Novel proteins for isothermal amplification

Abstract

The present invention relates to the field of isothermal amplification of nucleic acids, including DNA and RNA. In particular, the present invention relates to novel proteins for isothermal amplification as well as kits and methods comprising one or more proteins of the invention for amplification of target nucleic acids in laboratory and non-laboratory environments, in particular for diagnostic detection.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of isothermal amplification of nucleic acids, including DNA and RNA. In particular, the present invention relates to novel proteins for isothermal amplification as well as kits and methods comprising one or more proteins of the invention for amplifying target nucleic acids in laboratory as well as non-laboratory settings, in particular for diagnostic testing. BACKGROUND

[0002] Nucleic acid amplification techniques, including detection techniques, are widely used in research and medical diagnostics. Such techniques, like molecular real-time PCR assays, are generally very sensitive and provide target specificity, but they still have certain drawbacks. In PCR, primer annealing can only be performed by repeated cycles between high temperature, allowing thermal denaturation of double-stranded DNA (dsDNA), and low temperature, allowing primer annealing and polymerase extension of the primer. The inherent need for cycles of successive rapid cooling and heating, twenty to fifty cycles of two to three alternating temperatures, accurate temperature control and the use of high temperatures (> 90°C), require specialized thermal cyclers and are highly energy consuming. Thus, PCR-based DNA amplification methods have significant drawbacks, requiring well-equipped laboratories and well-trained personnel. Furthermore, the maximum possible efficiency of a PCR method is inevitably limited to one doubling per cycle, which does not allow rapid amplification (10 to 20 minutes) while maintaining high yield. The outbreak of SARS-CoV-2 has shown that PCR-based diagnostics by specialized laboratories is too slow and cumbersome for responsive monitoring of infection events. Thus, local virus outbreaks still cannot be reacted to quickly and virus spread effectively stopped. Attempts are made to meet the obvious demand for rapid and easy testing and point-of-need diagnostic tools by antigen-based diagnostic tools. However, detection of viral RNA or DNA, which is essential for accurate diagnosis of viral infection, is still slow and cumbersome. Therefore, there is an urgent need for new portable diagnostic solutions with high sensitivity and specificity and which can provide reliable results at the site of detection.

[0003] A solution that allows both rapid point-of-care diagnostics as well as detection of viral RNA or DNA can be a PCR alternative, namely the so-called isothermal amplification methods (for review see: Zanoli and Spoto, Biosensors (Basel), 2013 3(1): 18-43) or Glöckner et al., Critical Reviews in Biochemistry and Molecular Biology, 2021 56:6, 543-586). The main advantage compared to PCR is that isothermal nucleic acid amplification methods do not require any thermal cycling but can be performed at constant temperature. This makes the amplification process easier to handle and control than the PCR method and less energy consuming. This is particularly advantageous because the technical requirements for the construction of the testing device are reduced, which is necessary to provide a cost-effective but sensitive point-of-care testing. The constant temperature of the isothermal method additionally allows the use of completely closed microstructured devices, which reduces the risk of sample contamination and means low sample consumption, multiplex DNA analysis and the realization of portable devices. Finally, the constant temperature would be very preferable for a point-of-care and / or portable diagnostic device, as recently developed by the present applicant (DE 10 2020 109 744.1). As clearly demonstrated during the coronavirus pandemic from 2020 to 2022, there is still a worldwide need for rapid and reliable nucleic acid amplification technology, not only in specialized diagnostic centers but also at the location where the biological sample is taken from the examinee. Furthermore, it has been observed worldwide that the time to issue the result is crucial, because unless strict and preventive isolation rules are applied, there is a great risk that an infected but asymptomatic patient spreads the infectious disease before receiving the (reliable) test result, while currently the time to issue the result is generally 24 to 26 hours or more.

[0004] Currently, many isothermal amplification strategies are available, including sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple substitution amplification (MDA), recombinase polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR / EXPAR) (see: Zanoli and Spoto, 2013, Biosensors (Basel). March 2013; 3(1): 18-43; a comprehensive overview of existing RPA technologies: Li et al., Analyst, 2019, 144, 31, 31 to 67; or chain-invasive amplification (SIBA®, hereinafter referred to as SIBA) Hoser et al., 2014 PLoS ONE 9(11): e112656; or NEAR / EXPAR: Van Ness et al., 2003, PNAS, April 15, 2003, 100 (8)). (4504-4509). What these various isothermal amplification strategies have in common is that they all provide a specific method to allow the polymerase reaction to be initiated. In conventional PCR, this is achieved through thermal cycling, which allows the primers to anneal to single-stranded DNA (ssDNA) after denaturation.

[0005] Typical RPA systems for RNA amplification and detection require a staggering number of seven different proteins: recombinases, recombinase cofactors, single-stranded DNA-binding proteins, strand displacement polymerases, energy regeneration systems, exonucleases, and reverse transcriptases. This makes it difficult to produce cost-effective RPA kits and compositions for large-scale use. Furthermore, for point-of-care (POC) diagnostics and similar off-the-shelf applications, it is necessary, or at least highly desirable, to provide all enzymes as dry pellets that can be easily reconstituted in suitable buffers at the time of use. While enzyme granulation and reconstitution (especially for complex enzyme mixtures) is generally a challenging task, it is particularly critical for POC systems, which need to allow for immediate, robust, efficient, and reliable reactions, ideally also cost-effectively.

[0006] While RPA amplification and its components have been extensively optimized in the past, attempts to improve amplification have typically relied on incremental adjustments to established factors rather than replacing the enzymes involved. This implies a significant untapped and largely unexplored potential for novel factors that could significantly improve isothermal amplification.

