Thermostable RNA polymerase

Abstract

The present invention relates to a protein having at least 50% sequence identity to SEQ ID NO: 1 and its use in in vitro transcription methods. The present invention further relates to nucleic acid molecules encoding the protein and host cells expressing the protein.

Description

[Technical field]

[0001] Field The present invention is directed to novel thermostable RNA polymerases and their uses for in vitro transcription and in diagnostic methods. [Background technology]

[0002] 1 Introduction In vitro transcription (IVT) reactions usually require a template nucleic acid molecule containing a promoter, a buffer system containing ribonucleotide triphosphates, DTT and magnesium ions, and a suitable ribonucleic acid (RNA) polymerase. The RNA polymerase is often selected from single-subunit bacteriophage-derived RNA polymerases such as SP6, T3 and T7. Transcription is initiated by the binding of the RNA polymerase to its promoter sequence, followed by the donation of a template strand to the active site, the production of an RNA transcript, and the termination of the transcription reaction.

[0003] In addition to its use for IVT, RNA polymerase is often used in nucleic acid amplification methods, especially for diagnostic purposes. For example, nucleic acid sequence-based amplification (NASBA; U.S. Pat. No. 5,654,142A) and transcription-mediated amplification (TMA; WO 1991001384A1) involve alternating cycles in which RNA polymerase and reverse transcriptase are continuously generating each other's templates under isothermal conditions. After amplification, hybridization of complementary oligonucleotide probes can allow detection via detectable labels, for example when used in quantitative PCR, also called quantitative real-time PCR. Furthermore, certain clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) protein-based nucleic acid detection systems utilize RNA intermediates (Steens et al., 2021. Nature Comm 12:1-12).

[0004] It would be advantageous if the reaction temperature could be increased and the reaction rate could be improved. For example, if the reaction temperature of isothermal amplification is increased, it may be possible to amplify RNA with secondary structure. However, even if an RNA polymerase mutant that shows higher thermostability than the RNA polymerase derived from wild-type bacteriophage is identified (Boulain et al., 2013.Protein Eng Des Sel.26(11):725-734), there is currently no RNA polymerase commercially available that maintains activity after incubation at 65°C for more than 5 minutes. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for RNA polymerases that have improved stability and activity at higher reaction temperatures. [Means for solving the problem]

[0006] 2. Brief description of the invention The present invention provides a method comprising: incubating a template nucleic acid molecule with a protein having at least 50% sequence identity to SEQ ID NO:1 in the presence of ribonucleic acid nucleotides (rNTPs) and a suitable buffer; and transcribing at least a portion of the template nucleic acid molecule into an RNA molecule by carrying out a transcription reaction at a temperature between 30°C and 80°C. The template nucleic acid molecule is preferably a deoxyribonucleic acid (DNA) template molecule, either a single-stranded or double-stranded DNA template molecule. The buffer preferably contains 25 to 200 mM NaCl. The transcription reaction is preferably carried out at a temperature between 45°C and 75°C. The template nucleic acid molecule may be generated by any means, for example by a pre-amplification reaction.

[0007] The transcribed RNA molecule may further be incubated with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) protein-based nucleic acid detection system. The CRISPR-Cas nucleic acid detection system preferably comprises: a) an effector complex comprising a type III Cas protein and at least one CRISPR RNA (crRNA) that binds to a target RNA molecule; b) a means for directly or indirectly measuring the level of cyclic oligoadenylate (cOA). In a preferred method of the present invention, the pre-amplification reaction, the transcription reaction and the incubation with the nucleic acid detection system are all performed in a one-pot reaction.

[0008] The present invention further provides a protein having at least 50% sequence identity with SEQ ID NO: 1. The protein preferably comprises a suitable tag.

[0009] The present invention further provides a nucleic acid molecule encoding a protein of the invention, said nucleic acid molecule preferably being codon optimized for expression of said protein in a suitable host cell.

[0010] The present invention further provides host cells expressing the proteins of the invention.

[0011] The present invention further provides the use of the protein of the present invention for carrying out a transcription reaction to transcribe at least a part of a template nucleic acid molecule into an RNA molecule, said transcription reaction being preferably carried out at a temperature between 30°C and 80°C, preferably between 45°C and 75°C. [Brief description of the drawings]

[0012] 3 Brief description of the drawings [Figure 1] Substrate specificity of PhiFa_44 in the case of IVT. The marker used is NEB low range ssRNA (NEB#N0364S). [Diagram 2]Analysis of IVT performance of phiPa_44 under various NaCl concentrations and temperatures. A: Template only, no DNase. B: Template only, with DNase. C: PhiFa only, with DNase. [Diagram 3] SARS-COV-2 one-pot reaction. [Figure 4] In vitro transcription reactions using single-stranded (ss) DNA templates A and B. "+" denotes the presence of phiFa protein, whereas "-" denotes its absence. [Diagram 5] In vitro PhiFa-44 RNA transcription assay using 5' fluorescently labeled (Cy3) single-stranded DNA (50 nucleotides) as template. One reaction was subjected to RNase H treatment. All products were analyzed on a native 4-20% (w / v) polyacrylamide gel. Nucleic acids were selectively visualized using fluorescent gel scanning. Only the Cy3 filter shows the Cy3-labeled reaction products (left), whereas SYBR Gold staining followed by visualization with a Cy2 filter shows all nucleic acids (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 4 Detailed Description of the Invention 4.1 Definition The term "phiFa_44" as used herein refers to the protein having SEQ ID NO: 1. The protein originates from the bacteriophage phiFa (GenBank: MH673672.2; Taxonomic ID: 1400796), which was isolated near Mount Etna in Sicily, Italy (Severinov et al., 2014. Bacteriophage 4: e29399, DOI: 10.4161 / bact.29399).

[0014] The term "RNA polymerase," as used herein with reference to phiFa_44, refers to a DNA-dependent RNA polymerase that catalyzes the synthesis of a complementary strand of RNA from a DNA template.

[0015] The term "promoter" as used herein refers to a nucleotide sequence at the 5' end of a gene to which a transcription initiation mechanism, including an RNA polymerase such as DNA-dependent RNA polymerase, binds and initiates transcription. The promoter is often located 5 to 100 bp upstream of the start codon of the gene.

[0016] The term "clustered regularly interspaced short palindromic repeats (CRISPR)" as used herein refers to one or more specialized regions of DNA within the genome of a prokaryotic microorganism. These regions are characterized by the presence of nucleotide repeats interspersed with spacer sequences, typically directing DNA repeats of about 25 to about 38 bp separated by unique spacer sequences of similar length derived from previous encounters with invasive elements (Grissa et al., 2007. BMC Bioinformatics 8:172). They serve as memory to rapidly attack these invaders during the next infection. The genomic regions contain one or more genes encoding CRISPR-associated (Cas)-effector proteins located in the vicinity of the CRISPR locus.

[0017] The term "CRISPR RNA or crRNA" as used herein refers to an RNA molecule derived from a CRISPR, comprising a spacer sequence and at least a 5' repeat derived end. The crRNA preferably has a length of at least 30 nucleotides, more preferably at least 34 nucleotides, more preferably at least 40 nucleotides, more preferably at least 46 nucleotides. The crRNA is preferably less than 1000 nucleotides, preferably less than 200 nucleotides, preferably less than 100 nucleotides. The crRNA molecule may comprise ribonucleic acid nucleotide analogs, such as inosine, uridine, xanthine, hypoxanthine, 2,6-diaminopurine, and 6,8-diaminopurine based ribonucleotides and deoxyribonucleotides.

[0018] The term "CRISPR-associated (Cas) effector protein" as used herein refers to a protein that associates with crRNA. CRISPR / Cas systems are broadly divided into two classes here. Class 1 systems use a multi-subunit Cas complex, while class 2 systems use a single Cas protein to mediate their activity. Class 1, type III CRISPR-Cas systems have evolved to specifically target RNA sequences. The unique proteins in these systems are Cas3 in class 1, type I systems, Cas9 in class 2, type II systems, Cas10 in class 1, type III systems, Cas12 in class 2, type V systems, and Cas13 in class 2, type VI systems.

