set" refers to a plurality of moieties or sub-jects, e.g., analyte-specific probes that are different from each other and bind to independent regions of the analyte. A single analyte specific probe set is further characterized by the same unique identifier.

[0169] A "decoding oligonucleotide set" refers to a plurality of decoding oligonucleotides specific for a certain unique identifier needed to realize the encoding independent of the length of the code word. Each and all of the decoding oligonucleotides included in a "decoding oligonucleotide set" bind to the same unique identifier element (T) of the analyte-specific probe. In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides may be converted into a "code word." For example, the code words could be also "101" and "110" for an analyte, where a value of 1 represents binding and a value of 0 represents no binding. The code words may also have longer lengths in other embodiments (see Fig. 13). A code word can be directly related to a specific unique identifier sequence of an analyte-specific probe. Accordingly, different analytespecific probe may match certain code words, which can then be used to identify the different analytes of the analyte-specific probe based on the binding patterns of the decoding oligonucleotide. However, if no binding is evident, then the code word would be "000" in this example.

[0170] The values in each code word can also be assigned in different fashions in some embodiments. For example, a value of 0 could represent binding while a value of 1 represents no binding. Similarly, a value of 1 could represent binding of a secondary nucleic acid probe with one type of signaling entity while a value of 0 could represent binding of a secondary nucleic acid probe with another type of distinguishable signaling entity. These signaling entities could be distinguished, for example, via different colors of fluorescence. In some cases, values in code words need not be confined to 0 and 1. The values could also be drawn from larger alphabets, such as ternary (e.g., 0, 1, and 2) or quaternary (e.g., 0, 1, 2, and 3) systems. Each different value could, for example, be represented by a different distinguishable signaling entity, including (in some cases) one value that may be represented by the absence of signal.

[0171] The code words for each analyte may be assigned sequentially or may be assigned at random. For instance, a first analyte may be assigned to 101, while a second nucleic acid target may be assigned to 110. In addition, in some embodiments, the code words may be assigned using an error-detection system or an error- correcting 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). Such systems can be used to identify where errors have occurred, and in some cases, such systems can also be used to correct the errors and determine what the correct code word should have been. For example, a code word such as 001 may be detected as invalid and corrected using such a system to 101, e.g., if 001 is not previously assigned to a different target sequence. A variety of different errorcorrecting codes can be used, many of which have previously been developed for use within the computer industry; however, such error-correcting systems have not typically been used within biological systems. Additional examples of such error-correcting codes are discussed in more detail below.

[0172] T1 "Essentially complementary" means, when referring to two nucleotide sequences, that both sequences can specifically hybridize to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule with a sense and an antisense strand connected to each other via hydrogen bonds (Watson-and-Crick base pairs). "Essentially complementary" includes not only perfect basepairing along the entire strands, i.e., perfect complementary sequences but also imperfect complementary sequences which, however, still have the capability to hybridize to each other under stringent conditions. Among experts it is well accepted that an "essentially complementary" sequence has at least 88% sequence identity to a fully or perfectly complementary sequence.

[0173] "Percent sequence identity" or "percent identity" in turn means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the "Reference Sequence"). The percent identity is then determined according to the following formula: percent identity = 100 [1 -(C / R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein

[0174] (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and

[0175] (ii) each gap in the Reference Sequence and

[0176] (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

[0177] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity, then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.

[0178] In the "incubation" steps as understood herein the respective moieties or subjects such as probes or oligonucleotide, are brought into contact with each other under conditions well known to the skilled person allowing a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions etc. Such steps may, therefore, be preferably carried out in a liquid environment such as a buffer system which is well known in the art.

[0179] The "removing" steps according to the disclosure may include the washing away of the moieties or subjects to be removed such as the probes or oligonucleotides by certain conditions, e.g., pH, temperature, salt conditions etc., as known in the art.

[0180] It is understood that in an embodiment of the method according to the present disclosure a plurality of 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 differ 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 binds to analyte 2, the analyte-specific probes of set 3 binds to analyte 3, etc. In this embodiment also the use of different sets of decoding oligonucleotides is required in the methods according to the present disclosure.

[0181] The decoding oligonucleotides of a particular set differ from the decoding oligonucleotides of another set. This means, the decoding oligonucleotides of set 1 bind to the analyte-specific probes of above set 1 of analyte-specific probes, the decoding oligonucleotides of set 2 binds to the analyte-specific probes of above set 2 of analyte-specific probes, the decoding oligonucleotides of set 3 binds to the analytespecific probes of above set 3 of analyte-specific probes, etc.

[0182] In this embodiment where a plurality of analytes is to be encoded in parallel the different sets of analyte-specific probes may be provided as a premixture of different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided as a premixture of different sets of decoding oligonucleotides. Each mixture may be contained in a single vial. Alternatively, the different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided in steps singularly.

[0183] A "kit" is a combination of individual elements useful for carrying out the use and / or method of the disclosure, wherein the elements are optimized for use together in the methods. The kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the disclosure. Such kits unify all essential elements required to work the method according to the disclosure, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the present disclosure. The term "quencher" or "quencher dye" or "quencher molecule" refers to a dye or an equivalent molecule, such as nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which is capable of reducing the fluorescence of a fluorescent reporter dye or donor dye. A quencher dye may be a fluorescent dye or non-fluorescent dye. When the quencher is a fluorescent dye, its fluorescence wavelength is typically substantially different from that of the reporter dye and the quencher fluorescence is usually not monitored during an assay. Some embodiments of the present disclosure disclose signal oligonucleotides comprising a quencher and / or a quencher in combination with a signal element (see Fig. 14), and therefore the signal oligonucleotides is not detectable during imaging.

[0184] In an embodiment of the disclosure the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells. A biological sample may be derived from an organ, organoids, cell cultures, stem cells, cell suspensions, primary cells, samples infected by viruses, bacteria, or fungi, eukaryotic or prokaryotic samples, smears, disease samples, a tissue section.

[0185] The method is particularly qualified to encode, identify, detect, count, or quantify analytes or single analytes molecules in a biological sample, i.e., such as a sample which contains nucleic acids as said analytes. It is understood that the biological sample may be in a form as it is in its natural environment (i.e., liquid, semi-liquid, solid etc.), or processed, e.g., as a dried film on the surface of a device which may be re-liquefied before the method is carried out.

[0186] In another embodiment of the disclosure prior to step (2) the biological tissue and / or biological cells are fixed. For example, in some embodiments, the cell and / or the tissue is fixed prior to introducing the probes, e.g., to preserve the positions of the analytes like nucleic acids within the cell. Techniques for fixing cells are known to those of ordinary skill in the art. As non-limiting examples, a cell may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, or the like. In one embodiment, a cell may be fixed using HEPES-glutamic acid buffer- mediated organic solvent (HOPE).

[0187] This measure has the advantage that the analytes to be encoded, e.g., the nuclei acids, are immobilized and cannot escape. In doing so, the analytes then prepared for a better detection or encoding by the method according to the disclosure. In yet a further embodiment within the set of analyte-specific probes the individual analytespecific probes comprise binding elements (SI, S2, S3, S4, S5) which specifically interact with different sub-structures of one of the analytes to be encoded.

[0188] By this measure the method becomes even more robust and reliable because the signal intensity obtained at the end of the method or a cycle, respectively, is increased. It is understood that the individual probes of a set while binding to the same analyte differ in their binding position or binding site at or on the analyte. The binding elements SI, S2, S3, S4, S5 etc. of the first, second, third fourth, fifth etc. analyte-specific probes therefore bind to or at a different position which, however, may or may not overlap.

[0189] In an advantageous embodiment, the present disclosure pertains to kit for multiplex analyte encoding, comprising:

[0190] (A) at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising

[0191] (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and

[0192] (bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein 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), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and

[0193] (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 which 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

[0194] (bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and

[0195] (C) a set of signal oligonucleotides, each signal oligonucleotide comprising:

[0196] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and

[0197] (bb) a signal element.

[0198] A multiplex method or assay allow the simultaneously or sequential measurement of multiple analytes. According to the present disclosure it may be used to determine simultaneously or sequential the presence or absence of a plurality of predetermined (known) analytes like genomic as well as extra- genomic (e.g., mRNA) 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 the that target.

[0199] In some advantageous embodiments according to the present disclosure at least 20, in particular at least 25, in particular at least 30 different analytes are detected and / or quantified in a sample in parallel. For example, there may be 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 that are applied to a sample, e.g., simultaneously or sequentially.

[0200] In some advantageous embodiments for the multiplexing twenty (20) or more different sets of analytespecific probes for encoding of at least 20 different analytes or more are required, in particular more than 50, more than 100 or more than 200. In the multiplexing methods of the present disclosure, in particular at least 20 different groups of analytes (e.g., mRNA molecules) i.e., tags are targeted.

[0201] In some advantageous embodiments, at least 4 rounds to collect information for identification of the analyte are carried out, wherein multiple readout increases the accuracy of identification and avoids false positives. The unique tag can be identified by various techniques, including hybridization, e.g., with labeled probes, directly or indirectly or by sequencing (by synthesis, ligation). In particular, the identity of the tag can be encoded with one single signal (binary code), two or more signals, wherein the signal can be a fluorescent label (e.g., attached to an oligonucleotide).

[0202] In some advantageous embodiments according to the present disclosure, the kit does not comprise sets of analyte-specific probes as defined under item A).

[0203] Preferably, if the analyte in the kits or methods according to the present disclosure is a nucleic acid, each set of analyte-specific probes comprises at least five (10) analyte-specific probes, in particular at least fifteen (15) analyte-specific probes, in particular at least twenty (20) analyte-specific probes which specifically interact with different sub-structures of the same analyte. Nucleic acid analyte includes specific DNA molecules, e.g., genomic DNA, nuclear DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA etc., and specific mRNA molecules, e.g., hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, etc.

[0204] In some advantageous embodiments according to the present disclosure the kit comprises at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

[0205] In particular, the kit may comprise at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analytespecific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0206] In some embodiments the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets for at least one analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide. In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interacts with identical identifier elements (T) which are unique to a particular analyte. In particular, all sets of decoding oligonucleotides for the different analytes may comprise the same type(s) of translator element(s) (c).

[0207] In another aspect, the present disclosure is generally directed to methods including acts of exposing a sample to a plurality of analyte-specific probes; for each of the analyte-specific probes, determining binding of the analyte-specific probes within the sample; creating code words based on the binding of the analyte-specific probes, the decoding oligonucleotides and the signal oligonucleotides; and for at least some of the code words, matching the code word to a valid code word. In certain embodiments, this pattern of binding or hybridization of the analyte-specific probes, the decoding oligonucleotides and the signal oligonucleotides may be converted into a "code word." For example, for instance, the code words may 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 decoding oligonucleotides and / or the binding of signal oligonucleotides without and / or quenched signal element. The analyte in the detection round / cycle is therefore not detectable during imaging.