[0007] Therefore, the objective of this invention is to identify and provide novel proteins from different species to enhance the application potential of current isothermal amplification. Technically, replacing enzymes in known systems is highly complex, especially for complex RPA systems that require intricate interactions of multiple different proteins under overall reaction conditions. Furthermore, identifying new candidates from countless possible candidate proteins and hypothetical proteins known only through gene screening is an extremely challenging task. Efforts to identify and test novel proteins, rather than attempting to optimize already used proteins, require significant investment in screening, validation, and testing, and must discard several suboptimal candidates in the process. Despite these challenges, a key objective of this invention is to optimize available RPA methods, particularly for diagnostic amplification, to create more robust yet cost-effective diagnostic tools, especially providing inexpensive yet highly reliable and rapid POC detection tools. Summary of the Invention

[0008] The above objectives have been achieved by providing novel proteins (including RPA) that allow amplification reactions to be carried out in a more economical manner, requiring fewer enzymes, and / or being sufficiently efficient, thus enabling the method to be carried out without ATP or ATP analogs and / or energy regeneration systems and / or additional congestants, as the novel enzymes are much more efficient.

[0009] In one aspect, a protein or a functional fragment thereof is provided, preferably supporting isothermal amplification, wherein the protein comprises or is composed of: a) *Sphaerophyte* spp. Lutimaribacter SSB1, comprising or consisting of: the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it, preferably wherein the amino acid sequence has a serine residue at position 64 of SEQ ID NO 1 or 2; or SSB1 of the genus *S. spp.*, comprising or consisting of: the amino acid sequence of SEQ ID NO: 21, 22 or 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO: 21, 22 or 23, respectively; or b) SSB2 of the genus *S. spp.*, comprising or consisting of: SEQ ID NO: 1; The amino acid sequence of NO:2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or c) Shigella flexneri ( Shigella flexneri) SSB, comprising or consisting of: the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or d) λ phage β protein, comprising or consisting of: the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or e) *RecA1*, comprising or consisting of: the amino acid sequence of SEQ ID NO: 5 or 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or f) *RecA2*, comprising or consisting of: SEQ ID NO: The amino acid sequence of SEQ ID NO: 8, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or g) Shigella flexneri RecA, comprising or consisting of: the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or h) Escherichia coli ( Escherichia coli i) RecT, which comprises or consists of: the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or i) λ phage Orf, which comprises or consists of: the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it.

[0010] In some respects, a set of proteins or fragments thereof as defined above are disclosed.

[0011] In one embodiment, a fusion protein is provided comprising at least one first aspect protein and at least one additional functional portion, wherein at least two portions are optionally separated by at least one linker.

[0012] The second aspect further provides a fusion protein, wherein the fusion protein comprises at least one affinity tag, preferably at the C-terminus and / or N-terminus.

[0013] The invention also provides a fusion protein of the above aspects and embodiments, wherein the fusion protein comprises at least one GST tag, MBP tag, Strep tag and / or HIS tag, preferably comprising at least one HIS tag.

[0014] In a third aspect, a nucleic acid molecule is also provided that encodes at least the protein of the first aspect and / or at least one fusion protein of the second aspect.

[0015] In the fourth aspect, a vector or expression construct is provided that contains at least one nucleic acid molecule of the fourth aspect.

[0016] In a fifth aspect, a cell is provided that comprises at least one protein of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid molecule of the third aspect and / or at least one vector or expression construct of the fourth aspect.

[0017] In a sixth aspect, a kit is provided comprising at least one protein of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid molecule of the third aspect; and / or at least one vector or expression construct of the fourth aspect; and / or at least one cell of the fifth aspect. In one embodiment, the kit comprises (i) at least one single-stranded binding protein, optionally a single-stranded binding protein of the first aspect items a), b), c) and / or d), preferably a single-stranded binding protein of the first aspect item a), and / or at least one fusion protein comprising a second aspect thereof; (ii) at least one recombinase, optionally a recombinase of the first aspect items e), f), g) and / or h), and / or at least one fusion protein comprising a second aspect thereof; (iii) optionally at least one recombinase cofactor, optionally a recombinase cofactor of the first aspect item i), and / or at least one fusion protein comprising the aforementioned second aspect thereof; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) optionally at least one reverse transcriptase; (vii) a suitable reaction component comprising at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analog, and additional optional congestants; (viii) optionally at least one probe and additionally optional at least one enzyme for activating the probe.

[0018] On the other hand, the use of the first aspect of the protein; and / or the second aspect of the fusion protein; and / or the third aspect of the nucleic acid molecule; and / or the fourth aspect of the vector or expression construct; the fifth aspect of the cell; and / or the sixth aspect of the kit is provided for isothermal amplification of at least one target nucleic acid molecule, optionally for the detection of viral RNA and / or DNA, and / or for sample amplification for nucleic acid sequencing, preferably wherein the amplification is performed in vitro, and / or preferably wherein the amplification is performed with a field-available test, preferably using the kit of the sixth aspect.

[0019] In another aspect, a method is provided for isothermal amplification of at least one target sequence, preferably in a biological sample, the method comprising the steps of: (a) providing (i) at least one single-stranded binding protein, optionally a single-stranded binding protein of the first aspect items a), b), c) and / or d), preferably a single-stranded binding protein of the first aspect item a), and / or at least one fusion protein comprising a second aspect thereof; (ii) at least one recombinase, optionally a recombinase of the first aspect items e), f), g) and / or h), and / or at least one fusion protein comprising a second aspect thereof; (iii) optionally at least one recombinase cofactor, optionally a recombinase of the first aspect item i), and / or at least one fusion protein comprising a second aspect thereof; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) optionally at least one reverse transcriptase; (vii) a suitable reaction component comprising at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analogue, and further optionally a congesting agent; (b) (c) Provide at least one biological sample to be analyzed to detect the presence of at least one target sequence, and optionally provide a preferred sterile extraction kit to obtain the biological sample; (d) optionally: provide at least one probe and optionally provide at least one enzyme to activate said probe, thereby generating a detectable signal; (e) add initiating reagent to initiate an amplification reaction; and (f) obtain at least one amplified target sequence and / or obtain at least one detectable signal, each signal indicating successful amplification of the corresponding target sequence, wherein step (a) includes providing at least one protein of the first aspect and / or at least one fusion protein of the second aspect in substeps (i), (ii) and / or (ii).