[0019] The term "effector complex" as used herein refers to a CRISPR-Cas ribonucleoprotein complex that has (ribo)nuclease activity and can cleave and inactivate an invading nucleic acid sequence that contains a complementary sequence to a spacer sequence in the crRNA. The complex comprises at least one crRNA and at least one Cas effector protein.

[0020] The term "reverse transcription" as used herein refers to the generation of complementary DNA (cDNA) from an RNA template. Retroviruses and some retrotransposons encode an enzyme called reverse transcriptase to replicate their genomes. Retroviral reverse transcriptase has three sets of biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNAse H), and DNA-dependent DNA polymerase activity. Enzymes often used to reverse transcribe RNA are Moloney murine leukemia virus reverse transcriptase and avian myeloblastosis virus reverse transcriptase, and variants thereof, including heat-stable variants.

[0021] The term "isothermal amplification" as used herein refers to the exponential amplification of nucleic acid molecules in the absence of thermal cycling, as required for polymerase chain reaction (PCR). Polymerases with strand displacement activity are usually used in isothermal methods. Preferred single-tube isothermal reactions include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA) reaction, and nicking enzyme amplification reaction (NEAR). Preferred single-tube isothermal reaction is loop-mediated isothermal amplification (LAMP).

[0022] The term "type V effector protein" as used herein refers to class 2 effector proteins, characterized by a specific nuclease domain required for cleavage of even non-target desoxyribonucleic acid molecules. At least 13 subtypes are currently known: VA, VB to VM. Type V Cas proteins can be isolated from organisms such as Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., Prevotella sp., and some archeaebacteria. The length of the entire guide is preferably 42-44 nucleotides. The gRNA of a Cas12 V-type effector protein preferably comprises the dinucleotide TT as a protospacer adjacent motif (PAM) in the gRNA, more preferably a 5"-TTTN motif. Exemplary V-type effector proteins are Cpf1 (VA type) and C2c1 (VB type). Cas12 and Cas14 are prototypic V-type effector proteins.

[0023] The term "type VI effector protein" as used herein refers to a class 2 effector protein with non-specific ribonuclease activity. Four subtypes are currently known: subtypes VI-A, VI-B, VI-C, and VI-D. Type VI Cas proteins can be isolated from organisms such as Leptotrichia buccalis, Leptotrichia shahii, Ruminococcus flavefaciens, Bergeyella zoohelcum, Prevotella buccae, and Listeria seeligeri. The length of the entire guide is preferably 52-66 nucleotides. The PAM motif of Type VI effector proteins is variable, as known to those skilled in the art, and may contain 3'non-G, 5'non-C and 3'NAN or NNA for LshCas13a (BzCas13b), or none (RfCas13d).Cas13 is the prototypic Type VI effector protein.

[0024] The term "Type III Cas protein" as used herein refers to an RNA-targeting multi-subunit CRISPR-associated complex that contains at least the Cas10 protein.

[0025] The term "type IIIA Cas protein" as used herein refers to an RNA-targeting type III CRISPR / Cas protein complex that has non-specific deoxyribonuclease activity upon binding to a target RNA molecule. Type IIIA Cas proteins include, for example, type IIIA Csm protein complexes from Staphylococcus thermophilus, Thermus thermophilus, and Staphylococcus epidermis.

[0026] The term "type III-B Cas protein" as used herein refers to RNA-targeting type III CRISPR-Cas protein complexes, apart from type III-B Cas proteins from Thermus species, which also have non-specific deoxyribonuclease activity. The type III-B Cas complexes consist of six to seven individual proteins. Type III-B Cas proteins include, for example, type III-B Cmr protein complexes from Pyrococcus furiosus, Thermus thermophilus, and Sulfolobus solfataricus.

[0027] The terms "quenched, quencher, and quenching" as used herein refer to a process in which the signal intensity, preferably the fluorescence intensity, of a given substance is reduced. Fluorescence quenching is a physicochemical process that absorbs the emission from a fluorescent molecule. Fluorescence quenching can be used as an indicator in nucleic acid diagnostics when a fluorophore and a quencher molecule are attached to the end of a single-stranded nucleic acid molecule and are in close proximity to each other. When the nucleic acid molecule hybridizes to its target or is cleaved by a nuclease, the fluorophore-quencher complex is separated, allowing the fluorophore to emit light.

[0028] The term "dark quencher" as used herein refers to a quencher that absorbs the excitation energy from a fluorophore and dissipates the energy as heat. Therefore, dark quenchers do not emit light themselves. Dark quenchers are used in molecular biology in combination with fluorescent molecules.

[0029] The term "cyclic oligoadenylate (cOA)" as used herein refers to a ring structure containing 3-6 molecules of adenosine monophosphate (AMP). The formation of cOA is catalyzed by the cyclase domain of Cas10, which is part of the type III CRISPR / Cas effector system.

[0030] The term "PPi" or pyrophosphate, as used herein, refers to salts or esters of pyrophosphate. Alternative names are diphosphate and dipolyphosphate.

[0031] The term "inorganic pyrophosphatase," or inorganic diphosphatase, as used herein, refers to an enzyme that catalyzes the conversion of one pyrophosphate ion into two phosphate ions. The enzyme belongs to the enzyme class EC 3.6.1.1.

[0032] The term "cOA-dependent effector protein" as used herein refers to a protein whose activity depends on the amount of cOA. Examples include ribonucleases such as endoribonucleases that non-specifically degrade RNA using a HEPN (high eukaryotic and prokaryotic nucleotide binding) active site. These nucleases are activated by the binding of one or more cOA molecules using their CRISPR-associated Rossmann fold (CARF) domains. Examples of such non-specific effector endoribonucleases include the Cas accessory protein Csx1 from Pyrococcus furiosus and Csm6 from Mycobacterium tuberculosis.

[0033] The term "non-naturally occurring protein" as used herein refers to a protein that has an amino acid sequence and / or a post-translational modification pattern that differs from that of the protein in its natural state. For example, a non-naturally occurring protein may have one or more amino acid substitutions, deletions, or insertions at the N-terminus, C-terminus, and / or between the N-terminus and C-terminus of the protein. A "non-naturally occurring" protein may have an amino acid sequence that differs from a naturally occurring amino acid sequence but is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a naturally occurring amino acid sequence. In certain cases, a non-naturally occurring protein may contain an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, phosphorylation, etc.) when it is produced by a different (e.g., bacterial) cell. Additionally, a non-naturally occurring protein is a tagged protein that contains one or more specific tags by genetic engineering. Examples of such tags include, but are not limited to, c-myc domains, hemagglutinin tags, maltose binding proteins, glutathione-S-transferase, FLAG tag peptides, biotin acceptor peptides, streptavidin binding peptides, and calmodulin binding peptides, as presented in Chatterjee, 2006 (Chatterjee, 2006. Cur Opin Biotech 17:353-358).

[0034] The term "non-naturally occurring nucleic acid," as used herein, refers to a nucleic acid that contains: (1) a sequence of nucleotides that differs from that of a naturally occurring nucleic acid; (2) one or more non-naturally occurring nucleotide monomers; and / or (3) one or more other modifications, such as an added label or other moiety.

[0035] The term "non-naturally occurring composition" refers to a composition that includes: (1) a combination of components that are not naturally combined, e.g., because they are present in different locations, different cells, or different cellular compartments; (2) a combination of components that have relative concentrations not found in nature; (3) a combination of components that is devoid of one of the components that is normally associated with it in nature; (4) a combination of components that is not found in nature, e.g., in a dried, lyophilized, crystalline, aqueous, etc. form; and / or (5) a combination that contains a non-naturally occurring component. For example, the preparation may contain a non-naturally occurring buffer, detergent, dye, solvent, or preservative. The composition may be in any form, e.g., aqueous or lyophilized, and may be in any state, e.g., frozen or aqueous form.

