The term “complementary to at least a portion of the target RNA polynucleotide” as used herein, means that the annealing segment has a complementary overlap with said target RNA polynucleotide. For example, the catcher polynucleotide may be complementary to a target RNA polynucleotide or a polynucleotide comprising a target specific sequence as defined herein. The overlap may, for example, be an overlap of 5, 7, 10, 12, 15, 18, 20, 22, 25, 28 or 30 nucleotides, or any value in between the mentioned values. Also envisaged are longer overlaps. The overlap typically is at the 3′ end of catcher polynucleotide and at the 3′ end of the target RNA polynucleotide. Within said overlap the matching or complementarity between the complementary bases is preferably 100%. Alternatively, the matching is less than 100%, e.g. 99%, 95%, 90%, 85% or less than 85%.

The term “catcher polynucleotide”, as used herein, relates to a polynucleotide of a defined length which is designed to anneal with and thereby to bind to or to catch a corresponding target RNA polynucleotide. The catcher polynucleotide may have any suitable length. It may, for example, comprise between about 15 to 300 nucleotides, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, 150, 200, 250, or 300 nucleotides or any value in between the mentioned values. Also envisaged are longer catcher polynucleotides. It is particularly preferred that the catcher polynucleotide comprises about 15-25 nucleotides. The catcher polynucleotide may, in certain embodiments, further comprise additional elements such as one or more spacer elements, linker elements etc. The catcher polynucleotide may comprise one or more sections which are capable of binding or hybridizing to a target RNA polynucleotide and one or more segments which are not capable of binding or hybridizing to a target RNA polynucleotide. The latter ones may have structural functions or provide a spacer or distancing functionality. It is preferred that the catcher polynucleotide is a single strand DNA molecule. Also envisaged are RNA, PNA, CNA, HNA, LNA or ANA molecules or mixtures thereof as catcher polynucleotide.

The present invention further envisages that the catcher polynucleotide comprises a tag which is capable of binding to a cognate interactor. The interactor-tag binding may advantageously be used to pull said catcher polynucleotides together with any bound or associated RNA polynucleotide from a solution, or to catch a target RNA polynucleotide when moving around said catcher polynucleotide. Examples of suitable tags and interactors are a biotin tag on the catcher polynucleotide and a streptavidin interactor provided on a suitable surface, e.g. of a reaction vessel, or on a bead etc. Further examples include a magnetic bead being bound to a catcher polynucleotide and a magnetic separator attracting said beads, e.g. attached at a surface.

It is preferred that the catcher polynucleotide is immobilized on magnetic beads or via streptavidin-biotin binding.

In a preferred embodiment the catcher polynucleotide comprises a poly T-section at least partially complementary to the poly-A-tail of the target RNA polynucleotide. This type of catcher polynucleotide is specifically designed to bind to or catch a poly-A-segment containing target RNA polynucleotides, in particular an mRNA polynucleotide.

In a further preferred embodiment the catcher polynucleotide comprises a segment at least partially complementary to a specific target sequence on the target RNA polynucleotide. For example, the catcher polynucleotide matches with a predefined sequence stretch on the target RNA polynucleotide. This sequence stretch is typically not a poly-A-tail or segment. The segment may, for example, represent a conserved sequence motif in a specific group of RNA polynucleotides, e.g. 16S RNAs, mitochondrial RNAs etc.

It is envisaged that the complementary section between the catcher polynucleotide and the target RNA polynucleotide, e.g. poly-T/poly-A complementarity or sequence specific complementarity, comprises between 12 to 30 nucleotides, preferably 20 nucleotides.

In a next step a mature crRNA (CRISPR RNA) molecule is annealed to a specific target sequence on the target RNA polynucleotide. The specific target sequence for the crRNA is typically not identical to the specific target sequence for the catcher polynucleotide. It is also preferred that these sequence segments do not overlap.