[0020] In one embodiment, a method is provided, wherein the method does not involve the use of ATP or ATP analogues and / or an energy regeneration system.

[0021] In another embodiment, a method is provided, wherein the method is performed without recombinase cofactor. Attached Figure Description

[0022] Figure 1A The purification results of LuSSB1 are shown. 1: soluble fraction, 2: insoluble fraction, 3 and 4: flow-through, 5 to 8: washing fraction, 9 to 14: elution fraction, L: unstained protein standard (10-20 kDa, NEB)

[0023] Figure 1B The purification of LuSSB2 is shown. 1: Soluble fraction, 2: Insoluble fraction, 3: Eluent, 4: Wash fraction, 5 to 7: Elution fraction, L: Unstained protein standard (10-20 kDa, NEB)

[0024] Figure 1C The purification results of SfRecA are shown. 1: Soluble fraction, 2: Insoluble fraction, 3: Eluent, 4: Wash fraction, 5 to 7: Elution fraction, L: Unstained protein standard (10-20 kDa, NEB)

[0025] Figure 2A The results of the electrophoretic mobility variation assay (EMSA) for LuSSB2 are shown. The numbers indicate the amount of LuSSB2 protein added. "Oligonucleotide" indicates a negative control with no protein added.

[0026] Figure 2B The results of the electrophoretic mobility shift assay (EMSA) for SfSSB are shown. The numbers indicate the amount of SfSSB protein added. "Oligonucleotides" represent the negative control with no protein added.

[0027] Figure 2C The results of the electrophoretic mobility variation assay (EMSA) for LuSSB1 are shown. The numbers indicate the amount of LuSSB1 protein added. "Oligonucleotide" indicates a negative control with no protein added.

[0028] Figure 2D The results of the electrophoretic mobility shift assay (EMSA) for RecT are shown. The numbers indicate the amount of RecT protein added. "Oligonucleotide" indicates a negative control with no protein added.

[0029] Figure 2E The results of the electrophoretic mobility variation assay (EMSA) of β protein are shown. The numbers indicate the amount of β protein added. "Oligonucleotide" indicates a negative control with no protein added.

[0030] Figure 2F The results of the electrophoretic mobility variation assay (EMSA) for λOrf are shown. The numbers indicate the amount of λOrf protein added. "Oligonucleotide" indicates a negative control with no protein added.

[0031] Figure 3 Exemplary results of the RPA reaction are shown. “a” represents an RPA mixture using LuSSB1 as a single-chain binding protein. “b” represents an RPA mixture using RB69 GP32 as a single-chain binding protein. Additional curves represent negative controls. The Y-axis shows relative fluorescence units (Axxin T8 device), and the x-axis shows time in seconds.

[0032] Figure 4 Exemplary results of the RPA reaction are shown. “a” and “b” represent RPA mixtures with 330 ng / μl LuSSB1 as the single-chain binding protein. “c” and “d” represent RPA mixtures with 580 ng / μl RB69 GP32 as the single-chain binding protein. The Y-axis shows relative fluorescence units (Axxin T8 device), and the x-axis shows time in seconds.

[0033] Figure 5 Exemplary results of RPA reactions with symmetric and asymmetric primer concentrations are shown. Circles represent reaction mixture 1 with symmetric primer concentrations. Squares represent reaction mixture 1 with asymmetric primer concentrations of 1:4. Triangles represent reaction mixture 2 with asymmetric primer concentrations of 1:4. The Y-axis shows tt (time threshold), and the x-axis shows the number of DNA target molecules.

[0034] Figure 6 Results of various RPA tests performed on different instruments are shown, namely the Axxin Cycler, the midge medical's minoo, and the Corbett cycler. In all assays, the LuSSB1 of this invention exhibited superior performance.

[0035] Sequence Summary

[0036] Seq ID NO: 1 Amino acid sequence of single-chain binding protein 1 (LuSSB1) of *Staphylococcus aureus* genus EGI FJ00015

[0037] The amino acid sequence of single-chain binding protein 2 (LuSSB2) of *Staphylococcus aureus* genus EGI FJ00015.

[0038] Seq ID NO: 3 Amino acid sequence of Shigella flexneri single-chain binding protein (SfSSB)

[0039] Seq ID NO: 4 Amino acid sequence of λ phage β protein

[0040] The amino acid sequence of Seq ID NO: 5 *Staphylococcus aureus* genus EGI FJ00015 RecA1 (LuRecA1)

[0041] The amino acid sequence of Seq ID NO: 6 *Staphylococcus aureus* genus EGI FJ00015 RecA1 (LuRecA1) H64S

[0042] The amino acid sequence of Seq ID NO: 7, genus *Staphylococcus* EGI FJ00015 RecA2 (LuRecA2).

[0043] Amino acid sequence of Shigella flexneri RecA (SfRecA) Seq ID NO: 8

[0044] The amino acid sequence of Escherichia coli RecT, Seq ID NO: 9

[0045] The amino acid sequence of λ phage Orf (λOrf) with Seq ID NO: 10

[0046] Seq ID NO: 11 Nucleic acid sequence encoding single-stranded binding protein 1 (LuSSB1) of *Staphylococcus aureus* genus EGI FJ00015

[0047] Seq ID NO: 12 Nucleic acid sequence encoding single-stranded binding protein 2 (LuSSB2) of *Staphylococcus aureus* genus EGI FJ00015.

[0048] Seq ID NO: 13 Nucleic acid sequence encoding the Shigella flexneri single-strand binding protein (SfSSB)

[0049] Seq ID NO: 14 Nucleic acid sequence encoding λ phage β protein

[0050] The coding nucleic acid sequence of Seq ID NO: 15, EGI FJ00015 RecA1 (LuRecA1) of the genus *Staphylococcus*.