[0208] To create such a zero (0) in a code word for an individual analyte the kit may comprise:

[0209] (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

[0210] To create such a zero (0) in a code word for an individual analyte the kit may comprise:

[0211] (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and comprise a translator element that does not interact / bind to a signal oligonucleotide due to an instable binding sequence and / or due to the translator element is to short (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0212] In some advantageous embodiments, the kit comprises:

[0213] (D) at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

[0214] In some advantageous embodiments, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

[0215] Furthermore, in some advantageous embodiments the kit may comprise:

[0216] (E) a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising:

[0217] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0218] In some advantageous embodiments, the kit comprises:

[0219] (E) at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising:

[0220] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0221] In some advantageous embodiments, the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

[0222] Further, in some embodiments the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte. In some advantageous embodiments, the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately. In some advantageous embodiments, the different sets of analyte-specific probes may be comprised in a premixture of different sets of analyte-specific probes or exist separately. In some advantageous embodiments, the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0223] In some advantageous embodiments, a mixture of decoding oligonucleotides and / or multi-decoders is provided that specifically hybridize to the unique identifier sequences of the probe sets. In some embodiments, the decoding oligonucleotides comprise of at least two sequence elements, a first element that is complementary to the unique identifier sequences of the corresponding probe set and a second sequence element (translator element) that provides a sequence for the specific hybridization of a signal oligonucleotide, the translator element defines the type of signal that is recruited to the decoding oligonucleotide. In some embodiments multi-decoders comprising at least three sequence elements are used, a first element that is complementary to the unique identifier sequences of the corresponding probe set and at least to additional sequence elements (translator elements) that provide sequences for the specific hybridization of at least two different signal oligonucleotides The translator elements define the type of signals that are recruited to the multidecoder. Different possible structures of a multi-decoder can be seen in Fig. 15. Since a multi-decoder does recruit a full signal oligonucleotide per translator element, the brightness of the signals in each channel is not lower than the brightness of signals with decoding oligonucleotides.

[0224] The usage of multi-decoders increases further the efficiency of the encoding scheme. Figure 16 shows a possible encoding scheme using multi-decoders based upon the same conditions used for the examples with the decoding oligonucleotide with two sequence elements. One can clearly see that the multi-decoder based encoding scheme can create a higher hamming distance, with the same number of rounds and the same number of different signal oligonucleotides used in the example of Fig. 5.

[0225] As mentioned above the analyte to be encoded may be a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA, and / or mixtures thereof.

[0226] The present disclosure pertains to a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes, comprising the steps of: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising

[0227] (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and

[0228] (bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein 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), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and

[0229] (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:

[0230] (aa) an identifier connector element (t) comprising a nucleotide sequence which 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

[0231] (bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and

[0232] (C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:

[0233] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and

[0234] (bb) a signal element.

[0235] (D) Detecting the signal caused by the signal element; (E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded;

[0236] (F) Performing at least three (3) further cycles comprising steps B) to E) to generate an encoding scheme with a code word per analyte, wherein in particular the last cycle may stop with step (D).

[0237] As mentioned above, the method according to the present disclosure comprises selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analyte to be encoded. All steps are performed sequentially. However, some steps may be performed simultaneously or sequential, the contacting steps A) to C), in particular B) and C).

[0238] By this measure the requirements for another round / cycle of binding further decoding oligonucleotides to the same analyte-specific probes are established, thus finally resulting in a code, or encoding scheme comprising more than one signal. This step is realized by applying conditions and factors well known to the skilled person, e.g., pH, temperature, salt conditions, oligonucleotide concentration, polymers etc.

[0239] In another embodiment of the present disclosure, the method may comprise repeating steps (B)-(E) at least three times to generate an encoding scheme. With this measure a code of four signals in case of four cycles / rounds which are carried out by the user, where 'n' is an integer representing the number of rounds. The encoding capacity of the method according to the disclosure is herewith increased depending on the nature of the analyte and the needs of the operator. In an embodiment of the disclosure said encoding scheme is predetermined and allocated to the analyte to be encoded.

[0240] However, this measure enables a precise experimental set-up by providing the appropriate sequential order of the employed decoding and signal oligonucleotides and, therefore, allows the correct allocation of a specific analyte to a respective encoding scheme. The decoding oligonucleotides which are used in repeated steps (B)-(D2) may comprise a translator element (c2) which is identical with the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). In another embodiment of the disclosure decoding oligonucleotides are used in repeated steps (B)-(E) comprising a translator element (c2) which differs from the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). It is understood that the decoding elements may or may not be changed from round to round, i.e., in the second round (B)-(E) comprising the translator element c2, in the third round (B)-(E) comprising the translator element c3, in the fourth round (B)-(E) comprising the translator element c4 etc., wherein 'n' is an integer representing the number of rounds.

[0241] The signal oligonucleotides which are used in repeated steps (B)-(E) may comprise a signal element which is identical with the signal element of the decoding oligonucleotides used in previous steps (B)- (E). In a further embodiment of the disclosure signal oligonucleotides are used in repeated steps (B)- (E) comprising a signal element which differs from the signal element of the decoding oligonucleotides used in previous steps (B)-(E). In some embodiments no-signal oligonucleotides and / or no-signal decoding oligonucleotides for an individual analyte are used, resulting to the value 0 in the code word for this cycle / position. In some embodiments in a repeated cycle no decoding oligonucleotides for an individual analyte is contacted with the sample resulting also to the value 0 in the code word for this cycle / position.

[0242] By this measure each round the same or a different signal is provided resulting in an encoding scheme characterized by a signal sequence consisting of numerous different signals. This measure allows the creation of a unique code or code word which differs from all other code words of the encoding scheme. In another embodiment of the disclosure, the binding element (S) of the analyte-specific probe comprises a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0243] In some advantageous embodiments, all steps are automated, in particular wherein steps B) to F) are automated, in particular by using a robotic system and / or an optical multiplexing system according to the present disclosure. In some examples, the steps may be performed in a fluidic system.

[0244] As mentioned above, with the methods according to the present disclosure an encoding scheme with a code word per analyte is generated. Therefore, each analyte may be associated with a specific code word, wherein said code word comprise a number of positions, and wherein each position corresponds to one cycle resulting in a plurality of distinguishable encoding schemes with the plurality of code words. In particular, said encoding scheme may be predetermined and allocated to the analyte to be encoded.

[0245] In some advantageous embodiments, the code words obtained for the individual analytes in the performed cycles comprise the detected signals and additionally at least one element corresponding to no detected signal like 0,1 or 0,1,2 etc. (see also Fig. 13 and Fig. 14). No signal is detected for at least one analyte within at least one cycle if using a non-signal probe according to Figure 14, No. 2 to 4, or a non-signal decoding oligonucleotide as shown in Figure 14 No. 5, or if in one cycle no decoding oligonucleotide is contacted with the corresponding identifier sequence comprised on analyte-specific probe interacting with the corresponding analyte in the sample. In this cycle the position has the value zero (0).

[0246] In some advantageous embodiments, at least for one individual analyte a position of the code word is zero (0).ln particular, the code word zero (0) is generated by using no decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte. As mentioned above, in some embodiments, if at least for one individual analyte a position of the code word is zero (0) in this cycle no corresponding decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte are used.

[0247] Furthermore, in some advantageous embodiments the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

[0248] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0249] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein only one set of decoding oligonucleotides per analyte is used per cycle, and / or wherein different sets of decoding oligonucleotides are used in different cycles in combination with the corresponding set of signal oligonucleotides in the same cycle.

[0250] In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). All sets of decoding oligonucleotides for the different analytes may comprise the same type(s) of translator element(s) (c).

[0251] In some advantageous embodiments of the method according to the present disclosure, the sample is contacted with at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

[0252] As mentioned 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 analytespecific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

[0253] In some advantageous embodiments of the method according to the present disclosure, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately. Furthermore, in some advantageous embodiments of the method according to the present disclosure, the sample is contacted with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising:

[0254] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0255] In further embodiments, the sample may be contacted with: at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising:

[0256] (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0257] As mentioned above, the different sets of non-signal oligonucleotides may be comprised in a premixture of different sets of non-signal oligonucleotides or exist separately.

[0258] In further embodiments, the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

[0259] As mentioned above, the different sets of decoding oligonucleotides may be comprised in a premixture of different sets of decoding oligonucleotides or exist separately as well as the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately as well the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0260] In some advantageous embodiments of the method according to the present disclosure, the binding element (S) comprise a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0261] In some advantageous embodiments of the method according to the present disclosure, after step A) and before step B) the non-bound analyte-specific probes may be removed, in particular by washing, further after step B) and before step C) the non-bound decoding oligonucleotides may be removed, in particular by washing further, after step C) and before step D) the non-bound signal oligonucleotides may be removed, in particular by washing. In some advantageous embodiments of the method according to the present disclosure, the analyte specific probes may be incubated with the sample, thereby allowing a specific binding of the analyte specific probes to the analytes to be encoded, further the decoding oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the decoding oligonucleotides to identifier elements (T) of the respective analyte-specific probes, further the signal oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the signal oligonucleotides to translator elements (T) of the respective decoding oligonucleotides.

[0262] As mentioned above, the analyte to be encoded may be a nucleic acid, preferably DNA, PNA, RNA, in particular mRNA, or combinations thereof.

[0263] By this measure the method is further developed to such an extent that the encoded analytes can be detected by any means which is adapted to visualize the signal element. Examples of detectable physical features include e.g., light, chemical reactions, molecular mass, radioactivity, etc.

[0264] In some advantageous embodiments, the signal caused by the signal element, therefore in particular the binding of the signal oligonucleotides to the decoding oligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte is determined by:

[0265] (a) Imaging at least a portion of the sample; and / or

[0266] (b) Using an optical imaging technique; and / or

[0267] (c) Using a fluorescence imaging technique; and / or

[0268] (d) Multi-color fluorescence imaging technique; and / or

[0269] (e) Super-resolution fluorescence imaging technique.

[0270] The kits and method according to the present disclosure may be used ideally for in vitro methods for diagnosis of a disease selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases.