This method is, in general, based on the employment of the CRISPR/Cas system. The term “CRISPR/Cas system” as used herein relates to a biochemical method to specifically cut and modify nucleic acids, also known as genome editing. For example, genes in a genome can generally be inserted, removed or switched off with the CRISPR/Cas system, nucleotides in a gene or nucleic acid molecule can also be changed. The effect of the concept and activity steps of the CRISPR/Cas system has various similarities to that of RNA interference, since short RNA fragments of about 18 to 20 nucleotides mediate the binding to the target in both bacterial defense mechanisms. In the CRSIPR/Cas system typically RNA-guided nucleic acid-binding proteins, such as Cas proteins, bind certain RNA sequences as ribonucleoproteins. For example, a Cas endonuclease can bind to certain RNA sequences termed crRNA repeats and cut a nucleic acid in the immediate vicinity of these sequences. Without wishing to be bound by theory, it is believed that the crRNA repeat sequence forms a secondary RNA structure and is then bound by the nucleic acid-binding protein (e.g. Cas) which alters its protein folding allowing the target nucleic acid to be bound by the RNA.

In type VI CRISPR/Cas systems a single RNA-guided effector protein, Cas13, forms a crRNA-guided RNA-targeting effector complex when assembled with a crRNA. The CRISPR/Cas13 system accordingly comprises two main components: (i) the programmable single-effector RNA-guided RNase Cas13 and (ii) a 64-66 nucleotide CRISPR RNA (crRNA), which recognizes a 24-30 nucleotide sequence on the target RNA by means of a protospacer-flanking site (PFS). Also, all Cas13 proteins feature two enzymatically distinct ribonuclease activities: (i) nucleolytical processing of pre-crRNA pre-CRISPR-RNA by Cas proteins and/or host factors to generate mature crRNA by RNase and (ii) degradation of target RNA initialized by the RNase activity of two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains (Grandaros-Riveron and Aquino-Jarquin, 2018, Cancer Research, 78 (15), 4107-13).

The term “mature crRNA (CRSIPR RNA)” as used herein thus relates to an RNA molecule of the type VI CRISPR/Cas system which is capable of identifying and binding to specific RNA polynucleotide sequences. Typically, the mature crRNA comprises a target specific sequence which can be used to guide an RNA binding protein towards the binding site. This target specific sequence may have any suitable length. It is preferred that said length is between about 19 to 60 nucleotides. More preferably, the sequence has a length of 30 nucleotides.

As described in East-Seletsky et al., 2017, Mol Cell., 66(3), 373-383 mature crRNAs of the type VI CRISPR/Cas system according to the present invention comprise a stem segment, a loop segment, a bulge segment and a spacer segment which comprises the target specific sequence as defined above. Mature crRNAs typically are derived from pre-crRNA molecules via a maturation processing. They can also be provided as such and be used without being derived from said pre-crRNA stage. Further options for obtaining a mature crRNA can be derived from Smargon et al., 2017, Mol Cell., 16, 65(4), 618-630, or from Cox et al., 2017, Science, 10.1126/science.aaq0180. For example, a mature crRNA may be provided which comprises different forms of a stem structure, different loop structures, or in which the spacer sequence is differentially truncated etc. According to further embodiments of the present invention, the sequence and form of the mature crRNA may vary in accordance with the form or identity of the crRNA-guided RNA-binding protein, e.g. Cas protein. Accordingly, depending on the origin of said crRNA-guided RNA-binding protein, a different combination of sequence elements may be used. The present invention further envisages any future development in this context and includes any modification or improvement of the crRNA-RNA-binding protein interaction surpassing the information de-rivable from Smargon et al., 2017, Mol Cell., 16, 65(4), 618-630. Particularly preferred is the use of a mature crRNA for Cas13a, Cas13b, Cas13c or Cas13d. Also envisaged are mature crRNA forms from commercial suppliers, or individually prepared crRNAs.