[0051] The coding nucleic acid sequence of Seq ID NO: 16 *Sphaerocarpus spp.* EGI FJ00015 RecA1 (LuRecA1) H64S

[0052] The coding nucleic acid sequence of Seq ID NO: 17, genus *Staphylococcus* EGI FJ00015 RecA2 (LuRecA2).

[0053] The coding nucleic acid sequence of Shigella flexneri RecA (SfRecA) Seq ID NO: 18

[0054] The coding nucleic acid sequence of Escherichia coli RecT, Seq ID NO: 19

[0055] Seq ID NO: 20 Nucleic acid sequence encoding λ phage Orf (λOrf)

[0056] The amino acid sequence of Seq ID NO: 21, single-chain binding protein 1 (LuSSB1) T246H from the genus *Staphylococcus aureus* EGI FJ00015.

[0057] The amino acid sequence of Seq ID NO: 22, single-chain binding protein 1 (LuSSB1) A247H from the genus *Staphylococcus aureus* EGI FJ00015.

[0058] The amino acid sequence of Seq ID NO: 23, single-chain binding protein 1 (LuSSB1) A248H from the genus *Staphylococcus aureus* EGI FJ00015.

[0059] Seq ID NO: 24 Amino acid sequence of single-chain binding protein 1 (LuSSB1) of *Staphylococcus aureus* genus EGI FJ00015 with N-terminal and C-terminal hexa-HIS tags.

[0060] "Biological sample" should be interpreted broadly and includes any material isolated from a living organism containing genetic material from that organism and / or genetic material from pathogens (pathogenic prokaryotes or eukaryotes) or viruses (with RNA or DNA genomes, single-stranded or double-stranded, or retroviruses) that infect, invade, or coexist with that organism. Biological samples include, for example, swabs and saliva samples, including nasopharyngeal and oropharyngeal swabs or saliva, as well as sputum, blood, urine, and feces. Preferably, biological samples tested in a non-laboratory setting will be swabs, saliva, urine, or fecal samples that can be collected without the assistance of medical professionals.

[0061] As used herein, "congestant" is intended to define any reagent that helps provide an optimal reaction environment for an enzymatic reaction. The mode of action of a congestant is to create smaller reaction compartments, thereby increasing the probability of reaction between the substrate and the corresponding enzyme. Congestants typically added actively to isothermal amplification reactions include, for example, polyethylene glycol (PEG), dextran, and ficoll. The concentration of the congestant is typically from 1% to 12% by weight (w / v) of the reaction. In preferred embodiments, as disclosed herein, the concentration of the congestant used is less than 5% by weight (w / v). While all PEG polymers are acceptable, preferred PEG compounds disclosed include PEG1450, PEG3000, PEG8000, PEG10000, PEG compounds with a molecular weight of 15,000 to 20,000 (also known as Carbowax 20M), and combinations thereof.

[0062] Whenever this disclosure relates to the percentage of identity of nucleic acid or amino acid sequences, that identity is determined by aligning the sequence of interest (reference sequence, such as a SEQ ID NO disclosed herein) with another sequence (query sequence) over the full length of the reference sequence. Identity is obtained using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program or the EMBOSS Water Pairwise Sequence Alignments (protein) program (for amino acid sequences) (www.ebi.ac.uk / Tools / psa / emboss_water / ). The tools for local sequence alignment provided by the European Institute of Bioinformatics (EBI) of the European Molecular Biology Laboratory (EMBL) use the modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, MS “Identification of common molecular subsequences” Journal of Molecular Biology, 1981 147 (1):195-197). When performing alignment, the default parameters defined by EMBL-EBI are used. These parameters are (i) for amino acid sequences: matrix=BLOSUM62, vacancy opening penalty=10 and vacancy extension penalty=0.5, or (ii) for nucleic acid sequences: matrix=DNAfull, vacancy opening penalty=10 and vacancy extension penalty=0.5.

[0063] "Markers" can be chemical modifications or isotopic substitutions that enable the identification of molecules, groups, or atoms during metabolic transformation or transport processes, or particularly during the diagnostic or preparative assays of nucleic acid amplification.

[0064] As used herein, the term "probe" broadly refers to a molecule or atom used in molecular biology, particularly in the methods disclosed herein, to study the properties of another target molecule or structure or in a diagnostic setting. Those skilled in the art can use a variety of different probes that can be readily combined with the methods disclosed herein (see Molecular Probe Techniques, Tree Number(s) E05.601, Unique ID D015336, RDF UniqueIdentifier, http: / / id.nlm.nih.gov / mesh / D015336). For the use of the methods disclosed herein on the specific apparatus disclosed herein, it is preferable to provide probes that can detect and / or quantify signals, optionally activatable probes such as Förster / fluorescence resonance energy transfer (FRET) probes, etc. Given the methods disclosed herein, this can allow for rapid diagnostic results. The advantage it offers is that it includes a positive control associated with one probe and different probes associated with the actual target sequence, thereby significantly increasing the reliability of diagnostic results.

[0065] As used herein, a single protein is a protein consisting of a single continuous polypeptide chain or two or more closely interacting subunits (e.g., homodimers or heterodimers), wherein a single protein may have more than one different function.

[0066] As used herein, the term "functional portion" describes a portion of a protein that can perform at least one desired function of the full-length protein in the absence of the rest of the protein. For example, the functional portion of an SSB protein is the portion of the SSB protein that retains single-stranded (DNA) binding activity in the absence of the rest of the full-length SSB protein.

[0067] As used herein, a "fusion protein" refers to a single, continuous polypeptide chain, or two polypeptide chains linked by a non-peptide linker, which do not occur in this juxtaposition in their native state. Preferably, a fusion protein is a single, continuous polypeptide chain, such as two amino acid sequences linked by a peptide linker. Typically, fusion proteins are produced by expressing a DNA sequence containing an ORF encoding a fusion sequence. The design, expression, and / or production of fusion proteins are well known to those skilled in the art.