[0271] Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for diagnosis of a disease in plants selected from the group comprising: diseases caused by biotic stress, preferably by infectious and / or parasitic origin, or diseases caused by abiotic stress, preferably caused by nutritional deficiencies and / or unfavorable environment. Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for screening, identifying and / or testing a substance and / or drug comprising:

[0272] (a) contacting a test sample comprising a sample with a substance and / or drug

[0273] (b) detecting different analytes in a sample by sequential signal-encoding of said analytes with a method according to the present disclosure.

[0274] An optical multiplexing system suitable for the method according to the present disclosure, comprising at least: a reaction vessel for containing the kits or part of the kits according to the present disclosure; a detection unit comprising a microscope, in particular a fluorescence microscope; a camera; a liquid handling device.

[0275] In some embodiments, optical multiplexing system may comprise further a heat and cooling device and / or a robotic system.

[0276] In some embodiments, the method according to the present disclosure encodes a nucleic acid analyte, such as an mRNA, e.g., such an mRNA coding for a particular protein.

[0277] In some advantageous embodiments, the method described herein is used for specific detection of many different analytes in parallel. The technology allows to distinguish a higher number of analytes than different signals are available. The process includes at least four consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called "decoding"-oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides. In a specific embodiment, the method may comprise the steps of 1. providing one or more analyte specific probe sets, the set of analyte specific probes consist of one or more different probes, each differing in the binding moiety that specifically interacts with the analyte, all probes of a single probe set are tethered to a sequence element (unique identifier), that is unique to a single probe set and allows the specific hybridization of a decoding oligonucleotide, 2. specific binding of the probe sets to their target binding sites of the analyte, 3. eliminating non-bound probes (e.g. by a wash step), 4. providing a mixture of decoding oligonucleotides that specifically hybridize to the unique identifier sequences of the probe sets, the decoding oligonucleotides comprise of at least two sequence elements, a first element that is complementary to the unique identifier sequences of the corresponding probe set and a second sequence element (translator element) that provides a sequence for the specific hybridization of a signal oligonucleotide, the translator element defines the type of signal that is recruited to the decoding oligonucleotide, 5. specific hybridization of the decoding oligonucleotides to the unique identifier sequences provided by the bound probe sets, 6. eliminating non-bound decoding oligonucleotides (e.g. by washing step), 7. providing a mixture of signal oligonucleotides, comprising of a signal that can be detected and a nucleic acid sequence that specifically hybridizes to the translator element of one of the decoding oligonucleotides used in the former hybridization step, 8. specific hybridization of the signal oligonucleotides, 9. eliminating nonbound signal oligonucleotides, 10. detection of the signals, 11. selective release of decoding oligonucleotides and signal oligonucleotides while the binding of specific probe sets to the analyte is almost or completely unaffected, 12. eliminating released decoding oligonucleotide and signal oligonucleotides (e.g. by a washing step) while the binding of specific probes sets to the analytes is almost or completely unaffected , repeating the steps 4 to 12 at least three times until the detection of a sufficient number of signals to generate an encoding scheme for each different analyte of interest. It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respective case, but also in other combinations or in an isolated manner without departing from the scope of the disclosure.

[0278] The disclosure is now further explained by means of embodiments resulting in additional features, characteristics, and advantages of the disclosure. The embodiments are of pure illustrative nature and do not limit the scope or range of the disclosure. The features mentioned in the specific embodiments are general features of the disclosure which are not only applicable in the specific embodiment but also in an isolated manner in the context of any embodiment of the disclosure.

[0279] The method disclosed herein is used for specific detection of many different analytes in parallel. The technology allows distinguishing a higher number of analytes than different signals are available. The process preferably includes at least two consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called "decoding" oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides.

[0280] The present disclosure pertains further to methods of detecting an analyte, comprising: attaching a plurality of analyte-specific probes to the analyte, wherein the analyte-specific probes independently attach to the analyte and wherein the analyte-specific probes share a common identifier segment (T); annealing a plurality of first decoding oligonucleotides to the analyte-specific probes, wherein the first decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second common region; annealing a first signal oligonucleotide to at least one of the pluralities of first decoding oligonucleotides such that an oligo tethered to the first signal oligonucleotide is reverse complementary to the second common region; detecting the first signal oligonucleotide; removing the plurality of first decoding oligonucleotides; annealing a plurality of second decoding oligonucleotides to the analyte-specific probes, wherein the second decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second decoding oligonucleotides second common region that differs from the second common region of the first decoding; annealing a second signal oligonucleotide to at least one of the pluralities of second decoding oligonucleotides such that an oligo tethered to the signal oligonucleotide is reverse complementary to the second decoding oligonucleotide second common region; and detecting the second signal oligonucleotide. In the above-mentioned embodiment a second aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes. Furthermore, a first aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes.

[0281] In some embodiments, no second signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed. No third signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed.

[0282] In some further advantageous embodiments, the present disclosure pertains to a method of assigning an analyte to a position in an image, comprising assigning a fluorescence pattern to the analyte, observing the fluorescence pattern at the position in the image, and assigning the analyte to the position, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tagged oligo drawn from a re-accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tag-recruiting bridging oligo drawn from a re-accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant.

[0283] In some further advantageous embodiments, the present disclosure pertains to a composition comprising a cell having nucleic acids distributed therein, wherein a first nucleic acid is tagged by a first plurality of probes that target adjacent segments of the first nucleic acid and that share a common first tether segment; a second nucleic acid is tagged by a second plurality of probes that target adjacent segments of the second nucleic acid and that share a common second tether segment; and a third nucleic acid is tagged by a third plurality of probes that target adjacent segments of the third nucleic acid and that share a common third tether segment; a first adapter population comprising molecules having a first tether reverse complementary region and a first fluorophore adapter tether; a second adapter population comprising molecules having a second tether reverse complementary region and a second fluorophore adapter tether; a third adapter population comprising molecules having a third tether reverse complementary region and a first fluorophore adapter tether; a population of first fluorophores having a first tether reverse complementary region; and a population of second fluorophores having a second tether reverse complementary region.

[0284] In some further advantageous embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising subjecting the cell to a plurality of detection rounds, each detection round comprising contacting the cell to representatives of the same at least two populations of tagged fluorescence moieties, and removing the fluorescent moieties after a single excitation event, wherein the number of patterns detectable increases exponentially with the number of detection rounds, wherein the fluorescence moieties are not tagged with nucleic acid tags that are specific to the target nucleic acids, and wherein separate aliquots of common tagged fluorescence moieties are used across multiple detection rounds.

[0285] With the above notified method a total decoding efficiency of at least 30% may be achieved.

[0286] In some further advantageous embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising: contacting a target to a bipartite labeling probe, the bipartite labeling probe comprising a target-specific moiety and a fluorophore-specifying moiety; contacting the bipartite labeling probe to a first aliquot of a fluorophore reservoir comprising no more than two populations of fluorophores; replacing the fluorophore specifying moiety in the bipartite probe, and contacting the bipartite labeling probe to a second aliquot of the fluorophore reservoir comprising the same no more than two populations.

[0287] In some embodiments of the above notified method, replacing the fluorophore specifying moiety in the bipartite probe comprises denaturing a binding between a target-specific moiety and a fluorophore-specifying moiety after subjecting the bipartite labeling probe bound to a fluorophore of the fluorophore to excitation energy. Replacing the fluorophore specifying moiety in the bipartite probe comprises drawing from one of no more than two fluorophore specifying moiety reservoirs.

[0288] In some further advantageous embodiments, the present disclosure pertains to a method of detecting an analyte, comprising: attaching a plurality of probes to the analyte, in particular a nucleic acid, wherein the probes independently attach / anneal to the analyte and wherein the probes share a common identifier segment; annealing a plurality of first adapter segments to the probes, wherein the first adapter segments share a first common region that is reverse complementary to the common identifier segment and a second common region, in particular configured to accommodate a single reporter / selected from no more than two reporter categories; annealing a first reporter to at least one of the plurality of first adapter segments such that an oligo tethered to the first reporter is reverse complementary to the second common region; detecting the first reporter; removing the plurality of first adapter segments, in particular without annealing a second reporter to the at least one of the pluralities of first adapter segments; annealing a plurality of second adapter segments to the probes, wherein the second adapter segments share a first common region that is reverse complementary to the common identifier segment and a second adapter second common region that differs from the second common region of the first adapter segments, in particular configured to accommodate a single reporter / selected from no more than two reporter categories; annealing a second reporter to at least one of the pluralities of second adapter segments such that an oligo tethered to the second reporter is reverse complementary to the second adapter second common region; and detecting the second reporter, in particular without annealing a third reporter to the at least one of the pluralities of first adapter segments.

[0289] METHODS AND EXAMPLES

[0290] In an application variant, the analyte or target is nucleic acid, e.g., DNA or RNA, and the probe set comprises oligonucleotides that are partially or completely complementary to the whole sequence or a subsequence of the nucleic acid sequence to be detected (Figure 1). The nucleic acid sequence specific oligonucleotide probe sets comprising analyte-specific probes (1) including a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, and an identifier element (T) comprising a nucleotide sequence which is unique to said set of analyte-specific probes (unique identifier sequence).

[0291] In a further application variant, at least one analyte is a nucleic acid and at least a second analyte is a nucleic acid and at least the first probe set binds to the nucleic acid sequence and at least the second probe set binds specifically to the nucleic acid analyte. Other combinations are possible as well.

[0292] An Embodiments of the general method of the present disclosure may be: Step 1: Applying the at least 20 analyte- or target-specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides with sequences complementary to the target nucleic acid. In this example, a probe set of 5 different probes is shown, each comprising a sequence element complementary to an individual subsequence of the target nucleic acid sequence (SI to S5). In this example, the regions do not overlap. Each of the oligonucleotides targeting the same nucleic acid sequence comprises the identifier element or unique identifier sequence (T), respectively.

[0293] Step 2: Hybridization of the probe set. The probe set is hybridized to the target nucleic acid sequence under conditions allowing a specific hybridization. After the incubation, the probes are hybridized to their corresponding target sequences and provide the identifier element (T) for the next steps.

[0294] Step 3: Eliminating non-bound probes. After hybridization, the unbound oligonucleotides are eliminated, e.g., by washing steps.

[0295] Step 4: Applying the decoding oligonucleotides. The decoding oligonucleotides consisting of at least two sequence elements (t) and (c) are applied. While sequence element (t) is complementary to the unique identifier sequence (T), the sequence element (c) provides a region for the subsequent hybridization of signal oligonucleotides (translator element).

[0296] Step 5: Hybridization of decoding oligonucleotides. The decoding oligonucleotides are hybridized with the unique identifier sequences of the probes (T) via their complementary first sequence elements (t). After incubation, the decoding oligonucleotides provide the translator sequence element (c) for a subsequent hybridization step.