One of the central principles of the present invention is thus the use of a sequence binding to a target RNA section within the crRNA, wherein said binding sequence is specific for the target RNA polynucleotide and is accordingly able to identify said sequence and to distinguish it from other sequences.

In a further step a catalytically-inactive crRNA-guided RNA-binding protein is bound to the complex of step (iii), i.e. the complex of a mature crRNA (CRISPR RNA) molecule and the target RNA polynucleotide. This results in a protein-RNA polynucleotide complex. In accordance with the CRSPR/Cas approach as defined above, the sequence which has been identified by the mature crRNA as described above is thus indirectly bound (via the crRNA) by a cognate catalytically-inactive crRNA-guided RNA-binding protein.

The term “catalytically-inactive crRNA-guided RNA-binding protein” as used herein relates to an RNA binding protein, e.g. Cas protein, which is capable of binding to a specific stretch of RNA, but does not retain its endonuclease activity and thus does not cut or digest the RNA. This allows for a programmable tracking and identification of RNA molecules without damaging them. In a preferred embodiment, said catalytically-inactive crRNA-guided RNA-binding protein is a Cas protein, more preferably it is a member of the family of Cas12 or Cas13 proteins. Examples of proteins of the Cas12 family include Cas12a. The Cas12 protein is preferably a derivative of the naturally occurring Cas12 protein, in particular a derivative which has been modified, e.g. via protein engineering, to be capable of binding to RNA polynucleotides. It is particularly preferred that it is a Cas13 protein or a derivative thereof. Suitable subtypes may include, for example, Cas13a, Cas13b, Cas13c and Cas13d proteins. Further information on catalytically-inactive crRNA-guided RNA-binding protein may be derived from suitable literature sources such as Cox et al., 2017, Science, 10.1126/science.aaq0180.

Also envisaged are derivatives of the mentioned RNA-binding proteins, e.g. Cas proteins, or mutants thereof. The derivative is preferably a functional derivative of a crRNA-guided RNA-binding protein, which is able to bind to specific stretches of RNA but does no retain its endonuclease activity. In specific embodiments the invention particularly envisages the production of catalytically inactive Cas13 or Cas12 proteins or “dead” Cas proteins (dCas). These proteins can be generated according to any suitable method known to the skilled person. For example, they may be obtained by mutating catalytic arginine residues within the two conserved HEPN domains. Further information may be derived from Abudayyeh et al., 2017, Nature, 550(7675), 280-284.

The present invention also envisages the use of Cas proteins, e.g. Cas13, derived from different bacterial sources. For example, the Cas13 protein may be derived from Leptotrichia shahii, Leptotrichia wadei, Prevotella spp., or Streptococcus pyogenes.

Further details on the form and use of Cas proteins may be derived from suitable literature sources such as Granados-Riveron and Aquino-Jarquin, 2018, Cancer Research; Cox et al., 2017, Science, 10.1126/science.aaq0180; Gootenberg et al., 2018, Science 360, 439-444 or Abudayyeh et al., 2016, Science, 353 (6299).

In a subsequent step unbound mature crRNA molecules and unbound catalytically inactive crRNA-guided RNA-binding proteins are removed. The removal may be implemented with any suitable technique, e.g. by removing or relocating the reactants, inhibiting or destroying crRNA molecules or RNA binding proteins etc. In a typical and preferred embodiment, the removal is achieved by washing the complex as obtained in the previous step(s) of the method. The washing step may be performed once, or it may be repeated 1, 2, 3 or more times. It is preferred to perform the washing with nuclease-free water or with any other suitable solution containing appropriate ion concentration and/or having a suitable pH, as would be known to the skilled person.

In a further step of the method according to the present invention, an antibody binds to the protein-RNA polynucleotide complex of step (iv).