[0068] Reverse transcriptase can be any enzyme capable of generating a complementary DNA (cDNA) strand from a template RNA molecule. In the context of this invention, "function" refers to the ability of a particular enzyme to perform its function according to the invention. Thus, reverse transcriptase function refers to the ability to generate cDNA from target RNA in a reaction mixture, and DNA polymerase function refers to the ability to polymerize DNA onto a target DNA strand in a reaction mixture.

[0069] As used in this article, “single-stranded binding protein” (also known as single-stranded (DNA) binding protein) or “SSB” or “SSB protein” refers to proteins of the single-stranded binding protein class (for example, see James L. Keck (ed.), Single-StrandedDNA Binding Proteins: Methods and Protocols, Methods in Molecular Biology, Vol. 922, DOI 10.1007 / 978-1-62703-032-8_1, # Springer Science+Business Media, LLC 2012).

[0070] The term "tag" or "protein tag" refers to various polypeptide sequences or corresponding nucleic acid sequences encoding them, which can be incorporated into the target protein at different positions, preferably at the N-terminus and / or C-terminus, or between different functional domains, and perform several different functions. Protein tags can be, for example, affinity tags, such as chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tags, and glutathione S-transferase (GST) or His tags. Affinity tags can be used for the specific purification of the target protein. Another example of a protein tag is a solubilizing tag, which allows the acquisition of a soluble form of the target protein, particularly in bacterial cell cultures. Epitope tags can be used to provide binding sites for analytical antibodies. Luminescent tags (preferably fluorescent tags) offer the possibility of detecting the target protein in cell cultures. Tags or labels can also be used to label the target protein or enzyme or fragments thereof to allow for easy identification. This can be particularly important for diagnostic purposes. Detailed Implementation

[0071] Despite the tremendous progress made in nucleic acid amplification technology (including isothermal amplification strategies) over the past few decades, the key working enzymes in all these technologies are usually not replaced, or are only modified gradually in small steps.

[0072] Existing RPA amplification methods typically begin as follows: With the assistance of T4 UvsY as a loading factor, a phage-derived T4 UvsX protein, acting as the core recombinase, binds to a primer. This complex invades double-stranded (ds) DNA, forming what is known as a D-loop, where the primer hybridizes with the template strand, initiating a strand exchange reaction. The unwound complementary strand is stabilized by a single-strand binding (SSB) protein, typically the T4-derived gp32 protein. The primer is then incorporated into a strand-displacement DNA polymerase (usually...). Bsu (Bacillus subtilis) Bacillus subtilis (Source) or Sau (Staphylococcus aureus) Staphylococcus aureus The polymerase derived from the primer initiates synthesis from the free 3'-OH of the primer. As polymerization continues, the two parental strands continue to separate. Then, the incorporation of the forward and reverse primers allows strand synthesis to proceed simultaneously in both directions, ultimately resulting in an exponential accumulation of amplified double-stranded DNA, which consists of the sequence between the forward and reverse primers. Therefore, standard RPA requires a large number of protein / enzyme interaction partners to ensure efficient DNA / RNA amplification, which seems to explain why there is a reluctance to change the proteins involved.

[0073] As described in the examples below, through extensive sequence-based screening, structural analysis, and experimental assays, several bacterial recombinases and SSB proteins were ultimately identified. These are *S. marineensis* SSB1 (LuSSB1), *S. marineensis* SSB2 (LuSSB2), *Shigella flexneri* RecA (SfRecA), *S. marineensis* RecA1 (LuRecA1), *S. marineensis* RecA2 (LuRecA2), and *Shigella flexneri* SSB (SfSSB).

[0074] Phage recombinases can be subdivided into two groups: (i) UvsX and Gp2.5, which are found in virulent phages, and (ii) Sak, β, Erf, and Sak4, which are found in temperate or formerly temperate phages (for an overview of phage recombinases, see: (Lopes et al., 2010; doi: 10.1093 / nar / gkq096. Epub March 1, 2010). PMID: 20194117; PMCID: PMC2896510). The characteristics of the corresponding categories are as follows: UvsX is a RecA-like enzyme, specifically found in phage T4, and its orthologs also exist in other phages with large genomes and strict virulent life cycles. Gp2.5 recombinases are specifically encoded by virulent phages such as T7 and share functional and structural homology with SSB proteins. However, unlike SSB, it also possesses recombinase function. Sak, β, and Erf are classified into three distinct families based on their primary sequences and possess single-chain annealing activity. These recombinases differ from RecA-like enzymes in the following three significant characteristics: (i) they are ATP-independent; (ii) they exhibit short pairing times (40-50... (iii) High efficiency in incorporating long bp sequences (30), and the ability to incorporate short single-stranded DNA substrates into bacterial chromosomes. β is derived from λ phage and is part of a recombination system that also consists of Exo, Gam, and λOrf (or NinB) proteins. β appears to exhibit both SSB and recombinase activity. Typically, β works in conjunction with λOrf, which is a functional equivalent of UvsY. λOrf has previously been described as a substitute for RecFOR in recombination (Maxwell et al., PNAS). August 9, 2005; 102(32): 11260-5). However, it seems to apply only to λ recombination. This seems to be related to the fact that λOrf can only efficiently replace β from nucleic acids, but not with bacterial SSB. Therefore, RecA from bacteria can be combined with λOrf and β for the RPA method. The advantage of doing so is that the bacterial accessory protein RecFOR can be omitted. In addition, since β exhibits recombinase activity, RPA can function without RecA. A system very similar to the Red recombinase system is the RecT / E system. A particular advantage of β / Orf and RecT is that they function independently of ATP (Li et al., J Mol Biol. March 6, 1998; 276(4): 733-44; Rybalchenko et al., PNAS December 7, 2004; 101(49): 17056-60; Noirot and Kolodner, J Biol Chem. May 15, 1998; 273(20):12274-80).Based on these properties, β, λOrf, and RecT were further selected for protein synthesis and experimental evaluation.