[0036] The term "primer", as used herein, refers to an oligonucleotide, preferably 15-50 nucleotides in length, that is effective for annealing to a template nucleic acid molecule and transcription priming, e.g., reverse transcription, or replicating said template nucleic acid molecule with a polymerase. The oligonucleotide may comprise deoxyribonucleotides, ribonucleotides, or combinations or variants thereof. The variants include synthetic oligonucleotide analogs, e.g., phosphorothioate, phosphotriester, phosphorothioate 2-alkylated, and phosphoramidate analogs, analogs having modifications at the 2' position of the nucleoside sugar ring, such as 2'-fluoro, O-methyl, or methoxyethyl, peptide nucleic acids, bridged nucleic acids, and / or locked nucleic acid molecules.

[0037] 4.2 PhiFa_44 protein 4.2.1 Currently, there are no commercially available DNA-dependent RNA polymerases that remain active for more than 5 minutes at 65°C. Bacteriophages that can infect thermophilic bacteria such as Thermus thermophilus were analyzed for thermostable DNA-dependent RNA polymerases. Bacteriophage phiFa (GenBank: MH673672.2; Taxonomic ID: 1400796) was identified as a candidate phage. PhiFa is a yet unclassified Oshimavirus that belongs to the Siphoviridae phage with a long non-contractile tail. PhiFa encodes a hypothetical protein "phiFa_44" (GenBank: QKE11339.1; SEQ ID NO: 1) predicted to be an "RNA polymerase".

[0038] A BlastP search identified a hypothetical protein WP_164703602.1 from Escherichia coli as having a relatively high identity match of 56.79% over approximately 90% of the length of the phiFa_44 protein. However, upon more thorough inspection, WP_164703602.1 appears to be a contaminant in the sequencing analysis, since alignment to the E. Coli genome was not possible. Furthermore, the coding sequence has been uploaded to a database as a separate contig. The true origin of WP_164703602.1 is unknown. No homology was found between phFa_44 and the bacteriophage-derived RNA polymerases SP6, T3, and T7.

[0039] One further alignment was to a hypothetical protein from Planctomycetes bacterium (MBI5851759.1) with a score of about 30% over 56% of the length of the phiFa_44 protein. The final alignment was also to a hypothetical protein from E. Coli (WP_206306715.1) with a score of about 47% over only 10% of the length of the phiFa_44 protein.

[0040] A search in the Pfam database (Mistry et al., 2021. Nucleic Acids Res 49:D412-D419) with the PhiFa_44 amino acid sequence identified only one result spanning a length of 257 amino acid residues with a score of only 0.019. This score is not significant. The one result identified (RdRP; PF05183) belongs to the family of eukaryotic RNA-dependent RNA polymerases. These proteins are involved in post-transcriptional gene silencing when thought to amplify dsRNA templates.

[0041] A search using the HHpred tool in the Teubingen toolkit (available at toolkit.tuebingen.mpg.de / ) identified two sequences, 5FSW_C and 2J7N_B, with very low sequence similarity (25.8% and 17.5%, respectively). Both identified proteins are predicted to be RNA-dependent RNA polymerases. A sequence with even lower sequence similarity (<8%) was identified as a multisubunit DNA-dependent RNA polymerase.

[0042] Both types of RNA-dependent RNA polymerases and multisubunit RNA polymerases with non-significant scores are not considered possible in relation to phiFa_44.

[0043] In silico structural analysis was performed to gain further insight into the phiFa_44 protein. Initial results showed some similarity to the QDE1 RNA-dependent RNA polymerase, where the similarity was up to 22% sequence identity over a region of only 207 amino acid residues. However, this domain resembles a magnesium (Mg) binding domain. The predicted 3D structure (data not shown) confirmed these similarities, but indicated amino acid residues D366, D368 and D370 as residues in phiFa_44 that may be involved in Mg binding.

[0044] Given the absence of significant sequence similarity to known RNA polymerases such as bacteriophage-derived RNA polymerases SP6, T3 and T7, it was decided not to pursue phiFa_44 as a candidate thermostable RNA polymerase, however, one final IVT experiment performed (see Example 1) suggested that phiFa_44 could function as a thermostable RNA polymerase, despite the low sequence identity.

[0045] 4.2.2 Proteins having at least 50% sequence identity with SEQ ID NO:1 according to the invention can be expressed and purified from a suitable expression system.

[0046] Commonly used expression systems for the production of heterologous proteins include E. Coli, Bacillus spp., baculovirus, yeast, fungi, filamentous fungi or yeasts such as Saccharomyces cerevisiae and Pichia pastoris, eukaryotic cells such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells and PER.C6® cells (Thermo Fisher Scientific, MA, USA), and plants. The efficiency of recombinant protein expression in heterologous systems depends on many factors, both on the transcriptional and translational levels.

[0047] The protein having at least 50% sequence identity with SEQ ID NO:1 according to the present invention is preferably produced in a prokaryotic cell, preferably E. Coli. The protein is preferably produced by expression cloning of the protein in the prokaryotic cell of interest, preferably E. Coli. The expression construct, preferably DNA, is preferably produced by recombinant techniques including the use of polymerases, restriction enzymes and ligases, as known to those skilled in the art. Alternatively, the expression construct is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant techniques, as known to those skilled in the art.

[0048] Alternatively or additionally, a protein having at least 50% sequence identity with SEQ ID NO:1 according to the invention can be isolated from a thermophilic organism by expression of a tagged protein having at least 50% sequence identity with SEQ ID NO:1 according to the invention in said thermophilic organism and isolation of said protein based on the tag.

[0049] Said expression construct is preferably codon optimized to enhance expression of a protein having at least 50% sequence identity with SEQ ID NO: 1 according to the invention in a prokaryotic cell of interest, preferably E. Coli. Further optimization may include removal of cryptic splice sites, removal of cryptic polyA tails and / or removal of sequences that cause undesired folding of the mRNA. Furthermore, the expression construct preferably encodes a protein transport signal for secretion of a protein having at least 50% sequence identity with SEQ ID NO: 1 according to the invention from the cell into the periplasm of the prokaryotic organism, allowing efficient purification of a protein having at least 50% sequence identity with SEQ ID NO: 1 according to the invention.

[0050] Methods for purifying proteins having at least 50% sequence identity with SEQ ID NO:1 according to the present invention are known in the art and are generally based on chromatography, such as affinity chromatography and ion exchange chromatography, to remove contaminants. In addition to contaminants, it may also be necessary to remove undesirable derivatives of the product itself, such as degradation products and aggregates. Suitable purification process steps are presented in Berthold and Walter, 1994 (Berthold and Walter, 1994. Biologicals 22:135-150).

[0051] Alternatively or additionally, recombinant proteins having at least 50% sequence identity with SEQ ID NO:1 according to the present invention can be tagged with one or more specific tags by genetic engineering, allowing the protein to attach to a tag-specific column and thus be isolated from impurities. The purified protein is then exchanged from the affinity column by a decoupling reagent. The method is increasingly applied to purify recombinant proteins. Conventional tags for proteins, such as histidine tags, can be used with affinity columns that specifically capture the tag (e.g. Ni-IDA columns for histidine tags) to isolate the protein from other impurities. The protein is then exchanged from the column using a decoupling reagent that conforms to the specific tag (e.g. imidazole for histidine tags). This method is more specific compared to traditional purification methods.

[0052] Further suitable tags include c-myc domain, hemagglutinin tag, maltose binding protein, glutathione-S-transferase, FLAG tag peptide, biotin acceptor peptide, streptavidin binding peptide and calmodulin binding peptide, as presented in Chatterjee, 2006 (Chatterjee, 2006. Cur Opin Biotech 17:353-358). Methods for using these tags are known in the art and can be used to purify proteins having at least 50% sequence identity with SEQ ID NO: 1 according to the present invention.

[0053] Methods for expressing proteins in E. coli are known in the art and can be used to express and purify proteins having at least 50% sequence identity with SEQ ID NO:1 in accordance with the present invention.