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that immunospecifically binds an antigen, in particular specifically binds the protein-RNA polynucleotide complex as defined herein above, more specifically binds the catalytically inactive crRNA-guided RNA-binding protein being part of said complex. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgG1, IgG2, IgG3, lgG4, lgA1 and IgA2) or subclass of immunoglobulin molecule. The immunospecific binding refers to the immunospecific detection and binding of an antibody to an antigenic epitope of a catalytically inactive crRNA-guided RNA-binding protein as defined herein above. In further embodiments antibodies of the invention include polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, or whole immunoglobulin molecules.

It is preferred that the antibody is a monoclonal antibody. In a particularly preferred embodiment, the antibody according to the present invention is a monoclonal antibody capable of binding a catalytically inactive dCas13 protein.

The antibody may, in certain embodiments, also be or comprise an antibody substructure. The term “antibody substructure” as used herein refers to single chain antibodies, Fab fragments, Fab′ fragments, fragments produced by a Fab expression library, F(ab′)2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, and epitope-binding fragments of any of the above. Preferred are Fab, Fab′ and F(ab′)2, Fv, single-chain Fvs (scFv), sc(Fv)2, single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The term “Fab fragment” as used herein refers to antibody fragments consisting of the first constant domain of the heavy chain (CH1), the constant domain of the light chain (CL), the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of an intact immunoglobulin protein.

The term “F(ab′)2” or “F(ab′)2 fragment” as used herein refers to antibody fragments consisting of two first constant domains of the heavy chain (CH1), two constant domains of the light chain (CL), two variable domains of the heavy chain (VH) and two variable domains of the light chain (VL) of an intact immunoglobulin protein, i.e. it comprises two Fab fragments. Additionally, F(ab′)2 molecules comprise a S-S linkage in the antibody hinge region which combines the Fab fragments. The term “Fab′ fragment” as used herein refers to fragments derived from “F(ab′)2” molecules, preferably fragments comprising the S-S linkage in the antibody hinge region. The term “Fv fragments” as used herein refers to antibody fragments consisting of the two variable antibody domains VH and VL; further details may be derived from Skerra and Pluckthun, 1988, Science, 240: 1038-1041. The term “single chain Fv fragment (scFv)” as used herein relates to antibody fragments consisting of the two VH and VL domains linked together by a flexible peptide linker; further details may be derived from Bird and Walker, 1991, Trends Biotechnol., 9: 132-137.

Also envisaged are antibody forms such as diabodies or mini-bodies. Details may be derived from Hudson and Kortt, 1999, J. Immunol. Methods, 231(1-2):177-89 or Quiocho, 1993, Nature, 362(6418): 293-294.

The antibodies may be from any animal origin including birds and mammals. Preferably, the antibodies are murine (e.g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, chicken, or human.

The antibodies according to the present invention are typically monospecific. In certain specific embodiments, the antibody may also be bispecific, or of a greater multispecificity. Multispecific antibodies may be specific for different epitopes of a catalytically inactive crRNA-guided RNA-binding protein as defined herein above.

Antibodies of the present invention may also be described or specified in terms of the epitope(s) or portion(s) of a polypeptide which they recognize or specifically bind. The epitopes may preferably be present on a catalytically inactive crRNA-guided RNA-binding protein as defined herein above, more preferably on a catalytically inactive Cas12 or Cas13 protein, or a catalytically inactive Cas12a derivative capable of binding to RNA polynucleotides, or a catalytically inactive Cas13a, Cas13b, Cas13c or Cas13d protein.

In a further step of the method of the present invention, unbound antibodies are removed. The removal may be implemented with any suitable technique. In a typical and preferred embodiment, the removal is achieved by washing the antibody-bound complex as obtained in the previous step(s) of the method. The washing step may be performed once, or it may be repeated 1, 2, 3 or more times. It is preferred to perform the washing with nuclease-free water or with any other suitable solution containing appropriate ion concentration and/or having a suitable pH, as would be known to the skilled person.