[0297] Step 6: Eliminating the excess of decoding oligonucleotides. After hybridization, the unbound decoding oligonucleotides are eliminated, e.g., by washing steps.

[0298] Step 7: Applying the signal oligonucleotide. The signal oligonucleotides are applied. The signal oligonucleotides comprise 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 oligonucleotides. The signal oligonucleotides are hybridized via the complementary sequence connector element (C) to the translator element (c) of decoding oligonucleotide. After incubation, the signal oligonucleotides are hybridized to their corresponding decoding oligonucleotides and provide a signal (F) that can be detected.

[0299] Step 9: Eliminating the excess of signal oligonucleotides. After hybridization, the unbound signal oligonucleotides are eliminated, e.g., by washing steps.

[0300] Step 10: Signal detection. The signals provided by the signal oligonucleotides are detected.

[0301] The following steps (steps 11 and 12) are unnecessary for the last detection round.

[0302] Step 11: Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotides is dissolved. The destabilization can be achieved via different mechanisms well known to the trained person like for example: increased temperature, denaturing agents, etc. The target- or analyte-specific probes are not affected by this step.

[0303] Step 12: Eliminating the denatured decoding oligonucleotides. The denatured decoding oligonucleotides and signal oligonucleotides are eliminated (e.g., by washing steps) leaving the specific probe sets with free unique identifier sequences, reusable in a next round of hybridization and detection (steps 4 to 10). This detection cycle (steps 4 to 12) is repeated at least four times until the planed encoding scheme is completed.

[0304] Another Embodiment of the general method of the present disclosure using multi-decoders may be (Fig. 16):

[0305] Step 1: Target nucleic acids: In this example three different target nucleic acids (A), (B) and (C) must be detected and differentiated by using only two different types of signal oligonucleotides. Before starting the experiment, a certain encoding scheme is set. In this example, the three different nucleic acid sequences are encoded by three rounds of detection with three different signal types (1), (2) and (1 / 2) and a resulting hamming distance of 3 to allow for error detection. The planed code words are: sequence A: (1) - (1) - (2) sequence B: (2) - (2) - (1 / 2) sequence C: (1 / 2) - (1 / 2) - (1)

[0306] Step 2: Hybridization of the probe sets: For each target nucleic acid, an own probe set is applied, specifically hybridizing to the corresponding nucleic acid sequence of interest. Each probe set provides a unique identifier sequence (Tl), (T2) or (T3). This way each different target nucleic acid is uniquely labeled. In this example sequence (A) is labeled with (Tl), sequence (B) with (T2) and sequence (C) with (T3). The illustration in Fig. 16 summarizes Steps 1 to 3 of Fig. 3.

[0307] Step 3: Hybridization of the decoding oligonucleotides and multi-decoders: For each unique identifier present, a certain decoding oligonucleotide or multi-decoder is applied specifically hybridizing to the corresponding unique identifier sequence by its first sequence element (here (tl) to (Tl), (t2) to (T2) and (t3) to (T3)). Each of the decoding oligonucleotides or multidecoders provides a translator or two translator elements that define the signals that will be generated after hybridization of signal oligonucleotides. Here nucleic acid sequence (A) is labeled with (cl), (B) is labeled with (c2) and (C) is labeled with both translator elements (cl) and (c2) resulting in the signal (1 / 2). The illustration in Fig. 16 summarizes steps 4 to 6 of Fig. 3.

[0308] Step 4: Hybridization of signal oligonucleotides: For each type of translator element, a signal oligonucleotide with a certain signal, differentiable from signals of other signal oligonucleotides, is applied. This signal oligonucleotide can specifically hybridize to the corresponding translator element. The illustration in Fig. 16 summarizes steps 7 to 9 of Fig. 3.

[0309] Step 5: Signal detection for the encoding scheme: The different signals are detected. Note that in this example the nucleic acids (A), (B) and (C) can already be distinguished after the first round of detection. This is in contrast to the step 5 of Fig. 5 explained by the additional signal type (1 / 2) that can be realized due to multi-decoders. Although nucleic acid sequences can already be distinguished, the additional rounds contribute to the planned hamming distance of 3. The illustration in Fig. 16 corresponds to step 10 of Fig. 3.

[0310] Step 6: Selective denaturation: The decoding (and signal) oligonucleotides and / or multidecoders of all nucleic acid sequences to be detected are selectively denatured and eliminated as described in steps 11 and 12 of Fig.3. Afterwards the unique identifier sequences of the different probe sets can be used for the next round of hybridization and detection.

[0311] Step 7: Second round of detection: A next round of hybridization and detection is done as described in steps 3 to 5. Note that in this new round the mix of different decoding oligonucleotides and multi-decoders is changed. For example, decoding oligonucleotide of nucleic acid sequence (A) used in the first round comprised of sequence elements (tl) and (cl) while the new multi decoder of round 2 comprises of the sequence elements (tl), (cl) and (c2). Note that now a hamming distance of 2 is already given after 2 rounds, which is the final result of the example in Fig. 3 after 3 rounds.

[0312] Step 8: Third round of detection: Again, a new combination of decoding oligonucleotides and / or multi-decoders is 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 comprise a hamming distance of 3 to other code words. Due to the hamming distance, an error in the detection of the signals (signal exchange) would not result in a valid code word and therefore could be detected and because of hamming distance 3 also corrected, in contrast to the encoding scheme of Fig. 3. This way three different nucleic acids can be distinguished in three detection rounds with two different signals, allowing an error detection and correction.

[0313] Note that in every round of detection, the type of signal provided by a certain unique identifier is controlled by the use of a certain decoding oligonucleotide. As a result, the sequence of decoding oligonucleotides applied in the detection cycles transcribes the binding specificity of the probe set into a unique signal sequence.

[0314] The steps of decoding oligonucleotide hybridization (steps 4 to 6) and signal oligonucleotide hybridization (steps 7 to 9) can also be combined in two alternative ways as shown in Figure 4.

[0315] Opt. 1: Simultaneous hybridization. Instead of the steps 4 to 9 of Figure 3, specific hybridization of decoding oligonucleotides and signal oligonucleotides can also be done simultaneously or sequential leading to the same result as shown in step 9 of Figure 3, after eliminating the excess decoding- and signal oligonucleotides. Opt. 2: Preincubation. Additionally, to option 1 of Figure 3, decoding- and signal oligonucleotides can be preincubated in a separate reaction before being applied to the target nucleic acid with the already bound specific probe set.

[0316] 1. Example for signal encoding of three different nucleic acid sequences by two different signal types and three detection rounds

[0317] Figure 3 shows the general concept of generation and detection of specific signals mediated by decoding oligonucleotides. It does not show the general concept of 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 with three different nucleic acid sequences. In this example, the encoding scheme includes error detection.

[0318] Step 1: Target nucleic acids. In this example three different target nucleic acids (A), (B) and (C) have to be detected and differentiated by using only two different types of signals. Before starting the experiment, a certain encoding scheme is set. In this example, the three different nucleic acid sequences are encoded by three rounds of detection with two different signals (1) and (2) and a resulting hamming distance of 2 to allow for error detection. The planed code words are: sequence A: (1) - (2) - (2); sequence B: (1) - (1) - (1); sequence C: (2) - (1) - (2).

[0319] Step 2: Hybridization of the probe sets. For each target nucleic acid, an own probe set is applied, specifically hybridizing to the corresponding nucleic acid sequence of interest. Each probe set provides a unique identifier sequence (Tl), (T2) or (T3). This way each different target nucleic acid is uniquely labeled. In this example sequence (T) is labeled with (Tl), sequence (B) with (T2) and sequence (C) with (T3). The illustration summarizes steps 1 to 3 of Figure 3.

[0320] Step 3: Hybridization of the decoding oligonucleotides. For each unique identifier present, a certain decoding oligonucleotide is applied specifically hybridizing to the corresponding unique identifier sequence by its first sequence element (here (tl) to (Tl), (t2) to (T2) and (t3) to (T3)). Each of the decoding oligonucleotides provides a translator element that defines the signal that will be generated after hybridization of signal oligonucleotides. Here nucleic acid sequences (A) and (B) are labeled with the translator element (cl) and sequence (C) is labeled with (c2). The illustration summarizes steps 4 to 6 of Figure 3.

[0321] Step 4: Hybridization of signal oligonucleotides. For each type of translator element, a signal oligonucleotide with a certain signal (2), differentiable from signals of other signal oligonucleotides, is applied. This signal oligonucleotide can specifically hybridize to the corresponding translator element. The illustration summarizes steps 7 to 9 of Figure 3.

[0322] Step 5: Signal detection for the encoding scheme. The different signals are detected. Note that in this example the nucleic acid sequence (C) can be distinguished from the other sequences by the unique signal (2) it provides, while sequences (A) and (B) provide the same kind 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 of Figure 3.

[0323] Step 6: Selective denaturation. The decoding (and signal) oligonucleotides of all nucleic acid sequences to be detected are selectively denatured and eliminated as described in steps 11 and 12 of Figure 3. Afterwards the unique identifier sequences of the different probe sets can be used for the next round of hybridization and detection.

[0324] Step 7: Second round of detection. A next round of hybridization and detection is done as described in steps 3 to 5. Note that in this new round the mix of different decoding oligonucleotides is changed. For example, decoding oligonucleotide of nucleic acid sequence (A) used in the first round comprised of sequence elements (tl) and (cl) while the new decoding oligonucleotide comprises of the sequence elements (tl) and (c2). Note that now all three sequences can clearly be distinguished due to the unique combination of first and second round signals.

[0325] Step 8: Third round of detection. Again, a new combination of decoding oligonucleotides is 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 comprise a hamming distance of 2 to other code words. Due to the hamming distance, an error in the detection of the signals (signal exchange) would not result in a valid code word and therefore could be detected. By this way three different nucleic acids can be distinguished in three detection rounds with two different signals, allowing error detection. 2. Advantages over prior art technologies

[0326] Coding strategy

[0327] Compared to state-of-the-art methods, one particular advantage of the method according to the disclosure is the use of decoding oligonucleotides breaking the dependencies between the target specific probes and the signal oligonucleotides.

[0328] Without decoupling target specific probes and signal generation, two different signals can only be generated for a certain target if using two different molecular tags. Each of these molecular tags can only be used once. Multiple readouts of the same molecular tag do not increase the information about the target. In order to create an encoding scheme, a change of the target specific probe set after each round is required (SeqFISH) or multiple molecular tags must be present on the same probe set (like merFISH, intronSeqFISH).

[0329] Following the method according to the 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. This leads to several advantages in contrast to the other methods.