[0054] 4.3 Methods for transcribing nucleic acid templates The present invention provides a thermostable DNA-dependent RNA polymerase that allows in vitro transcription reactions to be carried out at elevated temperatures without debilitating catalytic activity loss.The elevated temperature can increase specific activity and aid in relaxing secondary structures from the resulting RNA molecules.Furthermore, the availability of thermostable RNA polymerases allows amplification at elevated temperatures, such as LAMP amplification, and nucleic acid detection methods that include synthesis of RNA strands, particularly one-pot reactions in the case of detection systems that include type III CRISPR-Cas systems.

[0055] The thermostable RNA polymerase according to the present invention has at least 50% sequence identity with SEQ ID NO: 1, preferably at least 60% sequence identity, preferably at least 70% sequence identity, preferably at least 80% sequence identity, preferably at least 90% sequence identity, preferably at least 91% sequence identity, preferably at least 92% sequence identity, preferably at least 93% sequence identity, preferably at least 94% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, preferably at least 99.5% sequence identity with SEQ ID NO: 1 means that one amino acid residue may differ between the thermostable RNA polymerase and SEQ ID NO: 1.

[0056] Suitable template nucleic acid molecules are or contain deoxynucleic acid nucleotides and include single-stranded (ss) DNA molecules, double-stranded (ds) DNA molecules, or hybrid ss-ds molecules. It has been found that the thermostable RNA polymerase according to the present invention can generally transcribe either single-stranded or double-stranded DNA molecules into RNA molecules. Such general low transcription activity does not apparently require a specific promoter region. As shown in Figure 1, the initiation of transcription of dsDNA templates by thermostable RNA polymerases such as phiFA_44 appears to occur randomly, resulting in a ladder of RNA products.

[0057] It has further been found that the thermostable RNA polymerase according to the invention, such as phiFA_44, specifically transcribes in particular single-stranded DNA molecules into RNA molecules, wherein the template strand comprises the consensus sequence GGGGCGG, preferably AGGGGCGG, more preferably TAGGGGCGG, more preferably TAGGGGCGGM, more preferably TAGGGGCGGMTA, where M represents either C or A, which may function as the phiFa_44 promoter sequence. [ka] is present in the ssDNA template A used for the IVT reaction shown in FIG. [ka] may function as a promoter sequence, resulting in an RNA transcript of approximately 70 nucleotides, as shown in Figure 4. Related sequences may drive transcription from separate transcription start sites, resulting in a more limited RNA product of approximately 300 nt in Figure 1.

[0058] In vitro transcription reactions involving a thermostable RNA polymerase according to the invention are carried out in a suitable buffer, with the pH maintained at an approximately constant value. The buffer may comprise phosphate, borate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), glycine, and / or [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS). The preferred reaction pH for the RNA polymerase is 6-10, preferably 7-9, e.g., 7.2, 7.5, 7.8, 8.0, 8.1, 8.5, 8.6 or 8.8.

[0059] A preferred buffer for the transcription reaction is or comprises Tris, preferably 10-100 mM Tris, which is preferably set at the desired pH by addition of an acid such as acetate and / or hydrogen chloride.

[0060] A suitable buffer for the transcription reaction should contain one or more 2+ or Mn 2+The divalent metal ion may be provided as a salt thereof, such as magnesium chloride, magnesium sulfate, and / or magnesium acetate. The concentration is preferably 0.5 to 20 mM, for example 1 to 10 mM, preferably about 2 mM, about 3 mM, about 5 mM, or about 7 mM.

[0061] Additional components of the buffer may include potassium ions, other salts such as, e.g., potassium chloride, ammonium sulfate, and / or betaine, ethylene glycol, 1,2-propanediol, and / or spermidine, e.g., 2-10% DMSO or 2-10% glycerol, to enhance transcription of the template nucleic acid molecule, as known in the art (Cheng et al., 1994. Proc Natl Acad Sci 91:5695-5699).

[0062] Preferably, additional components are included in the reaction buffer to stabilize enzyme activity, including gelatin, albumin, a reducing agent such as one or more of beta-mercaptoethanol, dithiothreitol (DTT), and / or tris(2-carboxyethyl)phosphine (TCEP), and / or a mild detergent such as, for example, TWEEN 20 or Triton-X100.

[0063] The transcription reaction is carried out in the presence of ribonucleotides (NTPs) that contain a ribose sugar group coupled to a nucleobase and a triphosphate group. The nucleobases include adenine, guanine, cytosine, uracil, and any modifications thereof. The term ribonucleotide includes reference to analogs of ribonucleotides such as fluorescent molecules, e.g., 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP), and / or other analogs such as inosine, xanthosine, N4-hydroxycytosine, N4-methoxycytosine, and 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (Suzuki et al., 2005. Nucleic Acids Symp Series 49:97-98).

[0064] The transcription reaction is carried out at a temperature of 30°C to 80°C, preferably 45°C to 75°C, for example, 50°C to 70°C, for example, 55°C, 60°C, and 65°C.

[0065] The transcription reaction is preferably carried out in the presence of 10-50 mM, preferably 20 mM Tris-HCl pH 8.8, 100-1000 nanomolar (nM), preferably about 500 nM of a thermostable RNA polymerase according to the present invention, 1-5 mM, preferably about 2.5 mM NTP mixture (NEB#N0466S), 1-10 mM, preferably about 2 mM MgCl, 10-250 mM, preferably about 50 mM NaCl, and a DNA template, preferably 1-100 ng, for example about 25 ng of a dsDNA template, in a total volume of 20 μL, at a temperature of 45° C.-75° C., for example 50° C.-70° C., for example 55° C., 60° C. and 65° C.

[0066] The IVT reaction is carried out for a period of time suitable for transcribing the DNA template, preferably the double-stranded DNA template, which period is preferably 0.01 to 1 hour, more preferably 0.1 to 0.5 hours, more preferably 0.2 to 0.4 hours.

[0067] 4.4 Diagnostic Assays 4.4.1 The method of the invention makes it possible to detect specific nucleic acid sequences that can be used in particular in human healthcare and animal diagnostics. For these purposes, nucleic acid material, including DNA and / or RNA, is preferably isolated from the sample. To this end, said nucleic acid material may be purified, for example, using a combination of physical and chemical methods. Preferably, commercially available systems for nucleic acid isolation are used, such as the NucliSENS® easyMAG® or NucliSENS® miniMAG® Nucleic Acid Extraction Systems (bioMerieux, Marcy l'Etoile, France), or the MagNA Pure 96 system (Roche Diagnostics, Almere, The Netherlands).

[0068] The sample may include biological fluids, e.g., upper respiratory tract specimens such as saliva, nasopharyngeal swabs, lower respiratory tract specimens such as sputum, nasopharyngeal secretions, oropharyngeal secretions, sweat, urine, stool, or blood. The term "blood" includes plasma, which has been prepared by removing red and white blood cells, e.g., by centrifugation, and serum, which has been prepared by forming a clot and removing the clot, e.g., by use of a centrifuge. Methods and compositions for isolating nucleic acid material from biological fluids, particularly swabs, preferably use aqueous solvents and not organic solvents and chaotropic salts.

[0069] To this end, RNA may be isolated from the sample by any technique known in the art, including suitable commercially available RNA isolation kits such as, but not limited to, Trizol (Invitrogen; Carlsbad, California), RNAqueous® (Applied Biosystems / Ambion, Austin, Tx), Qiazol® (Qiagen, Venlo, The Netherlands), Agilent Total RNA Isolation Lits (Agilent; Santa Clara, California), RNA-Bee® (Tel-Test. Friendswood, Texas), RNeasy mini kit (Qiagen, Venlo, The Netherlands), and Maxwell™ 16 Total RNA Purification Kit (Promega; Madison, Wisconsin).

[0070] The isolated RNA is preferably reverse transcribed into single-stranded or double-stranded complementary DNA (cDNA) with the aid of an RNA-dependent DNA polymerase using methods known to those skilled in the art. Reverse transcription can be primed by a universal primer, such as a random hexamer or nonamer, or by one or more specific primers, such as a virus-specific primer or even a gene-specific primer.

[0071] DNA, including genomic DNA, may be isolated from the sample by any technique known in the art, including suitable commercially available DNA isolation kits, such as, but not limited to, Quick-DNA Virus Kit (Zymo Research; Irvine, Calif.), NucleoSpin Dx Virus (Macherey-Nagel, Dueren, Germany), QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands), and EasyPure® Viral DNA / RNA Kit (Transgen Biotech, Beijing, China).