In a final step of the method of the present invention, the antibody-bound protein-RNA polynucleotide-complex as mentioned above is detected. The detection may be performed according to any suitable methodology known to the skilled person. The detection of the antibody-bound protein-RNA polynucleotide-complex as mentioned above is preferably based on an enzymatic reaction or a non-enzymatic reaction.

The non-enzymatic reaction may, for example, be performed via the identification of a label present on the antibody as defined herein. Such a label may, for example be any suitable label known to the skilled person, e.g. a radioactive label, a fluorescent label, a chemiluminescent label or a bioluminescent label. Examples of labels that can be conjugated to an antibody according to the invention include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodop-sin), chemiluminescent compounds (e.g. luminal, imidazole), fluorescent polypeptides (e.g. green fluorescent protein (GFP)) and bioluminescent proteins (e.g. luciferin, luciferase), and contrast agents such as USPIOS or 19Fluor. Further labels which may be used within the context of the present invention include 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine, TAMRA, Dabcyl, Black Hole Quencher, BHQ-1 or BHQ-2. The antibody may also be labeled with radioisotopes e.g. ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb, ⁶⁸Ga or ¹⁸F. Particularly preferred is a chemiluminescent detection.

More preferably, the detection is based on a chemiluminescent reaction of an acridinium ester. In general, acridinium esters can be stimulated to produce light in the presence of dilute alkaline hydrogen peroxide. Moreover, formed N-methylacridone molecules are typically resistant to quenching before radiation. The chemiluminescent label, in particular the acridinium ester, may be conjugated or covalently linked to the antibody as described herein at any suitable position.

The detection of the antibody-bound protein-RNA polynucleotide-complex as mentioned above via an enzymatic reaction may involve the presence of an enzymatic label. Preferred enzymatic activities or labels are horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOD) or esterase. The enzymatic activities may accordingly be conjugated or covalently linked to the antibody as described herein at any suitable position.

The enzyme-based detection further requires a suitable substrate which is converted by an enzymatic activity as mentioned above. For example, for horseradish peroxidase (HRP) there are several different types of substrates which may be chosen in accordance with the particular assay and mode of detection. One option is the use of chromogenic HRP substrates, which remain in solution and become colored after reaction with HRP. Typically used chromogenic HRP substrates include 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2′-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid](ABTS). After oxidation by HRP, some substrates precipitate. An oxidized HRP substrate may, for example, form a precipitate that is colored, electron-dense, or luminescent. An example of a suitable HRP substrate that is both, colored and electron-dense, which is envisaged by the present invention, is 3,3′-diaminobenzidine (DAB). Further envisaged is the use of the enhanced chemiluminescence (ECL) technology, e.g. as commercialized by BioRad Inc.

Suitable substrates for alkaline phosphatase (AP) include p-Nitrophenyl phosphate (PNPP) and 4-Methylumbelliferyl phosphate (4-MUP). The use of PNPP is based on the principle that when alkaline phosphatase and PNPP are reacted, a yellow water-soluble reaction product is formed. This reaction product can be detected since it absorbs light at 405 nm. 4-MUP is a substrate for alkaline phosphatase that forms the soluble fluorescent reaction product methylumbelliferone. Fluorescence can be measured with excitation at 360 nm and emission at 440 nm. The fluorescent end product may, for example, be observed using a UV light source.

The enzymatic test with glucose oxidase (GOD} is based on the oxidation of glucose by glucose oxidase, i.e. the oxygen-dependent oxidation of the C1 carbon atom of glucose, resulting in the provision of gluconolactone and hydrogen peroxide. Hydrogen peroxide may subsequently be reduced in a downstream color reaction performed by a peroxidase (POD) such as HRP. Alternatively, the produced hydrogen peroxide may be measured electrochemically and thus be quantified.