[0330] (1) The encoding scheme is not defined by the target specific probe set as it is the case for all other methods of prior art. Here the encoding scheme is transcribed by the decoding oligonucleotides. This leads to a much higher flexibility concerning the number of rounds and the freedom in signal choice for the code words. Looking on the methods of prior art (e.g., merFISH or intronSeqFISH), the encoding scheme (number, type and sequence of detectable signals) for all target sequences is predefined by the presence of the different tag sequences on the specific probe sets (4 of 16 different tags per probe set in the case of merFISH and 5 of 60 different tags in the case of intron FISH). In order to produce a sufficient number of different tags per probe set, the methods use rather complex oligonucleotide designs with several tags present on one target specific oligonucleotide. In order to change the encoding scheme for a certain target nucleic acid, the specific probe set has to be replaced. The method according to the disclosure describes the use of a single unique tag sequence (unique identifier) per analyte, because it can be reused in every detection round to produce a new information. The encoding scheme is defined by the order of decoding oligonucleotides that are used in the detection rounds. Therefore, the encoding scheme is not predefined by the specific probes (or the unique tag sequence) but can be adjusted to different needs, even during the experiment. This is achieved by simply changing the decoding oligonucleotides used in the detection rounds or adding additional detection rounds.

[0331] (2) The number of different signal oligonucleotides must match the number of different tag sequences with methods of prior art (16 in the case of merFISH and 60 in the case of intronSeqFISH). Using the method according to the disclosure, the number of different signal oligonucleotides matches the number of different signals used. Due to this, the number of signal oligonucleotides stays constant for the method described here and never exceeds the number of different signals but increases with the complexity of the encoding scheme in the methods of prior art (more detection rounds more different signal oligonucleotides needed). As a result, the method described here leads to a much lower complexity (unintended interactions of signal oligonucleotides with environment or with each other) and dramatically reduces the cost of the assay since the major cost factor are the signal oligonucleotides.

[0332] (3) In the methods of prior art, the number of different signals generated by a target specific probe set is restricted by the number of different tag sequences the probe set can provide. Since each additional tag sequence increases the total size of the target specific probe, there is a limitation to the number of different tags a single probe can provide. This limitation is given by the size dependent increase of several problems (unintended inter- and intramolecular interactions, costs, diffusion rate, stability, errors during synthesis etc.). Additionally, there is a limitation of the total number of target specific probes that can be applied to a certain analyte. In case of nucleic acids, this limitation is given by the length of the target sequence and the proportion of suitable binding sites. These factors lead to severe limitations in the number of different signals a probe set can provide (4 signals in the case of merFISH and 5 signals in the case of intronSeqFISH). This limitation substantially affects the number of different code words that can be produced with a certain number of detection rounds. In the approach of the disclosure only one tag is needed and can be freely reused in every detection round. This leads to a low oligonucleotide complexity / length and at the same time to the maximum encoding efficiency possible (number of colorsnumber of rounds). The vast differences of coding capacity of our method compared to the other methods is shown in Figures 1 and 5. Due to this in approach of the disclosure a much lower number of detection rounds is needed to produce the same amount of information. A lower number of detection rounds is connected to lower cost, lower experimental time, lower complexity, higher stability and success rate, lower amount of data to be collected and analyzed and a higher accuracy of the results.

[0333] Coding capacity

[0334] All three methods compared in the Table 1 below use specific probe sets that are not denatured between different rounds of detection. For intronSeqFISH there are four detection rounds needed to produce the pseudo colors of one coding round, therefore data is only given for rounds 4, 8,12,16 and 20. The merFISH-method uses a constant number of 4 signals, therefore the data starts with the smallest number of rounds possible. After 8 detection rounds our method exceeds the maximum coding capacity reached with 20 rounds of merFISH (depicted with one asterisk) and after 12 rounds of detection the maximum coding capacity of intron FISH is exceeded (depicted with two asterisks). For the method according to the disclosure usage of 3 different signals is assumed (as is with intronSeqFISH).

[0335] Table 1: Comparison of coding capacity

[0336] As shown in Figure 6 the number of code words for merFISH does not exponentially increase with the number of detection cycles but gets less effective with each added round. In contrast, the number of code words for intronSeqFISH in the method according to the disclosure increases exponentially. The slope of the curve for the proposed method is much higher than that of intron FISH, leading to more than 10,000 times more code words usable after 20 rounds of detection.

[0337] Note that this maximum efficiency of coding capacity is also reached in case of seqFISH, where specific probes are denatured after every detection round and a new probe set is specifically hybridized to the target sequence for each detection round. However, this method has major downsides to technologies using only one specific hybridization for their encoding scheme (all other methods):

[0338] (1) For the efficient denaturation of the specific probes, rather crude conditions must be used (high temperatures, high concentrations of denaturing agent, long incubation times) leading to much higher probability for the loss or the damage of the analyte.

[0339] (2) For each round of detection an own probe set has to be used for every target nucleic acid sequence. Therefore, the number of specific probes needed for the experiment scales with the number of different signals needed for the encoding scheme. This dramatically increases the complexity and the cost of the assay.

[0340] (3) Because the hybridization efficiency of every target nucleic acid molecule is subject to some probabilistic effects, the fluctuations of signal intensity between the different detection rounds is much higher than in methods using only one specific hybridization event, reducing the proportion of complete codes.

[0341] (4) The time needed for the specific hybridization is much longer than for the hybridization of signal oligonucleotides or decoding oligonucleotides (as can be seen in the method parts of the intronSeqFISH, merFISH and seqFISH publications), which dramatically increases the time needed to complete an experiment. Due to these reasons all other methods use a single specific hybridization event and accept the major downside of lower code complexity and therefore the need of more detection rounds and a higher oligonucleotide design complexity.

[0342] The method according to the disclosure combines the advantages of seqFISH (mainly complete freedom concerning the encoding scheme) with all advantages of methods using only one specific hybridization event while eliminating the major problems of such methods.

[0343] Note that the high numbers of code words produced after 20 rounds can also be used to introduce higher hamming distances (differences) between different code words, allowing error detection of 1, 2 or even more errors and even error corrections. Therefore, even very high coding capacities are still practically relevant.

[0344] As mentioned above, the usage of multi-decoders further increases the coding capacity of the encoding scheme. Instead of being limited to the having exactly the same number of different signal types as different signal oligonucleotides and corresponding translator elements, the use of multidecoders increases the signal types that can be used to: (N x (N+l)) / 2 (with N being the number of different signal oligonucleotides used). For the code used in table 1 with 3 different signal oligonucleotides this would mean the following 7 different signal types could be used: (S1),(S2),(S3),(S1 / S2),(S1 / S3),(S2 / S3),(S1 / S2 / S3). The effect to the coding efficiency can be seen in Table lb and Figure 17.

[0345] Table lb shows the coding capacity of the four methods. All four methods compared in the table use specific probe sets that are not denatured between different rounds of detection. For intronSeqFISH there are four detection rounds needed to produce the pseudo colors of one coding round, therefore data is only given for rounds 4, 8,12,16 and 20. The merFISH-method uses a constant number of 4 signals, therefore the data starts with the smallest number of rounds possible. After 4 detection rounds the method with multi-decoders as described here exceeds the maximum coding capacity reached with 20 rounds of merFISH (depicted with one asterisk), after 7 rounds of detection the maximum coding capacity of intron FISH is exceeded (depicted with two asterisks) and after 12 rounds of detection the maximum coding capacity of the method of the present disclosure is exceeded (depicted with three asterisks). The usage of 3 different signal oligonucleotides is assumed (as is with intronSeqFISH).

[0346] 3. Selective denaturation, oligonucleotide assembly and reuse of unique identifiers are surprisingly efficient

[0347] A key factor of the method according to the disclosure is the consecutive process of decoding oligonucleotide binding, signal oligonucleotide binding, signal detection and selective denaturation. In order to generate an encoding scheme, this process has to be repeated several times (depending on the length of the code word). Because the same unique identifier is reused in every detection cycle, all events from the first to the last detection cycle are depending on each other. Additionally, the selective denaturation depends on two different events: While the decoding oligonucleotide must be dissolved from the unique identifier with highest efficiency, specific probes have to stay hybridized with highest efficiency.

[0348] Due to this the efficiency E of the whole encoding process can be described by the following equation:

[0349] E = total efficiency

[0350] Bsp= binding of specific probes

[0351] Bde= binding of decoding oligonucleotides Bsj= binding of signal oligonucleotides Ede= elimination of decoding oligonucleotides Ssp= stability of specific probes during elimination process n = number of detection cycles

[0352] Based on this equation the efficiency of each single step can be estimated for a given total efficiency of the method. The calculation is hereby based on the assumption, that each process has the same efficiency. The total efficiency describes the portion of successfully decodable signals of the total signals present.

[0353] The total efficiency of the method is dependent on the efficiency of each single step of the different factors described by the equation. Under the assumption of an equally distributed efficiency the total efficiency can be plotted against the single step efficiency as shown in Figure 7. As can be seen, a practically relevant total efficiency for an encoding scheme with 5 detection cycles can only be achieved with single step efficiencies clearly above 90%. For example, to achieve a total efficiency of 50% an average efficiency within each single step of 97.8% is needed. These calculations are even based on the assumption of a 100% signal detection and analysis efficiency. Due to broad DNA melting curves of oligonucleotides of a variety of sequences, the inventors assumed prior to experiments that the selective denaturation would work less efficient for denaturation of decoding oligonucleotides and that sequence specific binding probes are not stable enough. In contrast to this assumption, we found a surprising effectiveness of all steps and a high stability of sequence specific probes during selective denaturation.

[0354] Experimentally, the inventors achieved a total decoding efficiency of about 30% to 65% based on 5 detection cycles. A calculation of the efficiency of each single step (Bsp, Bde, Bsi, Ede, Ssp) by the formula given above revealed an average efficiency of about 94.4% to 98%. These high efficiencies are very surprising and cannot easily be anticipated by a well-trained person in this field.

[0355] 4. Experimental data

[0356] Background

[0357] The experiment shows the specific detection of 10 to 50 different mRNAs species in parallel with single molecule resolution. It is based on 5 detection cycles, 3 different fluorescent signals and an encoding scheme without signal gaps and a hamming distance of 2 (error detection). The experiment proofs the enablement and functionality of the method according to the disclosure.

[0358] Oligonucleotides and their sequences

[0359] All oligonucleotide sequences used in the experiment (target specific probes, decoding oligonucleotides, signal oligonucleotides) are listed in the sequence listing of the appendix. The 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. The stock solutions (100 pM) were stored at -20°C.