[0075] A key feature of this invention is its ability to reliably amplify any type of nucleic acid sequence, particularly linear target molecules. The applicability of this method to detect linear targets is crucial for methods used as diagnostic tools for viral infections. Different viruses contain various forms of genetic material, such as linear or circular, double-stranded or single-stranded DNA or RNA. Therefore, isothermal amplification methods for detecting viral nucleic acid material need to be able to detect all possible forms of viral nucleic acid material. Detection of linear nucleic acid targets is necessary for viruses with linear DNA and for all RNA viruses, as RNA must be reverse transcribed into linear complementary DNA (cDNA). Furthermore, the need to detect linear nucleic acid targets to detect RNA transcripts also exists in DNA viruses.

[0076] In a first aspect, a protein or a functional fragment thereof is provided, preferably supporting isothermal amplification, wherein the protein comprises or consists of: a) *S. spp.* SSB1, comprising or consisting of: the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity therewith, preferably wherein the amino acid sequence has a serine residue at position 64 of SEQ ID NO: 1 or 2; or *S. spp.* SSB1, comprising or consisting of: the amino acid sequence of SEQ ID NO: 21, 22 or 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO: 21, 22 or 23, respectively; or b) *S. spp.* SSB2, comprising or consisting of: SEQ ID NO: The amino acid sequence of SEQ ID NO: 2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or c) Shigella flexneri SSB, comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or d) λ phage β protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or e) *Sphaerobacterium* RecA1, comprising or consisting of: SEQ ID NO: The amino acid sequence of SEQ ID NO: 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or f) *Staphylococcus aureus* RecA2, comprising or consisting of the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it; or g) *Shigella flexneri* RecA, comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it;Or h) *Escherichia coli* RecT, comprising or consisting of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or i) λ phage Orf, comprising or consisting of the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it.

[0077] In some embodiments, a set of proteins or functional fragments thereof of the first aspect may be provided. Preferably, at least one of the proteins in the set will be LuSSB1 or a variant or mutant thereof, for example, a solubility-enhancing mutant as shown in SEQ ID NO:21 to 23, or a combination of these mutations, i.e. (refer to SEQ ID NO:1) namely: T246H, A247H, A248A, T246H / A247H, T246 / A248H, A247H / A248H, or T246H / A247H / A248H.

[0078] The proteins of this invention can be expressed and purified using standard methods known in the art. Exemplary and non-limiting examples of expression and purification protocols are shown in Example 2 below.

[0079] In a second aspect, a fusion protein comprising at least one protein of the first aspect and at least one additional functional portion is provided, wherein at least two portions are optionally separated by at least one linker.

[0080] As used herein, a linker refers to a molecule, typically a segment of polypeptide (linker peptide) that can be inserted between different parts of a protein (particularly a fusion protein). Typically, a linker is located between two different polypeptides that fuse together (via the linker) to form a fusion protein, for example, by fusing a tag to a protein. The length of peptide linkers used in this invention can range from 1 to more than 100 amino acids. Linkers can be flexible linkers, rigid linkers, and / or cleavable linkers. A peptide linker can be, for example, a GS linker, but any peptide linker described in the art can be used in this invention.

[0081] In one embodiment, the fusion protein includes at least one affinity tag, preferably at the C-terminus and / or N-terminus. As shown in SEQ ID NO:24, the tag may also be present at both the N-terminus and C-terminus.

[0082] In one embodiment, the fusion protein includes at least one GST tag, an MBP tag, a Strep tag, and / or an HIS tag, preferably including at least one HIS tag.

[0083] As is known to those skilled in the art, creating fusion proteins (e.g., fusing a tag to a protein) may require tests for solubility, stability, activity, etc., at different linker sites of the fusion component (e.g., the fusion tag) and for linkers of different types and / or lengths to achieve optimal results. Methods for designing, expressing, and testing fusion proteins are well known in the art and readily available to those skilled in the art. Assessing the activity of proteins used for isothermal amplification can be performed in accordance with methods known in the art and / or disclosed herein.

[0084] In one implementation, the GST tag, MBP tag, Strep tag, and / or HIS tag are fused to the fusion protein via a cleavable linker.

[0085] In a third aspect, a nucleic acid molecule is provided that encodes at least one protein of the first aspect; and / or at least one fusion protein of any one of the second aspects.

[0086] In the fourth aspect, a nucleic acid or expression construct is provided, which comprises at least one nucleic acid molecule of the third aspect.

[0087] The vector or expression construct of the fourth aspect may contain regulatory sequences, including all other nucleic acid sequences required for expression and replication in the desired target cells, such as promoters and terminators operatively linked to the sequence to be expressed, origin of replication, and / or selection markers. Those skilled in the art are well aware that the selection of regulatory sequences depends on the choice of target cells. For a variety of cell types, the expression of recombinant proteins, as well as suitable vectors and expression systems, promoters, and further regulatory sequences, have been established.

[0088] The nucleic acid of the third aspect or the vector or expression construct of the fourth aspect may contain intron-free cDNA or coding sequences that encode the acyltransferase and / or fusion protein of the present invention, for example for expression in prokaryotic cells.

[0089] In one embodiment, the nucleic acid of the third aspect or the vector or expression construct of the fourth aspect may comprise a sequence selected from SEQ ID NO:11 to 20, or a sequence having 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0090] The nucleic acid sequence contained in the nucleic acid of the third aspect and the vector or expression construct of the fourth aspect encodes at least the protein of the first aspect; and / or at least one fusion protein of any of the second aspects, which may be codon-optimized for expression in a desired host cell.

[0091] In a fifth aspect, a cell is provided comprising at least one protein of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid molecule of the third aspect; and / or at least one vector or expression construct of the fourth aspect.

[0092] Cells can be, for example, cells from commonly used cell expression systems, including prokaryotic expression systems and eukaryotic expression systems.

[0093] In a sixth aspect, a kit is provided comprising at least one protein of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid molecule of the third aspect; and / or at least one vector or expression construct of the fourth aspect; and / or at least one cell of the fifth aspect.