Suitable substrates for esterase based tests include 5-Bromo-4-chloro-3-indolyl acetate, 4-Methylumbelliferyl acetate, 1-Naphthyl acetate, 1-Naphthyl butyrate, 4-Nitrophenyl dedecanoate, 4-Nitrophenyl myristate, 4-Nitrophenyl butyrate p-aminophenylbutyrate, 5-(and 6-)carboxy-2′,7′-dichlorofluorescein diacetate or 4-Nitrophenyl octanoate.

The detection may be performed according to any suitable assay format, in particular an immunobiological assay. Examples of envisaged immunobiological assays in which the target RNA polynucleotide according to the present invention may be used include competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassay like RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays and electrochemiluminescence immunoassay (ECLIA) or suitable derivatives thereof. Details and further features of such assays would be known to the skilled person or can be derived from suitable literature sources such as the ebook Assay Guidance Manual, edited by G. Sitta Sittampalam, Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004 at ncbi.nlm.nih.gov/books/NBK53196/, in particular section Immunoassay Methods, Karen L. Cox et al., Eli Lilly & Company, Indianapolis, IN, of 2012, or later updated versions thereof. In specific embodiments, the assay format is an ELISA. The term “ELISA” or “ELISA assay” as used herein relates to a solid-phase enzyme immunoassay (EIA) which is generally used to detect the presence of a compound in a sample, which is typically liquid, or has been brought into solution, using one or more antibodies. The ELISA can be used as a diagnostic tool, e.g. in medicine, or as quality-control check.

Typically, antigens from the sample, i.e. antigens present on the catalytically-inactive crRNA-guided RNA-binding protein bound to the target RNA polynucleotide as described herein, are attached to a solid phase such as a surface or a magnetic bead which can be attracted to a compatible surface, e.g. via the binding of the RNA polynucleotide to the catcher polynucleotides which is complementary to at least a portion of a target RNA polynucleotide as described herein above. It is preferred to use magnetic beads. Subsequently, a specific antibody is applied over the surface or the beads so it can bind to the antigen. This antibody may be linked to an enzyme, e.g. as defined herein above, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, for example a color change in the substrate.

In particularly preferred embodiments the assay is performed in accordance with and/or on immunoassay platforms such as Centaur or Atellica as commercialized by Siemens Healthineers. Further information on these platforms may be derived, for example, from healthcare.siemens.de/immunoassay/systems/advia-centaur-xpt (visited on Oct. 24, 2018) or healthcare.siemens.com/integrated-chemistry/systems/atellica-solution-analyzers (visited on Oct. 24, 2018), or from the document “ADVIA Centaur XPT Immunoassay System Technical Specifications” which can be derived from suitable internet repositories such as https://static.healthcare.siemens.com/siemens_hwem-hwem_ssxa_websites-context-root/wcm/idc/groups/public/@global/@lab/documents/download/m da2/otmw/~edisp/170168-gc1_updates_advia_centaur_xpt_ss_fnl2-03931885.pdf (visited on Oct. 25, 2018) or from the document “Atellica IM 1300 Analyzer and Atellica IM 1600 Analyzer Technical Specifica-tions” which can be derived from suitable internet repositories such as https://static.healthcare.siemens.com/siemens_hwem-hwem_ssxa_websites-context-root/wcm/idc/groups/public/@global/@lab/@corelab/documents/d ownload/mda4/mtgy/~edisp/30-17-10702-01-76_atellica_im_1300-1600_ss_final_v2-05269699.pdf (visited on Oct. 25, 2018).

The detection according to the present invention may be a qualitative detection, a semi-quantitative detection or a quantitative detection.

The term “qualitative detection” as used herein means that the method of the present invention is capable of indicating whether a specific target RNA polynucleotide is present or not. Accordingly, by detecting a signal via the bound antibody, it can de deduced that the target RNA polynucleotide is present, e.g. in a mixture of different RNA polynucleotides as described above. In the preferably envisaged ELISA a qualitative assay means that the assay can be used to achieve a yes or no answer indicating whether a target RNA polynucleotide (via the catalytically-inactive crRNA-guided RNA-binding protein as defined herein above) is present in a sample. This may be achieved via a comparison versus a sample containing no target RNA polynucleotide or an unrelated control target RNA polynucleotide.