[0360] Experimental overview

[0361] The 50 different target specific probe sets are divided into 5 groups. The name of the transcript to be detected and the name of the target specific probe set are the same (transcript variant names of www.ensemble.org). The term "new" indicates a revised probe design. All oligonucleotide sequences of the probe sets can be found in the sequence listing. The table lists the unique identifier name of the probe set as well as the names of the decoding oligonucleotides used in the different detection cycles. The resulting code shows the sequence of fluorescent signals generated during the 5 detection cycles (G(reen)=Alexa Fluor 488, O(range)= Atto 594, Y(ellow)= Alexa Fluor 546).

[0362]

[0363] Table 2: Experimental overview

[0364] Variations of the experiment

[0365] Some variations of the experiment have been performed. Experiments 1 to 4 mainly differ in the number of transcripts detected in parallel. The groups listed as target specific probe sets refer to table 6. Experiments 5 to 8 are single round, single target controls for comparison with the decoded signals.

[0366] Table 3: Variations of the experiment

[0367] Experimental details

[0368] A. Seeding and cultivation of cells

[0369] Tumor cells were grown in tumor cell culture medium to nearly 100% confluency. The tumor cell culture medium comprises DMEM (Thermo Fisher, Cat.: 31885) with 10% FCS (Biochrom, Cat.: S0415), 1% Penicillin-Streptomycin (Sigma-Adrich, Cat.: P0781) and 1% MEM Non- Essential Amino Acids Solution (Thermo Fisher, Cat.: 11140035). After aspiration of cell culture medium, cells were trypsinized by incubation with trypsin EDTA solution (Sigma-Aldrich, Cat.: T3924) for 5 min at 37°C after a washing step with PBS (1,424 g / l NazHPO^HjO, 0,276 g / l, NaH2PO4*2H2O, 8,19 g / l NaCI in water, pH 7,4). Cells were then seeded on the wells of a p- Slide 8 Well ibidi-Treat (Ibidi, Cat.: 80826). The number of cells per well was adjusted to reach about 50% confluency after adhesion of the cells. Cells were incubated over night with 200 pl tumor cell culture medium per well.

[0370] B. Fixation of cells

[0371] After aspiration of cell culture medium and two washing steps with 200 pl 37°C warm PBS per well, cells were fixed with 200 pl precooled methanol (-20°C, Roth, Cat.: 0082.1) for 10 min at -20°C.

[0372] C. Counterstaining with Sudan Black

[0373] Methanol was aspirated and 150 pl of 0.2% Sudan Black-solution diluted in 70% ethanol were added to each well. Wells were incubated for 5 min in the dark at room temperature. After incubation cells were washed three times with 400 pl 70% ethanol per well to eliminate the excess of Sudan Black-solution.

[0374] D. Hybridization of analyte / target-specific probes

[0375] Before hybridization, cells were equilibrated with 200 pl sm-wash-buffer. The sm-wash-buffer comprises 30 mM NasCitrate, 300 mM NaCI, pH7, 10% formamide (Roth, Cat.:P040.1) and 5mM Ribonucleoside Vanadyl Complex (NEB, Cat.: S1402S). For each target-specific probe set 1 pl of a 100 pM oligonucleotide stock solution was added to the mixture. The oligonucleotide stock solution comprises equimolar amounts of all target's specific oligonucleotides of the corresponding target specific probe set. The total volume of the mixture was adjusted to 100 pl with water and mixed with 100 pl of a 2x concentrated hybridization buffer solution. The 2x concentrated hybridization buffer comprises 120 mM NasCitrate, 1200 mM NaCI, pH7, 20% formamide and 20 mM Ribonucleoside Vanadyl Complex. The resulting 200 pl hybridization mixture was added to the corresponding well and incubated at 37°C for 2 h. Afterwards cells were washed three times with 200 pl per well for 10 min with target probe wash buffer at 37°C. The target probe wash buffer comprises 30 mM NasCitrate, 300 mM NaCI, pH7, 20% formamide and 5 mM Ribonucleoside Vanadyl Complex.

[0376] E. Hybridization of decoding oligonucleotides

[0377] Before hybridization, cells were equilibrated with 200 pl sm-wash-buffer. For each decoding oligonucleotide 1,5 pl of a 5 pM stock solution were added to the mixture. The total volume of the mixture was adjusted to 75 pl with water and mixed with 75 pl of a 2x concentrated hybridization buffer solution. The resulting 150 pl decoding oligonucleotide hybridization mixture was added to the corresponding well and incubated at room temperature for 45 min. Afterwards cells were washed three times with 200 pl per well for 2 min with sm-wash-buffer at room temperature. F. Hybridization of signal oligonucleotides

[0378] Before hybridization, cells were equilibrated with 200 pl sm-wash-buffer. The signal oligonucleotide hybridization mixture was the same for all rounds of experiments 1 to 4 and comprised 0,3 pM of each signal oligonucleotide (see table A3) in lx concentrated hybridization buffer solution. In each round 150 pl of this solution were added per well and incubated at room temperature for 45 min. The procedure was the same for experiments 5 to 8 with the exception that the final concentration of each signal oligonucleotide was 0,15 pM. Afterwards cells were washed three times with 200 pl per well for 2 min with sm-wash-buffer at room temperature.

[0379] G. Fluorescence and white light imaging

[0380] Cells were washed once with 200 pl of imaging buffer per well at room temperature. In experiments without Trolox (see table 7, last column) imaging buffer comprises 30 mM NasCitrate, 300 mM NaCI, pH7 and 5mM Ribonucleoside Vanadyl Complex. In experiments with Trolox, imaging buffer additionally contains 10 % VectaCell Trolox Antifade Reagent (Vector laboratories, Cat.: CB-1000), resulting in a final Trolox concentration of 10 mM.

[0381] A Zeiss Axiovert 200M microscope with a 63x immersion oil objective (Zeiss, apochromat) with 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 of the regions. Filter sets and LED-wavelengths were adjusted to the different optima of the fluorophores used. Illumination times per image were 1000 ms for Alexa Fluor 546 and Atto 594 and 400 ms for Alexa Fluor 488.

[0382] In each experiment, three regions were randomly chosen for imaging. For each region, a z- stack of 32 images was detected with a z-step size of 350 nm. Additionally, one white light image was taken from the regions. In experiments with more than one detection cycle, the regions of the first detection round were found back and imaged in every subsequent round. H. Selective denaturation

[0383] For selective denaturation, every well was incubated with 200 pl of sm-wash-buffer at 42°C for 6 min. This procedure was repeated six times.

[0384] Steps (E) to (H) were repeated 5 times in experiments 1 to 4. Step (H) was omitted for the 5thdetection cycle.

[0385] / . Analysis

[0386] Based on custom ImageJ-plugins a semi-automated analysis of the raw data was performed to distinguish the specific fluorescent signals from the background. The resulting 3D-point clouds of all three fluorescent channels were combined in silico with a custom VBA script. The resulting combined 3D-point clouds of the 5 detection cycles were aligned to each other on the basis of a VBA script. The resulting alignments revealed the code words for each unique signal detected. Successfully decoded signals were used for quantitative and spatial analysis of the experiments based on custom VBA-scripts and ImageJ-plugins.

[0387] Results

[0388] I. Absolute numbers of decoded signals

[0389] The absolute numbers of successfully decoded signals for all transcripts are listed for each region of each experiment in the following Table 4. In summary, the sum of correct codes depicts the total number of decoded signals that were assigned to transcripts detectable in the corresponding experiment, while the sum of incorrect codes it the total number of decoded signals not detectable in the corresponding experiment. The total number of signals comprises successfully decoded as well as unsuccessfully decoded signals.

[0390]

[0391] Table 4: Absolute numbers of decoded signals

[0392] Table 4 shows a very low number of incorrectly decoded signals compared to the number of correctly decoded signals. The absolute values for decoded signals of a certain transcript are very similar between different regions of one experiment. The fraction of the total number of signals that can be successfully decoded is between 27.1 % and 64.5 %. This fraction depends on the number of transcripts and / or the total number of signals present in the respective region / experiment.

[0393] Conclusion

[0394] The method according to the disclosure produces a low amount of incorrectly assigned code words and can therefore be considered specific. The fraction of successfully decodable signals is very high, even with very high numbers of signals per region and very high numbers of transcripts detected in parallel. The high fraction of assignable signals and the high specificity make the method practically useful.

[0395] Comparison of relative transcript abundancies between different experiments

[0396] As shown in Figure 8 for both comparisons (A and B) the overlap of detected transcripts between the experiments is used for the analysis. Each bar represents the mean abundance of all three regions of an experiment. The standard deviation between these regions is also indicated.

[0397] Correlation of relative transcript abundancies between different experiments

[0398] As can be seen in Figure 9 the mean relative abundances of transcripts from experiment 1 are correlated to the abundances of the overlapping transcripts of experiment 3, 4 and 2. The correlation coefficient as well as the formula for the linear regression are indicated for each correlation.

[0399] Figure 8 shows low standard deviations, indicating low variations of relative abundances between different regions of one experiment. The differences of relative abundances between transcripts from different experiments are also very low. This is the case for the comparison of transcripts from group 1 (Fig. 8A), that were detected in experiments 1, 2 and 3. It is also the case for the comparison of the transcripts from groups 2, 3 and 4 that were overlapping 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 the abundances of the other multi round experiments. The correlation factors are between 0.88 and 0.91, while the slope of the linear regressions is between 0.97 and 1.05.

[0400] Conclusion

[0401] The relative abundancies of transcripts correlate very well between different regions of one experiment but also between different experiments. This can be clearly seen by the comparisons of Figures 3 and 4. The main difference between the experiments is the number of different targets and hence the total number of signals detected. Therefore, the number of transcripts detected as well as the number and density of signals does not interfere with the ability of the method to accurately quantify the number of transcripts. The very good correlations further support the specificity and stability of the method, even with very high numbers of signals.

[0402] Comparison of intercellular distribution of signals

[0403] In Figure 10 the 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 assigned to SPOCK1, C: region 1 of experiment 8 (single round, single transcript experiment detecting THRAP3), D: 2D-projection of all selected signals from experiment 1, region 1 assigned to THRAP3.

[0404] Comparison of intracellular distribution of signals

[0405] In Figure 11 the maximum projections of image stacks are shown. Magnified sub regions of the corresponding regions are shown. A: region 1 of experiment 8 (single round, single transcript experiment detecting THRAP3), B: 2D-projection of selected signals from experiment 1, region 1 assigned to THRAP3, C: region 1 of experiment 5 (single round, single transcript experiment detecting

[0406] DDX5), D: 2D-projection of all selected signals from experiment 1, region 1 assigned to DDX5.