[0094] In one embodiment, the kit comprises (i) at least one single-stranded binding protein, optionally a protein of the first aspect a), b), c) and / or d), preferably a protein of the first aspect a), and / or at least one fusion protein containing a second aspect thereof; (ii) at least one recombinase, optionally a protein of the first aspect e), f), g) and / or h), and / or at least one fusion protein containing a second aspect thereof; (iii) optionally at least one recombinase cofactor, optionally a protein of the first aspect i) and / or at least one fusion protein containing a second aspect thereof; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) optionally at least one reverse transcriptase; (vii) suitable reaction components, including at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analog, and additionally optional congesting agents; (viii) optionally at least one probe and additionally optional at least one enzyme for activating the probe.

[0095] In one embodiment, the β protein of protein d) of the first aspect or the fusion protein of the second aspect comprising it performs the functions of the SSB protein of component (i) and the recombinase of component (ii). Therefore, the β protein of protein d) of the first aspect or the fusion protein of the second aspect comprising it can simultaneously constitute components (i) and (ii) of the kit of the sixth aspect.

[0096] In embodiments where components (i) and / or components (ii) of the kit in the eighth aspect contain the β protein of the protein d) of the first aspect or a fusion protein of the second aspect containing therein, or are composed of therein, the recombinase cofactor of component (iii) preferably contains λOrf of the protein i) of the first aspect or a fusion protein of the second aspect containing therein, or is composed of therein.

[0097] In one embodiment, the kit of the sixth aspect comprises (i) and / or (ii) a β protein of the protein d) of the first aspect or a fusion protein of the second aspect comprising therein, or is composed of therein, and the kit does not contain ATP or ATP analogues and / or does not contain an energy regeneration system.

[0098] In one embodiment, the sixth aspect of the kit component (ii) comprises RecT of the first aspect protein h) or a second aspect fusion protein containing thereof, or is composed of thereof, and the kit does not contain ATP or ATP analogues and / or does not contain an energy regeneration system.

[0099] In one embodiment, the kit does not contain a recombinase cofactor for proteins.

[0100] It may be desirable to provide proteins in the form of pellets or powders, such as lyophilized pellets or powders that can be reconstituted in a suitable buffer solution.

[0101] All reaction mixtures disclosed herein may be provided, for example, in liquid form or as lyophilized pellets.

[0102] In a preferred embodiment, the kit contains an amount and / or concentration of a crowding agent such that the concentration of the crowding agent in the final reaction mixture is less than 5%, for example, about 1%, about 2%, about 3%, about 3.5%, about 4%, or about 4.5%, wherein the concentration is by weight (w / v).

[0103] The crowding agent can be selected, for example, from polyethylene glycol (PEG), dextran, ficoll, PEG1450, PEG3000, PEG8000, and PEG10000.

[0104] In some implementations, the kit does not contain a crowding agent.

[0105] In one embodiment, the method does not include ATP or ATP analogues and / or an energy regeneration system; and / or the method does not include recombinase cofactors.

[0106] In one embodiment, the method includes ATP or ATP analogues, but does not include an energy regeneration system.

[0107] In embodiments involving RNA amplification, the kit may contain reverse transcriptase. The kit may include proteins that function as both DNA polymerase and reverse transcriptase, allowing a single protein to perform both functions.

[0108] In some embodiments, the kit may also contain at least one reducing agent, preferably dithiothreitol (DTT). The concentration of DTT may be from 1 mM to 50 mM.

[0109] In some embodiments, the kit may further contain at least one RNase inhibitor.

[0110] In one embodiment, at least one forward primer and at least one reverse primer are provided as target sequence-specific primers, wherein at least one of these primers, preferably a reverse primer, is provided in a higher amount and / or concentration relative to the other primer, wherein the amount and / or concentration may be, for example, more than 1.1, 1.2, 1.25, 1.5, 1.75, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 to 2.5, 2.6 to 2.9, 3 to 4, 5 to 6, 7 to 8, or 9 to 10 times.

[0111] The kit may also include instructions for use.

[0112] In some embodiments, the kit contains at least one component configured to be suitable for a probe analysis system (such a system is disclosed in European patent application EP20201885.9).

[0113] In some embodiments, the kit further comprises a second part, which includes (i), (ii), optional (iii), (iv), (v), optional (vi), (vii), and optional (viii), wherein the second part includes a predetermined second target sequence as a positive control and at least one, preferably at least two, primers specific to the second target sequence, and preferably a second probe.

[0114] Further details regarding the suitable reaction components, primer design, and concentrations to be achieved in the final reaction mixture are disclosed in aspect eight, and also apply to aspect six. Of course, when using a kit, the concentrations of the components contained in the provided kit may differ from the concentrations in the final reaction mixture.

[0115] In a laboratory setting, skilled molecular biologists or trained personnel can readily perform the methods disclosed herein, design primers and / or probes for the target nucleic acid to be amplified, and easily modify suitable reaction conditions if necessary. Of course, performing this method in a non-laboratory setting presents a greater challenge. Particularly in the latter case, it may be preferable to provide a kit containing all necessary components in readily available sterile vials or tubes, allowing for easy reactivation of (bio)chemical reagents and components (for enzymes, e.g., after lyophilization), and enabling the reaction to be initiated in a simple manner.

[0116] In the seventh embodiment, the use of a protein of the first aspect; and / or a fusion protein of the second aspect; and / or a nucleic acid molecule of the third aspect; and / or a vector or expression construct of the fourth aspect; a cell of the fifth aspect; and / or a kit of the sixth aspect for isothermal amplification of at least one target nucleic acid molecule is provided.