[0072] 4.4.2 The resulting RNA, DNA or cDNA may be used directly in a diagnostic CRISPR / Cas effector protein-based assay according to the invention, but is preferably amplified prior to detection to increase the level of detection. Amplification may be performed by any suitable amplification system, including, for example, ligase chain reaction (LCR), isothermal ribonucleic acid amplification systems, such as nucleic acid sequence-based amplification (NASBA), cleavage-based RNA signal amplification (Zhao et al., 2013. Nature Comm 4:1493), transcription-mediated amplification, strand displacement amplification, and polymerase chain reaction (PCR).

[0073] Preferred amplification reactions are single-tube isothermal reactions such as NASBA, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) reactions, and nicking enzyme amplification reactions (NEAR). Preferred single-tube isothermal reactions are loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) reactions.

[0074] In LAMP, six primers are typically used that recognize different target sequences on the template strand. Four of these primers are "internal primers" (LF, LB, FIP and BIP) that are designed to synthesize a new DNA strand. The external primers (F3 and B3) anneal to the template strand, also generating new DNA. These primers are accompanied by a DNA polymerase that assists in strand displacement and releases the newly formed DNA strand. This amplification is done under isothermal conditions at 60-65°C in less than an hour, e.g. in 20 minutes.

[0075] The RPA process (TwistDx Ltd., Cambridge, UK) uses a recombinase, a single-stranded DNA binding protein (SSB) and a strand-displacing polymerase, preferably supplemented with a reverse transcriptase, more preferably a reverse transcriptase and a DNA-dependent RNA polymerase such as T7 polymerase. The amplification is carried out under isothermal conditions at 37-42°C for less than one hour.

[0076] 4.4.3 The thermostable RNA polymerase according to the invention may replace DNA-dependent RNA polymerases in amplification reactions such as NASBA, cleavage-based RNA signal amplification (Zhao et al., 2013. Nature Comm 4:1493), transcription-mediated amplification, or RPA, thereby allowing said amplification reactions to be performed at higher temperatures.

[0077] 4.5 CRISPR / Cas-based detection systems 4.5.1 The thermostable RNA polymerase according to the invention can further be used in a clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection system comprising a CRISPR-associated effector protein (Cas) and at least one CRISPR RNA (crRNA) that binds to a target nucleic acid molecule.

[0078] Said CRISPR / Cas based detection system is preferably provided as a sensing device or "biosensor" comprising a CRISPR based nucleic acid system according to the invention. The signal, e.g. colorimetric, fluorometric, fluorescent or bioluminescent signal, generated when the CRISPR based nucleic acid system interacts with a nucleic acid molecule complementary to the crRNA, may be coupled to a transducer to allow quantification of the signal. The signal may be directly visualized or alternatively or additionally, may be converted into a measurable electrical parameter such as current or potential using a suitable transducer.

[0079] 4.5.2 Binding of crRNA to the target sequence results in activation of Cas effector proteins, which can be detected, for example, by collateral cleavage of reporter molecules, e.g., mediated by Cas12, Cas13 and Cas14, and / or by the generation of cyclic oligoadenylates (cOA), e.g., catalyzed by the cyclase domain of Cas10.

[0080] Crispr / Cas class 1 systems use multi-protein effector complexes (Koonin et al., 2017. Curr Opin Microbiol 37:67-78) and include type I systems, type III systems such as Cas10, and type IV systems. Crispr / Cas class 2 systems use single protein effectors and include type II CRISPR systems such as Cas9, type V systems such as Cas12 (also known as cpf1) and Cas14 (Harrington et al., 2019, Science 362:839-842), and type VI systems such as Cas13 (Makarova et al., 2017. Cell 168:328-328).

[0081] 4.5.3 Binding of the crRNA to its target nucleic acid sequence activates the collateral trans-acting nuclease activity of the Type V or Type VI CRISPR / Cas effector protein, which allows the use of a single-stranded "reporter" molecule to score the activation of the Type V or Type VI CRISPR / Cas effector protein.

[0082] The reporter for activated type V or type VI Cas is preferably a single-stranded nucleic acid molecule of at least 9 nucleotides, the cleavage of which by activated type V or type VI Cas can be detected. The single-stranded nucleic acid molecule preferably comprises 9-50 nucleotides, more preferably 10-25 nucleotides or even 11-20 nucleotides, such as 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, or 19 nucleotides. The optimal length of the reporter is about 12 nucleotides.

[0083] The reporters include either single-stranded DNA for detecting activated Cas12 or Cas14 effector proteins, or single-stranded RNA for detecting activated Cas13 effector proteins. The reporters may include deoxyribonucleotide or ribonucleotide analogs, such as inosine, uridine, xanthine, hypoxanthine, 2,6-diaminopurine, and 6,8-diaminopurine-based ribonucleotides and deoxyribonucleotides.

[0084] Detection of reporter cleavage may be performed by any method known in the art. For example, detection may be performed directly by mass spectrometry, e.g., ultra-high performance liquid chromatography (UHPLC) (LC-MS / MS) coupled with tandem mass spectrometry in positive electrospray ionization mode. LC-MS / MS analysis may be performed, for example, by using a high-end UHPLC chromatography system coupled with a triple-quadrupole mass spectrometer. Detection may further be performed by liquid-liquid phase separation (LLPS; Spoelstra et al., 2018. BioRXiv, CSHL (doi.org / 10.1101 / 471482), or by colorimetric, fluorometric, fluorescent or bioluminescent detection methods known to those skilled in the art.

[0085] Suitable reporters are single-stranded RNA (Cas13) or DNA (Cas12 and Cas14) molecules tagged with a fluorescent label at one end and a quencher at the other end. The proximity of the reporter and the quencher prevents the detection of their fluorescence. Cleavage of the substrate after activation of the collateral trans-acting nuclease activity of type V or type VI CRISPR / Cas effector proteins destroys the proximity of the reporter-quencher, thus allowing the unquenched emission of fluorescence, which can be detected after excitation with a laser.

[0086] Preferred fluorescent labels include Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto647N (ATTO-TEC GmbH, Siegen, Germany), YakimaYellow (Epoch Biosciences Inc, Bothell, WA, USA), Cal610 (BioSearch Technologies, Petaluma, CA, USA), Cal635 (BioSearch Technologies, Petaluma, CA, USA), FAM (Thermo Fisher Scientific Inc., Waltham, MA USA), TET (Thermo Fisher Scientific Inc., Waltham, MA USA), HEX (Thermo Fisher Scientific Inc., Waltham, MA USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, Cy7 (Thermo Fisher Scientific Inc., Waltham, MA USA), Alexa dyes (Thermo Fisher Scientific Inc., Waltham, MA USA), and the like. The substrate may be selected from: Fluorescein isothiocyanate (FITC, Thermo Fisher Scientific Inc., Waltham, MA USA), Tamra (Thermo Fisher Scientific Inc., Waltham, MA USA), ROX (Thermo Fisher Scientific Inc., Waltham, MA USA), JOE (Thermo Fisher Scientific Inc., Waltham, MA USA), fluorescein isothiocyanate (FITC, Thermo Fisher Scientific Inc., Waltham, MA USA), Yakima Yellow® (YY; Epoch Biosciences, Bothell, Washington) and tetramethylrhodamine (TRITC, Thermo Fisher Scientific Inc., Waltham, MA USA). The substrate is preferably labeled at the 5' end with a detectable label, preferably a fluorescent label.

[0087] Quenchers, such as tetramethylrhodamine TAMRA, a dihydrocyclopyrroloindole tripeptide minor groove binder, are known in the art. Preferred quenchers include Black Hole Quencher®-1 (BHQ1) and BHQ2 (Biosearch Technologies, Petaluma, CA, USA). The BHQ1 dark quencher has strong absorption at 480 nm to 580 nm and provides quenching of fluorophores that fluoresce within this range, such as FAM, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes. The BHQ2 dark quencher has strong absorption between 599 nm and 670 nm and provides quenching of fluorophores that fluoresce within this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes. BHQ1 and BHQ2 can quench fluorescence by both FRET and static quenching mechanisms.