[0407] Figure 10 shows huge differences of intercellular distributions between different transcripts. SPOCK1 seems to be highly abundant in some cells but nearly absent in other cells (Figure 10 A). THRAP3 shows a more uniform distribution over all cells of a region (Figure 10 C). These spatial distribution patterns can also clearly be observed with the point clouds assigned to the corresponding transcripts from experiment 1 (Figure 10 B and D).

[0408] Figure 11 shows huge differences of intracellular distributions between different transcripts. THRAP3 can be mainly observed in the periphery (cytoplasm) of the cells (Figure 11 A), while DDX5 shows a higher abundance in the center (nucleus) of the cells (Figure 11 C). These intracellular distributions can also be observed with the point clouds of experiment 1 assigned to THRAP3 and DDX5 (Figure 11 B and D).

[0409] Conclusion

[0410] Next to the reliability of quantification, the point clouds of multi round experiments also show the same intracellular and intercellular distribution patterns of transcripts. This is clearly proven by the direct comparison of the assigned point clouds with signals from single round experiments detecting only one characteristic mRNA-species.

[0411] Distribution pattern of different cell cycle dependent transcripts

[0412] All images of Figure 12 show region 1 of experiment 1. In each image, a point cloud is shown, that is assigned to a certain transcript, A: CCNA2, B: CENPE, C: CCNE1, D: all transcripts. Figure 12 shows the transcripts of three different cell cycle dependent proteins. CENPE (Figure 12 B) is also known as Centromere protein E and accumulates during G2 phase. It is proposed to be responsible for spindle elongation and for chromosome movement. It is not present during interphase. CCNA2 (Figure 12 A) is also known as Cyclin A2. It regulates the cell cycle progression by interacting with CDK1 during transition from G2 to M-phase. Interestingly there is an obvious colocalization of both mRNA-species. They are mainly present in the three central cells of region 1. CCNE1 (Figure 12 C) is also known as Cyclin El. This cyclin interacts with CDK2 and is responsible for the transition from G1 to S-phase. Figure 12 shows clearly, that the transcripts of this gene are not present in the three central cells, but quite equally distributed over the other cells. It therefore shows an anti-localization to the other two transcripts. The data for the corresponding point-clouds are derived from a point cloud with a very high number of points and a very high point density (Figure 12 D gives an impression). Conclusion

[0413] The three decoded point clouds of cell cycle dependent proteins shown in Figure 12, show distribution patterns that can be explained by their corresponding function. These data strongly suggest that our method reliably produces biological relevant data, even with a low number of signals per cell (Figure 12 C) and with very high signal densities (Figure 12 D).

[0414] Fig 20 shows a fluorescence image of the analytic probes binding to genomic elements within cells. Standard Molecular Cartography was performed on human liver samples using defined probes for specific markers. Note that 1, 2 and 3 different signals in the area shown here can be recognized depending on the genomic situation. Also, cells without any signal are recognized. For example, we found between 0 and 6 AOX1 gene signals per nucleus in a total of 2488 signals. 92% of signals could be mapped to the nucleus.

[0415] Liver is a tissue comprising cells that are not only diploid. Cells from liver can also have a multitude of genome equivalents due to endoreplication without a subsequent cell division. Therefore, liver cells can contain also more than two genomic signals as seen in figure 20. . The variance of the signal number is caused by three reasons:

[0416] 1) Liver cells undergo endoreplication of the genome without a cell division.

[0417] 2) Only actively transcribed genome site are detectable by the technology because probes are designed to bind to the RNA and no denaturation step (which is needed for DNA detection) was performed. So, genome copies that are not transcribed are not detectable by this method.

[0418] 3) Variance can be derived by the tissue sectioning as well. Here, 10 pm sections were used for Molecular Cartography of human genome elements. Since the nucleus size in polyploid cells is about 10 pm as well, the majority of nuclei (about ~65% according to Poisson distribution) are not completely represented in a 10 pm tissue section so that not all genome copies of a certain marker may be detectable. Some of the genome elements are lost during tissue section because they are simply located in a different sectioning plane which was not used for the analysis.

Claims

CLAIMS1. A method for simultaneously or sequential detecting, counting, localizing at least two nucleic acid elements selected from nuclear genome sites and / or extra-genomic nucleic acid elements such as RNA sequences, wherein the method is defined by an indirect detection process that includes a coding system.

2. The method according to claim 1 wherein the nucleic acid element of claim 1 is detected by in s / tu-hybridization and comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the detected nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein.

3. The method according to any of the previous claims comprising a detection and / or coding process comprising multiple rounds of detection to cover a coding of sites detected simultaneously or sequential.

4. The method according to any of the previous claims comprising a detection and / or coding process which is performed in situ.

5. The method according to any of the previous claims comprising a detection and / or coding process comprising multiple rounds of detection to allow detection of multiple sites simultaneously or sequential.

6. The method according to any of the previous claims comprising a detection and / or coding process comprising in spatially resolved images where copies of a certain transcribed genome element can be counted and localized.

7. The method according to any of the previous claims comprising a detection and / or coding process comprising detection of genome elements simultaneously or sequential because of the coding system.

8. The method according to any of the previous claims comprising the steps of:

1. Selecting a transcribed genome element of interest;2. Designing probes that are specific and complementary to RNA molecules but that preferably exclude exon areas;3. Providing and preparing a sample;4. Immobilizing the tissue section on a holder;5. Hybridizing specific probes to the tissue section;6. Building up a coding process by repeating round of switching the detectable label;7. Imaging the tissue section after colorization in each repeating round;8. Performing a computer aided decoding process of all images that were taken; and / or9) Counting specific signals and / or measure the intensity.

9. A method for an in s / tu-hybridization multiplex reaction characterized by comprising the steps of: a.) adding an analyte-specific probe oligonucleotide comprising a binding element which is essentially complementary to an analyte nucleic acid element to be detected, which is an nucleic acid element comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein; , and comprising an identifier element (T) comprising a nucleotide sequence which is unique to the analyte nucleotide sequence; and b.) adding a decoding oligonucleotide comprising an identifier element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique set identifier sequence of the identifier element (T) of the correspondingbinding element, and a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; and / or c.) adding a signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and a signal element facilitating a signal which is specific for the polymorphic analyte; and wherein the monitoring probe is added before, during and / or after adding the decoding oligonucleotide in step b).

10. The method according to claim 9, wherein the nucleic acid element comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein;; optionally up to 100% of the genome element is a non-coding acid element, optionally said nucleic acid element is an intron.

11. The method according to any of claims 9 or 10, comprising the steps:(A) contacting the analyte to be detected, which is a nucleic acid element comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein; with at least five (5) different sets of analyte-specific probes for encoding of at least five (5) different analytes, each setof analyte-specific probes interacting with a different analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein 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), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and contacting the sample with at least two different sets of analyte-specific probes for at least one analyte and a variation thereof, wherein the analyte-specific probes comprised in these different sets interacting with the same analyte, but specifically interact with different sub-structures of the same analyte, wherein the analyte-specific probes of the first set of analyte-specific probes interacts with a sub-structure which is comprised in all variations of an analyte, wherein the analyte-specific probes of the second set of analyte-specific probes (subgroup-specific probes) interacts with a sub-structure which is comprised only in a specific variation of the analyte, wherein the analyte-specific probes of the first set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), andwherein the analyte-specific probes of the second set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the identifier elements (T) of the analyte-specific probes of the first set of analytespecific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes are different for binding 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 which 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(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and(C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element.(D) Detecting the signal caused by the signal element;(E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded;(F) Performing at least one (1) further cycle comprising steps B) to E) to generate an encoding scheme with a code word per analyte. wherein in particular the cycle may stop with step (D).

12. The method according to any of claims 9 or 10, wherein the set of analyte-specific probes comprises at least five (5) subgroup-specific probes which specifically interact with different substructures of the same variation of an analyte.

13. The method according to any of the previous claims, wherein if the analyte is a nucleic acid, each set of analyte-specific probes comprises at least ten (10) analyte-specific probes, in particular at least fifteen (15) analyte-specific probes, in particular at least twenty (20) analytespecific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe.

14. The method according to any of the previous claims, wherein contacting a subgroup of at least one analyte with a set of at least five (5) subgroup-specific probes which differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T).

15. The method according to any of the previous claims, wherein all steps are automated, in particular wherein steps B) to G) are automated, in particular by using a robotic system.

16. The method according to any of the previous claims, wherein all steps are performed in a fluidic system such as a FACS-system.

17. The method according to any of the previous claims, wherein each analyte is associated with a specific code word, wherein said code word comprise a number of positions, and whereineach position corresponds to one cycle resulting in a plurality of distinguishable encoding schemes with the plurality of code words.

18. The method according to any of the previous claims, wherein said encoding scheme is predetermined and allocated to the analyte to be encoded.

19. The method according to any of the previous claims, wherein the code words obtained for the individual analytes in the performed cycles comprise the detected signals and additionally at least one element corresponding to no detected signal.

20. The method according to any of the previous claims, wherein no signal is detected for at least one analyte within at least one cycle.

21. The method according to any of the previous claims, wherein for at least for one individual analyte a position of the code word is zero (0).

22. The method according to any of the previous claims, wherein the code word zero (0) is generated by using no decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte.

23. The method according to any of the previous claims, wherein if at least for one individual analyte a position of the code word is zero (0) in this cycle no corresponding decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte are used.

24. The method according to any of the previous claims, wherein the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

25. The method according to any of the previous claims, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

26. The method according to any of the previous claims, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein only one set of decoding oligonucleotides per analyte is used per cycle, and / or wherein different sets of decoding oligonucleotides are used in different cycles in combination with the corresponding set of signal oligonucleotides in the same cycle.

27. The method according to any of the previous claims, wherein the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C).

28. The method according to any of the previous claims, wherein all sets of decoding oligonucleotides for the different analytes comprise the same type(s) of translator element(s) (c).

29. The method according to any of the previous claims, wherein the sample is contacted withat least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

30. The method according to any of the previous claims, wherein the sample is contacted with: at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

31. The method according to any of the previous claims, wherein the different sets of non- signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

32. The method according to any of the previous claims, wherein 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 which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

33. The method according to any of the previous claims, wherein the sample is contacted with: at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

34. The method according to any of the previous claims, wherein the different sets of nonsignal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

35. The method according to any of the previous claims, wherein the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

36. The method according to any of the previous claims, wherein the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately.

37. The method according to any of the previous claims, wherein the different sets of analytespecific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately.

38. The method according to any of the previous claims, wherein the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

39. The method according to any of the previous claims, wherein the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells and / or extracts and / or part of cells.