The term “semi-quantitative detection” as used herein means that the method of the present invention is capable of indicating whether a specific target RNA polynucleotide is present above a certain threshold with respect to its numbers or amount or concentration in a solution. The threshold may be suitably defined as would be known to the skilled person. For example, by detecting a signal in an assay on the basis of the antibody as described herein, it can de deduced that the target RNA polynucleotide which is present, e.g. in a mixture of different RNA polynucleotides as described above, has surpassed a certain predefined limit with respect to its numbers, amount or concentration. The format also allows to compare relative levels of target RNA polynucleotides between samples, in particular between samples used within the same assay format, e.g. ELISA, or assays performed on the platforms Centaur or Atellica as mentioned herein above.

The term “quantitative detection” as used herein means that the method of the present invention is capable of indicating the approximate or exact numbers, amount or concentration of a specific target RNA polynucleotide used for the method. For the quantitative detection suitable control and/or calibration steps are required. Typically, the quantitative de-tection involves the interpretation of signals in comparison to a standard curve (e.g. a serial dilution of a known, purified target RNA polynucleotide) in order to precisely calculate the concentrations of target RNA polynucleotide in various samples. For example, in an ELISA format, the signal data obtained may be graphed with optical density vs. log concentration to produce a sigmoidal curve. Known concentrations of target RNA polynucleotide may be used to produce a standard curve. This data may subsequently be used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. This can advantageously be done directly on the graph or with suitable curve fitting software known to the skilled person.

It is further preferred that the quantitative detection is performed by averaging the triplicate of the standards readings and by deducting the reading of the blank control sample. Subsequently, a standard curve is plotted and the line of best fit is identified so that the concentration of the samples can be determined. Any dilutions made need to be adjusted for at this stage. Alternatively, the signal data may be plotted using semi-log, log/log, log/logit or derivatives thereof in 4 or 5 parameter logistic models. Using software based/automated solutions suitable graphing approaches may be implemented. The approach further envisages the use of linear regression, e.g. within a software package, which allows for additional control possibilities. Further details would be known to the skilled person or can be derived from suitable literature sources such as the document Laboratory Procedure Manual: Complex PSA using Advia Centaur in Serum NHANES 2007-2008 of the University of Washington Medical Centre.

In very specific embodiments detected target RNA polynucleotides, i.e. RNA polynucleotides comprising a target specific sequence, may subsequently be extracted, optionally be purified, stored and/or used for additional activities.

The present invention further envisages, in specific embodiments, the optional use or implementation of one or more additional washing or removal steps between or after any one or more of the above mentioned steps as mentioned above. These removal steps may be implemented with any suitable technique, e.g. by removing or relocating the reactants, inhibiting or destroying molecules. In a typical and preferred embodiment, the removal is achieved by washing as described herein above. The washing step may be performed once, or it may be repeated 1, 2, 3 or more times. It is preferred to perform the washing with nuclease-free water or with any other suitable solution containing appropriate ion concentration and/or having a suitable pH, as would be known to the skilled person.

In preferred embodiments, one or more of the removal or washing steps as mentioned herein above, including the removal steps for the unbound mature crRNAs, unbound catalytically inactive crRNA-guided RNA-binding protein or the unbound antibodies, may be carried out with the help of a magnet capable of collecting magnetic beads, e.g. connected to the target RNA polynucleotide as mentioned above. The beads may subsequently be washed with a continuous water flow, followed by the creation of vacuum which removes the water.

It is particularly preferred that the washing steps, including any washing step as mentioned herein above, are performed in accordance with procedures described for the Centaur or Atellica assay platforms of Siemens Healthineers as described herein above or known to the skilled person.