[0088] Further preferred reporters allow detection in lateral flow assays (LFA). The principle of LFA is that the reaction product, in this case unreacted reporter, is immobilized at a first point, called the control line, while the cleaved reporter is immobilized at a second point, called the test line. LFA typically consists of a nitrocellulose membrane, a sample pad, a conjugate pad, a wicking or absorbing pad, and a backing pad (Jauset-Rubio et al., 2016. Sci Rep 6:37732). Nitrocellulose membranes are most commonly used because they facilitate a support that can be used for both reaction and detection, when capture biomolecules, such as antibodies, are deposited on the nitrocellulose to form test and control lines through a combination of electrostatic interactions, hydrogen bonds, and / or hydrophobic interactions (Jauset-Rubio et al., 2016. Sci Rep 6:37732).

[0089] 4.5.4 Alternatively, binding of the crRNA to its target nucleic acid sequence activates the cyclase domain of a type III effector system such as Cas10. Cas10-based ribonucleic acid detection systems are preferably derived from thermophilic organisms such as Pyrococcus furiosus, Sulfolobus solfataricus or Thermus thermophilus. For this system, the target DNA molecule needs to be transcribed into a target RNA molecule, for example by a thermostable RNA polymerase according to the invention, thereby allowing the transcription reaction at higher temperatures. A further advantage is that all three steps of amplification, transcription and detection can be used at elevated temperatures, for example around 65°C, making all steps compatible with one-pot reactions.

[0090] A further advantage of the CRISPR / Cas-based ribonucleic acid detection system from thermophilic organisms is that the detection can be carried out at elevated temperatures, for example, 40°C to 80°C, preferably 50°C to 70°C, for example, 55°C to 65°C, preferably about 65°C. Incubation at this temperature can accelerate the cOA synthesis reaction, compared to incubation at lower temperatures. Furthermore, the elevated temperature can inactivate nucleases, for example deoxyribonucleases or ribonucleases, or proteases present in the sample.

[0091] Furthermore, the advantages of a CRISPR / Cas-based ribonucleic acid detection system from thermophilic organisms may provide the system with increased stability, such as the ability to be stored for longer periods of time, compared to a CRISPR / Cas-based ribonucleic acid detection system from mesophilic organisms.

[0092] Recent results show that the presence of cOA causes a large increase in ribonuclease activity by Csm6 or Csx1 family members. Although these proteins are often encoded in type III loci, they do not directly associate with ribonucleoprotein complexes. Instead, recognition of invader RNA transcripts leads to the generation of cOA that triggers targeted RNA degradation by Cas7, non-specific DNA degradation by class I, type IIIA Cas10, and activation of Csm6 or Csx1, which can cause collateral cleavage of other nearby single-stranded RNA molecules.

[0093] 4.5.5 Methods for directly measuring the level of cOA preferably include methods for measuring the level of pyrophosphate or PPi. The formation of pyrophosphate is coupled to the formation of cOA from ATP by a CRISPR / Cas associated protein such as Cas10. The formation of cOA consisting of 3-6 AMP units leads to the simultaneous formation of 3-6 molecules of PPi.

[0094] 4.5.6 Alternatively, a detection system according to the invention may comprise inorganic pyrophosphatase, which leads to the degradation of PPi and the formation of two inorganic phosphate molecules for each PPi molecule. Thus, by measuring the level of inorganic phosphate, the number of detectable molecules is amplified from 1 molecule of cOA to 6-12 molecules of Pi.

[0095] A preferred inorganic pyrophosphatase is an enzyme that is active at the same or similar temperature as the type III CRISPR / Cas-based RNA detection system. Moreover, the inorganic pyrophosphatase is preferably active under the same or similar conditions as the type III CRISPR / Cas-based RNA detection system, for example at the same or similar pH and the same or similar salt concentration. For example, if the type III CRISPR / Cas-based RNA detection system has an optimal activity at 50°C, the inorganic pyrophosphatase is preferably active at this temperature. Preferably, the activity of the inorganic pyrophosphatase at 50°C is such that essentially all PPi molecules are decomposed into inorganic phosphate molecules. The decomposition is preferably instantaneous. Similarly, the activity of the inorganic pyrophosphatase at the selected pH and salt concentration is such that essentially all PPi molecules are decomposed into inorganic phosphate molecules. The decomposition is preferably instantaneous.

[0096] Preferred inorganic pyrophosphatases are derived from thermophylic organisms such as Pyrococcus furiosus, Sulfolobus solfataricus, and Thermus thermophilus, allowing for simultaneous isothermal detection. Thus, the levels of cOA can be measured directly by measuring the levels of Pi.

[0097] 4.5.7 PPi and Pi can be detected using methods known in the art, including colorimetric, fluorometric, fluorescent or bioluminescence based assays.

[0098] Suitable methods for measuring levels of PPi include pyrophosphate (PPi) assay kits for fluorimetry and / or colorimetry (Biovision Inc., Milpitas, CA); EnzChek® Pyrophosphate Detection Kit for fluorescence (ThermoFisher Scientific; Waltham, MA); Pyrophosphate Assay Kit for fluorimetry (Sigma Aldrich, Saint Louis, MO); and PPiLight™ Assay for luminescence (Lonza Group AG, Bazel, Switzerland).

[0099] Suitable methods for measuring inorganic phosphate or Pi levels include the colorimetric PiColorLock™ assay (Expedeon, Cambridge, UK); the colorimetric Malachite Green Phosphate Assay Kit (SigmaAldrich, Saint Louis, MO); a fluorescent phosphate sensor (ThermoFisher Scientific; Waltham, MA); luminescence readout after conversion of ADP to ATP (US Patent Publication No. 20140273036A); a fluorescent chemosensor (Meng et al., 2015. RSC Advances 5:53189-53197); and photoluminescent graphene quantum dots combined with europium ions (Bai et al., 2013. Chemistry 19:3822-3826).

[0100] Methods and means for directly measuring levels of cyclic oligoadenylate (cOA) preferably comprise at least one substrate and, where necessary, an enzyme enabling at least one of the specified detection methods for PPi or Pi.

[0101] A preferred method is a colorimetric method, such as the Malachite Green Phosphate Assay Kit, which allows rapid measurement of PPi or Pi levels as a direct measure of cOA levels.

[0102] 4.5.8 Methods for indirectly measuring the level of cOA preferably include methods for measuring the activity of a cOA-dependent non-specific effector nuclease, such as CRISPR-assisted nuclease 1 (Can1) or Can2, and preferably methods for measuring the activity of a cOA-dependent non-specific effector endoribonuclease, such as Csx1. Thus, a detection system according to any of the present invention preferably comprises a cOA-dependent non-specific effector endoribonuclease, such as Csx1.

[0103] The cOA-dependent non-specific effector nuclease, preferably an endoribonuclease, is preferably an enzyme that is active at the same or similar temperature as the type III CRISPR / Cas-based RNA detection system. Furthermore, the cOA-dependent non-specific effector endoribonuclease is preferably active under the same or similar conditions as the type III CRISPR / Cas-based RNA detection system, for example at the same or similar pH and the same or similar salt concentration. For example, if the type III CRISPR / Cas-based RNA detection system has an optimal activity at 65°C, it is preferred that the cOA-dependent non-specific effector endoribonuclease is active at this temperature. Preferably, the activity of the cOA-dependent non-specific effector endoribonuclease at 65°C is such that the cOA-induced activity of the cOA-dependent non-specific effector endoribonuclease results in the production of a detectable amount of a reaction product of a substrate of the cOA-dependent non-specific effector endoribonuclease. Similarly, the activity of a cOA-dependent nonspecific effector endoribonuclease at a selected pH and salt concentration is the degree to which cOA-induced activity of the cOA-dependent nonspecific effector endoribonuclease results in the production of a detectable amount of a reaction product of a substrate of said cOA-dependent nonspecific effector endoribonuclease.