40. The method according to any of the previous claims, wherein the cell is a prokaryotic cell or a eukaryotic cell, in particular a mammalian cell, in particular a human cell.

41. The method according to any of the previous claims, wherein the biological tissue, biological cells, extracts and / or part of cells are fixed.

42. The method according to any of the previous claims, wherein the analytes are fixed in a permeabilized sample, such as a cell-containing sample.

43. The method according to any of the previous claims, wherein the binding element (S) comprise a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

44. The method according to any of the previous claims, wherein after step A) and before stepB) the non-bound analyte-specific probes are removed, in particular by washing.

45. The method according to any of the previous claims, wherein after step B) and before stepC) the non-bound decoding oligonucleotides are removed, in particular by washing.

46. The method according to any of the previous claims, wherein after step C) and before stepD) the non-bound signal oligonucleotides are removed, in particular by washing.

47. The method according to any of the previous claims, wherein the analyte specific probes are incubated with the sample, thereby allowing a specific binding of the analyte specific probes to the analytes to be encoded.

48. The method according to any of the previous claims, wherein the decoding oligonucleotides are incubated with the sample, thereby allowing a specific hybridization of the decoding oligonucleotides to identifier elements (T) of the respective analyte-specific probes.

49. The method according to any of the previous claims, wherein the signal oligonucleotides are incubated with the sample, thereby allowing a specific hybridization of the signal oligonucleotides to translator elements (T) of the respective decoding oligonucleotides.

50. The method according to any of the previous claims, wherein the analyte to be encoded is a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA.

51. The method according to any of the previous claims, wherein the binding element (S) comprise an amino acid sequence allowing a specific binding to the analyte to be encoded.

52. The method according to any of the previous claims, wherein the signal caused by the signal element, therefore in particular the binding of the signal oligonucleotides to the decodingoligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte is determined by:(a) Imaging at least a portion of the sample; and / or(b) Using an optical imaging technique; and / or(c) Using a fluorescence imaging technique; and / or(d) Multi-color fluorescence imaging technique; and / or(e) Super-resolution fluorescence imaging technique.

53. The method according to any of the previous claims, wherein the decoding oligonucleotides in at least one set of decoding oligonucleotides are multi-decoders comprising(aa) an identifier connector element (t) comprising a nucleotide sequence which 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(bb) at least two translator elements (c), wherein the translator elements comprising different nucleotide sequences allowing a specific hybridization of a different signal oligonucleotide.

54. The method according to any of the previous claims, wherein the different signal oligonucleotides comprise a different signal element and comprise a different connector element (C).

55. A kit, comprising a.) an analyte-specific probe oligonucleotide comprising a binding element which is essentially complementary to an analyte nucleic acid element to be detected, which is an nucleic acid element comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein;, and comprising an identifier element (T)comprising a nucleotide sequence which is unique to the analyte nucleotide sequence; and b.) a decoding oligonucleotide comprising an identifier element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique set identifier sequence of the identifier element (T) of the corresponding binding element, and a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; and / or c.) a signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and a signal element facilitating a signal which is specific for the polymorphic analyte.

56. The kit according to claim 55, wherein the a nucleic acid element comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of a non-coding nucleic acid element and, thus, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, about 0% of the nucleic acid element will be present in the final functional RNA molecule and / or translated into the final peptide or protein;; optionally up to 100% of the genome element is a non-coding nucleic acid element, optionally said nucleic acid element is an intron.

57. The kit according to any of claims 55 or 56, comprising(A) at least five (5) different sets of analyte-specific probes for encoding of at least ten (10) different analytes, each set of analyte-specific probes interacting with a different analyte, wherein, optionally, if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures 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, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),wherein 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), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(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 which 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(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and(C) a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element.

58. The kit according to any of claims 55 - 57, wherein the kit comprises at least two different sets of analyte-specific probes for an analyte, wherein the analyte-specific probes comprised in these different sets interacting with the same analyte, but specifically interact with different sub-structures of the same analyte, wherein the analyte-specific probes of the first set of analyte-specific probes interacts with a sub-structure which is comprised in all variations of an analyte,wherein the analyte-specific probes of the second set of analyte-specific probes (subgroup-specific probes) interacts with a sub-structure which is comprised only in a specific variation of the analyte, wherein the analyte-specific probes of the first set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), and wherein the analyte-specific probes of the second set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the identifier elements (T) of the analyte-specific probes of the first set of analytespecific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes are different.

59. The kit according to any of claims 55 - 58, wherein the kit comprises at least five (5) sets of subgroup-specific probes that differ from the analyte-specific probes of another set of analytespecific probes in the nucleotide sequence of the identifier element (T).

60. The kit according to any of claims 55 - 59, wherein the kit does not comprise sets of analytespecific probes and / or subgroup-specific probes as defined in claim 47.

61. The kit according to any of claims 55 - 60, wherein if the analyte is a nucleic acid, each set of analyte-specific probes comprises at least ten (10) analyte-specific probes, in particular at least fifteen (15) analyte-specific probes, in particular at least twenty (20) analyte-specific probes which specifically interact with different sub-structures of the same analyte.

62. The kit according to any of claims 55 - 61, wherein the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentiallycomplementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

63. The kit according to any of claims 55 - 62, wherein the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which 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 wherein the decoding oligonucleotides of the different sets for at least one analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

64. The kit according to any of claims 55 - 63, wherein the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C).

65. The kit according to any of claims 55 - 64, wherein the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

66. The kit according to any of claims 55 - 65, wherein all sets of decoding oligonucleotides for the different analytes comprise the same type(s) of translator element(s) (c).

67. The kit according to any of claims 55 - 66, wherein the kit comprises:(D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T),wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

68. The kit according to any of claims 55 - 67, wherein 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 analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which 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 allowing a specific hybridization of a signal oligonucleotide.

69. The kit according to any of claims 55 - 68, wherein the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

70. The kit according to any of claims 55 - 69, wherein the kit comprises:(E) a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

71. The kit according to any of claims 55 -70, wherein the kit comprises:(E) at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and(bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

72. The kit according to any of claims 55 - 71, wherein the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

73. The kit according to any of claims 55 - 72, wherein the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

74. The kit according to any of claims 55 - 73, wherein the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately.

75. The kit according to any of claims 55 - 74, wherein the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately.

76. The kit according to any of claims 55 - 75, wherein the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

77. The kit according to any of claims 55 - 76, wherein the analyte to be encoded is a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA.

78. The kit according to any of claims 55 - 77, wherein the decoding oligonucleotides in at least one set of decoding oligonucleotides are multi-decoders comprising(aa) an identifier connector element (t) comprising a nucleotide sequence which 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(bb) at least two translator elements (c), wherein the translator elements comprising different nucleotide sequences allowing a specific hybridization of a different signal oligonucleotide.

79. The kit according to any of claims 55 - 78, wherein the different signal oligonucleotides comprise a different signal element and comprise a different connector element (C).

80. An in vitro method for diagnosis of a disease selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases and prenatal diseases comprising the use of the multiplex method according to any one of claims 1 to 54.

81. An in vitro method for diagnosis of a disease in plants selected from the group comprising: diseases caused by biotic stress, preferably by infectious and / or parasitic origin, or diseases caused by abiotic stress, preferably caused by nutritional deficiencies and / or unfavorable environment, said method comprising the use of the multiplex method according to any one of claims 1 to 54.

82. An optical multiplexing system suitable for the method according to any one of claims 1 to 54, comprising at least: one reaction vessel for containing the kits or part of the kits according to any one of claims 55 to 79; a detection unit comprising a microscope, in particular a fluorescence microscope a camera a liquid handling device.

83. The optical multiplexing system according to claim 82, wherein the system comprises further a heat and cooling device.

84. The optical multiplexing system according to any one of claim 82 to 83, wherein the system comprises further a robotic system.

85. An in vitro method for screening, identifying and / or testing a substance and / or drug comprising:(a) contacting a test sample comprising a sample with a substance and / or drug(b) detecting different analytes in a sample by sequential signal-encoding of said analytes with a method according to any one of claims 1 to 54.

86. The in vitro method according to claim 85, wherein the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells, in particular wherein the cell is a prokaryotic cells or a eukaryotic cell, in particular a mammalian cell, in particular a human cell.

87. A method of assaying for nucleic acid localization, comprising contacting a sample to a first probe set that targets segments of loci that are not included in mature mRNA molecules transcribed from the loci, contacting the sample to a second probe set that targets segments of loci that are included in mature mRNA molecules, and determining localization patterns for the first probe set and for the second probe set.

88. The method of claim 87, wherein determining localization patterns for the first probe set and for the second probe set comprises indirect labeling using a coding approach.

89. The method of claim 88, wherein determining localization patterns for the first probe set comprises determining the localization pattern for the first probe set without relying upon second probe set data.

90. The method of claim 89, wherein determining localization for target molecules of the second probe set comprises determining the localization pattern for the first probe set, determining the localization pattern for the second probe set, and subtracting the localization pattern for the first probe set from the localization pattern for the second probe set to identify localization of targets identified by the second probe set and not identified by the first probe set.

91. The method of claim 89, wherein target molecules of the second probe set comprise processed mRNA molecules.

92. The method of claim 89, wherein target molecules of the second probe set comprise spliced RNA molecules.

93. The method of claim 89, wherein target molecules of the second probe set comprise viral particle nucleic acids.

94. The method of claim 89, wherein target molecules of the second probe set comprise retrotransposon transcripts.

95. The method of claim 89, wherein target molecules of the first probe set comprise nucleic acid segments corresponding to introns.

96. The method of claim 89, wherein target molecules of the first probe set comprise nucleic acid segments corresponding to promoter elements.

97. The method of claim 89, wherein target molecules of the first probe set comprise nucleic acid segments corresponding to enhancer elements.

98. The method of claim 89, wherein target molecules of the first probe set comprise nucleic acid segments corresponding to repressor elements.

99. A method of assaying for transcriptional activity, comprising contacting a sample to a probe that targets a region of an RNA molecule that is present in a nascent transcript but is absent from the mature RNA molecule subsequent to processing.

100. The method of claim 99, wherein the probe is assayed using an indirect labeling coding approach.

101. The method of claim 100, wherein the region of the RNA molecule is spliced out of the nascent transcript pursuant to processing to generate the mature RNA molecule.

101. The method of claim 100, wherein the nascent RNA molecule is an hnRNA molecule.

102. The method of claim 99 or claim 100, wherein the sample is not subjected to double stranded DNA denaturing conditions sufficient to denature chromosomes during the contacting.

103. The method of claim 99 or claim 100, wherein the probe does not bind to DNA in the sample.

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