[0104] The substrate of cOA-dependent non-specific effector endoribonuclease is preferably an RNA molecule, the cleavage of which can be detected. Detection can be carried out by any method known in the art. For example, detection can be carried out directly by mass spectrometry, for example ultra-high performance liquid chromatography (UHPLC) (LC-MS / MS) coupled with tandem mass spectrometry in positive electrospray ionization mode. LC-MS / MS analysis can be carried out, for example, by using a high-end UHPLC chromatography system coupled with a triple-quadrupole mass spectrometer.

[0105] Detection may further be performed by liquid-liquid phase separation (LLPS; Spoelstra et al., 2018. BioRXiv, CSHL (doi.org / 10.1101 / 471482).

[0106] A preferred substrate for cOA-dependent nonspecific effector endoribonuclease is an RNA molecule tagged with a fluorescent reporter molecule at one end and a quencher at the other end. The proximity of the reporter and the quencher prevents the detection of its fluorescence. Cleavage of the substrate by activation of cOA-dependent nonspecific effector endoribonuclease destroys the proximity of the reporter-quencher, thus allowing the unquenched emission of fluorescence, which can be detected after excitation with a laser. Thus, an increase in the activity of cOA-dependent nonspecific effector endoribonuclease causes a proportional increase in fluorescence due to cleavage of the substrate and removal of the quencher that quenches the fluorescent reporter.

[0107] Alternatively or additionally to the activation of non-specific effector endoribonuclease, the level of target recognition may also be measured by measuring the activation of non-specific effector deoxyribonuclease activity present in the type IIIA CRISPR / Cas effector complex using a suitable substrate for said nuclease. The suitable substrate is preferably a DNA molecule tagged with a fluorescent label at one end and a quencher at the other end. Examples of suitable fluorescent labels and quenchers are listed herein above. The proximity of the reporter and quencher prevents the detection of their fluorescence. The cleavage of the substrate by the activation of cOA-dependent non-specific effector deoxyribonuclease destroys the proximity of the reporter-quencher, thus allowing the unquenched emission of fluorescence, which can be detected after excitation with a laser. Thus, the increase in the activity of cOA-dependent nonspecific effector deoxyribonuclease causes a proportional increase in fluorescence due to the cleavage of the substrate and the removal of the quencher that quenches the fluorescent reporter.The use of both the activation of nonspecific effector endoribonuclease and the activation of nonspecific effector deoxyribonuclease allows the determination of the presence or absence of two independent target RNA molecules in one single assay, provided that two independent ribonucleoprotein complexes are used, one of which specifically activates nonspecific effector deoxyribonuclease, while the other specifically allows the indirect or direct measurement of cOA levels.Those skilled in the art will understand that in this case, the fluorescent labels present on the substrate due to the activation of cOA-dependent nonspecific effector endoribonuclease and the activation of nonspecific effector deoxyribonuclease should be sufficiently different to allow the measurement of the level of each activity as a measure for measuring the level of cOA.

[0108] A suitable commercially available substrate is provided by the RNaseAlert® Lab test kit v2 (ThermoFisher Scientific; Waltham, Mass.).

[0109] Preferred cOA-dependent nonspecific effector endoribonucleases are derived from thermophylic organisms such as Pyrococcus furiosus, Sulfolobus solfataricus and Thermus thermophilus, allowing for simultaneous isothermal detection of cOA-dependent nonspecific effector endoribonuclease activity.

[0110] The methods and means for indirectly measuring the level of cyclic oligoadenylate (cOA) preferably comprise a cOA-dependent non-specific effector endoribonuclease and a substrate for said cOA-dependent non-specific effector endoribonuclease.

[0111] Although features are described herein as part of the same or separate embodiments for purposes of clarity and concise description, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features. EXAMPLES

[0112] 5. Working Example Example 1 Materials and Methods Protein purification The nucleotide sequence of the protein of interest was codon-optimized for E. Coli (see Table 1) and placed in a bicistronic expression plasmid containing a Strep-tag at the N-terminus (Nieuwkoop et al., 2019. Microbiol Biotechnol 12:173-179). The expression plasmid was transformed into E. Coli strain Bl21(DE3) and grown in the desired culture volume (2-12 L) at 37 °C until it reached an OD600 of approximately 0.6. The culture was then placed on ice for 1 h, after which IPTG was added to a final concentration of 0.5 mM. The culture was then incubated at 20 °C for approximately 16 h (overnight). The cells were collected and lysed in buffer A (100 mM Tris-HCl, 150 mM NaCl, pH 8) by sonication, followed by centrifugation at 30.000 g for 45 min. The cleared lysate was filtered (0.45 μM) and purified using a StrepTrap FPLC column. The protein of interest was eluted using Buffer B (100 mM Tris-HCl, 150 mM NaCl, and 2.5 mM desthiobitin, pH 8.0). A heparin purification step was then performed using Buffer C (100 mM Tris-HCl, 125 mM NaCl, pH 8) as the binding and washing buffer. A subsequent gradient elution was performed using Buffer D (100 mM Tris-HCl, 1 M NaCl, pH 8). Finally, size exclusion chromatography was performed using a Superdex 200 Increased 10 / 300 GL column (Cytiva; Marlborough, Mass.) in Buffer A.

[0113] IVT assay IVT assays were performed with the following basic composition in a total volume of 20 μL: 1x isothermal amplification buffer (NEB#B0537S), 250 nM phiFa_44, 1.875 mM rNTP / dNTP, 80 ng dsDNA template. These reactions were incubated at 65°C for 1 hour followed by incubation at 95°C for 10 minutes. Then, 2.3 μL of DNase I buffer and 1 μL of DNase I enzyme (NEB#B0303, #M0303, respectively) were added and the reactions were incubated at 37°C for 30 minutes. The reaction mixtures were resolved on a 10% urea-PAGE gel and stained with SybrGold (Thermo #S11494).

[0114] Optimal analysis of temperature and NaCl concentration IVT assays were performed with a base composition of 20 mM Tris-HCl pH 8.8, 500 nM phiFa_44, 2.5 mM rNTP mix (NEB#N0466S), 2 mM MgCl, 50 mM NaCl, and 25 ng dsDNA template in a total volume of 20 μL. Reaction mixtures were matched with varying NaCl concentrations only to determine optimal NaCl concentrations. Reactions were incubated at 65°C for 1 hour, after which reactions were extensively washed with Zymo RNA Clean and Concentrator Kit (Zymo#R1017). Subsequent DNase treatment was performed at 37°C for 1 hour in a total reaction volume of 20 μL (NEB#M0303S). Reaction mixtures were resolved on a 10% urea-PAGE gel and stained with SybrGold (Thermo #S11494).

[0115] ScopeDx Assay The ScopeDx assay mixture consisted of 1x Warmstart LAMP master mix (NEB#E1700), primers (FIP / BIP 1.6μM, F3 / B3 0.2μM, LoopF / LoopB 0.4μM), 62.5nM reconstituted TtCmr46 complex with E-gene crRNA (Steens et al., 2021. Nature Comm 12:1-12), 1μM TtHB144 (Steens et al., 2021. Nature Comm 12:1-12), 250nM rNTP mix (NEB#N0466), 500nM RNaseAlert V2 (Thermo #4479769) and 175nM phiFa_44. Varying amounts of Twist SARS-CoV-2 synthetic genome MN908947.3 were added to the reaction mixture and incubated at 65°C while time point measurements were taken for the FAM channel.

[0116] result In vitro transcription assay To determine whether dsDNA is a suitable template, a section from the phiFa phage genome was designated as a synthetic gene. The region containing the intergenic region at the end of phiFa-45 and the start of the phiFa_44 gene was selected (see Table 1). In vitro transcription assays designed to determine whether the polymerase uses rNTPs or dNTPs as substrates and whether DNA or RNA is produced were performed as shown in FIG. 1.

[0117] From these results, the inventors concluded that the template used was dsDNA, which, together with the rNTP substrate, led to the production of an RNA product (see FIG. 1, lanes 10-13).

[0118] Optimal analysis of temperature and NaCl concentration IVT assays performed using various NaCl concentrations and temperatures indicate that the optimal NaCl concentration is 25-200 mM and the optimal temperature range is 45-75 °C.

[0119] One-pot ScopeDx In the ScopeDx 1.0 tool described in Steens et al., 2021. Nature Comm 12:1-12, the inventors were able to link LAMP pre-amplification (65°C) to CRISPR readout (65°C). However, due to the incompatibility of the commercially available Hi-T7 RNA polymerase used with the extended incubation at 65°C, the inventors had to add a step in the protocol. First, LAMP pre-amplification was performed at 65°C for about 30 minutes, and then the Hi-T7+CRISPR mix was added to the reaction and incubated at 65°C for an additional 10-20 minutes. In this way, the Hi-T7 RNA polymerase, although limited in its duration of activity, can transcribe the dsDNA created by the pre-amplification into RNA, which can then be detected by the CRISPR system.

[0120] Since phiFa_44 is able to transcribe DNA to RNA at 65°C, the inventors decided to test it in a one-pot, one-step assay, ScopeDx2.0. The assay was performed as a single step at 65°C on a synthetic SARS-CoV-2 RNA genome (Figure 2). For this new assay, a detection limit of 200 copies was reached in less than 25 minutes, showing a significant improvement in time vs. signal. Furthermore, the fact that it is now a one-step protocol significantly reduces the contamination risk, which can be done if required to open the tube between steps, improving high throughput and automation capabilities.

[0121] In silico structural analysis In silico structural analysis was performed to gain more insight into the phiFa_44 protein. Initial results show similarities to the QDE1 polymerase-like enzyme. The dimerization behavior of QDE1 may similarly resemble the mode of action of phiFa_44. Based on the amino acid alignment (see FIG. 4), at this stage, a magnesium-binding domain can be identified. The predicted 3D structure confirms these similarities and indicates D366, D368 and D370 as residues in phiFa_44 involved in Mg binding.

[0122] Example 2 Materials and Methods IVT assay In vitro transcription (IVT) assays were performed with the following building blocks in a total volume of 20 μL: 1× isothermal amplification buffer (NEB#B0537S), 250 nanomolar PhiFa-44, 1.875 mM rNTP / dNTP, 80 ng double-stranded (ds) DNA template or 100 nM (final concentration) single-stranded (ss) DNA oligos. The reactions were incubated at 65° C. for 1 hour, followed by incubation at 95° C. for 10 minutes. A total of 2.3 μL and 1 μL of DNase I buffer and DNase I enzyme (NEB#B0303, #M0303), respectively, were added and the reactions were incubated at 37° C. for 30 minutes. The reaction mixtures were resolved on a 10% urea-PAGE gel and stained with SybrGold (Thermo #S11494).

[0123] ssDNA template A is [ka] It was.

[0124] ssDNA template B is [ka] It was.

[0125] The dsDNA templates consisted of annealed ssDNA template A and ssDNA template B.

[0126] result PhiFa IVT ssDNA template IVT reactions were performed using 90 nt long ssDNA templates, with template B being complementary to template A (see FIG. 4). This region of the PhiFa genome was selected because it contains the candidate motif of interest. From the results, it can be clearly seen that ssDNA template A resulted in a clear ssRNA transcript of approximately 75-80 nt, while ssDNA template B did not result in a clear transcript. This result indicates that ssDNA template A contains the motif of interest that is preferred by PhiFa44 polymerase.

[0127] The manner in which phage PhiFA expresses its genes is largely unknown: typically, phages use host RNA polymerases to express them, but since phage PhiFA encodes its own RNA polymerase, it is reasonable to assume that (at least) some of its genes are expressed by this particular polymerase (encoded by the PhiFA_44 gene).

[0128] Example 3 Materials and Methods In vitro RNA transcription assay PhiFa-44 was purified with a (His)6 tag in 50 mM NaCl, 100 mM Tris-HCl pH 8.0, and single-stranded DNA template (50 nt; SEQ ID NO: 12) was directed 5' labeling with Cy3 fluorescent dye. The ssDNA template was purified from denaturing PAGE using the "ZR Small-RNA PAGE Recovery Kit" (ZymoResearch, R1070). In vitro RNA transcription assays were performed in PhiFa-44 reaction buffer (35 mM NaCl, 20 mM Tris-HCl pH 8.8, 2 mM MgCl2, and 25 mM rNTPs) and MilliQ was added to a final volume of 20 μl. When required, ssDNA template and PhiFa-44 were added to the reaction mixture to a final concentration of 62 μM and 1 μM, respectively.

[0129] All reactions were incubated at 65°C for 2 hours, followed by RNA / DNA cleanup using the "RNA clean & Concentrator kit" (ZymoResearch, R1017). After adding RNase H (NEB, 5U / μl) to selected purified samples, all reactions were incubated at 37°C for 30 minutes. To analyze the reaction products, 2x RNA loading dye (200 mM Tris-HCl pH 8.0, 30% glycerol, 900 mM NaCl) was added to all incubation samples, which were then loaded onto a native 4-20% (w / v) gradient polyacrylamide gel. Gels were visualized using a fluorescent gel scanner (GE Amersham Typhoon). A Cy3 filter (560-580 nm) was used to visualize Cy3-labeled reaction products, while staining with SYBR Gold and application of a Cy2 filter (515-535 nm) showed that all nucleic acids were present on the gel.

[0130] result As shown in Figure 5, in vitro transcription using PhiFa-44 and ssDNA as a template resulted in the formation of complementary RNA, as indicated by a shift in the ssDNA template band. No change was observed when no MgCl was added and no incubation was performed (negative control). Similarly, addition of RNase H, enzymatic cleavage of the RNA in the RNA:DNA duplex, abrogated the shift in the ssDNA template band.

[0131] These results demonstrate that PhiFa-44 can transcribe a single-stranded template DNA into RNA.

[0132] [Table 1]

[0133] [Table 2]

[0134] [Table 3]

Claims

1. incubating a template nucleic acid molecule with a protein having at least 50% sequence identity to SEQ ID NO:1 in the presence of ribonucleoside nucleotides (rNTPs) and a suitable buffer; transcribing at least a portion of the template nucleic acid molecule into an RNA molecule by carrying out a transcription reaction at a temperature of 30°C to 80°C; A method comprising:

2. 2. The method of claim 1, wherein the template nucleic acid molecule is a deoxyribonucleic acid (DNA) template molecule.

3. 3. The method of claim 1, wherein the buffer contains 25 to 200 mM NaCl.

4. 3. The method of claim 1, wherein the transcription reaction is carried out at a temperature between 45°C and 75°C.

5. The method of claim 1 or 2, wherein the template nucleic acid molecule was generated by a pre-amplification reaction.

6. 2. The method of claim 1, wherein the transcribed RNA molecules are further incubated with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) protein-based nucleic acid detection system.

7. The CRISPR-Cas nucleic acid detection system a) an effector complex comprising a Type III Cas protein and at least one CRISPR RNA (crRNA) that binds to a target RNA molecule; b) Means for directly or indirectly measuring levels of cyclic oligoadenylate (cOA) The method of claim 6, comprising:

8. The method of claim 6 or 7, wherein the pre-amplification reaction, transcription reaction and incubation with the nucleic acid detection system are all carried out in a one-pot reaction.

9. A protein having at least 50% sequence identity with SEQ ID NO:

1.

10. 10. The protein of claim 9, comprising a suitable tag.

11. A nucleic acid molecule encoding the protein of claim 9 or 10.

12. 12. The nucleic acid molecule of claim 11, which is codon-optimized for expression of the protein in a suitable host cell.

13. A host cell expressing the protein of claim 9 or 10.

14. 11. Use of a protein according to claim 9 or 10 for carrying out a transcription reaction to transcribe at least a part of a template nucleic acid molecule into an RNA molecule.

15. The use according to claim 14, wherein the transcription reaction is carried out at a temperature of 30°C to 80°C.

16. The use described in claim 14, wherein the transcription reaction is carried out at a temperature of 45°C to 80°C.

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