[0117] In an eighth aspect, a method is provided for isothermal amplification of at least one target sequence, preferably in a biological sample, the method comprising the steps of: (a) providing (i) at least one single-stranded binding protein, optionally a protein of the first aspect (a), (b), (c), and / or d), preferably a protein of the first aspect (a), and / or at least one fusion protein containing a second aspect thereof; (ii) at least one recombinase, optionally a protein of the first aspect (e), (f), (g), and / or h), and / or at least one fusion protein containing a second aspect thereof; (iii) optionally at least one recombinase cofactor, optionally a protein of the first aspect (i) and / or at least one fusion protein containing a second aspect thereof; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) optionally at least one reverse transcriptase; (vii) a suitable reverse transcriptase; The amplification reaction comprises: (a) at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analog, and additional optional congestants; (b) at least one biological sample to be analyzed to detect the presence of at least one target sequence, and optionally a preferred sterile extraction kit to obtain the biological sample; (c) optionally: at least one probe and optionally at least one enzyme to activate the probe to generate a detectable signal; (d) adding an initiating reagent to initiate the amplification reaction; and (e) obtaining at least one amplified target sequence and / or obtaining at least one detectable signal, each signal indicating successful amplification of the corresponding target sequence, wherein step (a) includes, in substeps (i), (ii), and / or (ii), providing at least one protein of the first aspect and / or at least one fusion protein of the second aspect.

[0118] In one embodiment, the β protein of the first aspect d) or the fusion protein of the second aspect comprising the first aspect implements the function of the SSB protein in step (a)(i) and the function of the recombinase in step (a)(ii). Therefore, providing the β protein of the first aspect d) or the fusion protein of the second aspect comprising the first aspect can implement steps (a)(i) and (a)(ii) of the method of the eighth aspect.

[0119] In embodiments where the β protein of the first aspect protein d) or a fusion protein of the second aspect comprising it is provided in steps (a), (i) and / or (ii) of the method of the eighth aspect, the optional at least one recombinase cofactor in step (iii) is preferably λOrf of the first aspect protein i) or a fusion protein of the second aspect comprising it.

[0120] In one embodiment, the β protein of the first aspect protein d) or the fusion protein of the second aspect comprising it is provided in steps (a), (i) and / or (ii) of the method of the eighth aspect, and the method does not include providing ATP or ATP analogues and / or does not include an energy regeneration system.

[0121] In one embodiment, the method of the eighth aspect provides a protein (h) of the first aspect or a fusion protein of the second aspect comprising therein, RecT, in steps (a) and (ii) of the method of the eighth aspect, and the method does not involve the addition of ATP or an ATP analog and / or does not involve an energy regeneration system.

[0122] In one embodiment, the kit does not contain recombinase cofactor.

[0123] In a preferred embodiment, the concentration of the congestant is less than 5%, for example about 1%, about 2%, about 3%, about 3.5%, about 4%, or about 4.5%, wherein the concentration is by weight (w / v).

[0124] Typical concentrations of congestants used in isothermal reactions are higher than 5% by weight (w / v). However, such concentrations of congestants (e.g., PEG) can distort fluorescence measurements used as a tool for detecting amplification. Therefore, fluorescently labeled systems cannot reliably detect amplification, which is particularly problematic for diagnostic kits and systems. Conversely, this problem has not been observed with lower concentrations of congestants. The inventors have discovered that the proteins of this invention (particularly LuSSB1) allow for rapid and reliable amplification reactions while reducing the concentration of congestants. Therefore, the proteins of this invention offer the advantage that they solve the problem of congestants interfering with fluorescence measurements by allowing for lower concentrations of congestants.

[0125] The crowding agent can be selected, for example, from the group consisting of polyethylene glycol (PEG), dextran, ficoll, PEG1450, PEG3000, PEG8000, and PEG10000.

[0126] In some implementations, the method does not include a congestant.

[0127] In one embodiment, the method does not include ATP or ATP analogues and / or an energy regeneration system; and / or the method does not include recombinase cofactors.

[0128] In one embodiment, the method includes ATP or ATP analogues, but does not include an energy regeneration system.

[0129] In another embodiment, at least one energy regeneration system can be provided. Many systems are available and routinely used in isothermal RPA reactions and similar reactions for energy cycling. One such system is the creatine kinase / creatine phosphate system, or chicken myokinase, which converts one AMP molecule and one ATP molecule into two ADP molecules. The creatine kinase / creatine phosphate system is then used to convert ADP back to ATP (Li et al., see above). For this type of enzyme, creatine phosphate di(tri) salts or buffer solutions may be suitable (1 mM to 100 mM).

[0130] It has also been found that simply providing excess ATP to the reaction system is perfectly sufficient to carry out a reliable amplification reaction in a single vial within a short time, thus providing reliable results. Therefore, particularly when using the kits and / or diagnostic handheld systems of the present invention to perform the methods disclosed herein, it may be preferable to provide excess ATP at concentrations higher than 2 mM, or higher than 5 mM, or higher than 7.5 mM, or higher than 15 mM, up to a maximum of 100 mM. For preparative purposes (e.g., DNA synthesis), higher concentrations of ATP are required compared to those used for diagnostic purposes (where only a specified amount of a relatively short fragment between two primers is amplified). Studies have found that providing high concentrations of ATP will be sufficient to successfully drive the amplification reaction, which may be preferred in several respects: (i) the reaction will be cheaper because no additional enzymes are required, and (ii) the complexity of the system will be reduced, and therefore more stable, and (iii) it will be robust in its use, with particular advantages if used in a home setting and performed almost automatically, rather than by laboratory technicians.

[0131] The DNA polymerase used in any aspect of the present invention may be a DNA polymerase having strand displacement activity, that is, in a polymerization reaction, it is capable of displacing a DNA strand that hybridizes or partially hybridizes with the DNA strand on which the polymerase polymerizes.

[0132] In some embodiments of the method of the present invention, at least one additional recombinase cofactor may be provided, preferably prior to step (d). Such enzymes or chemicals perform the function of a coenzyme for the recombinase, and / or chemical factors activate, stabilize, or otherwise enhance the activity of the recombinase. For the RPA reaction, UvsY is typically such a recombinase coenzyme or loading factor, which facilitates the initiation of nucleoprotein filament construction or helps stabilize nucleoprotein complexes containing any type of recombinase (Li et al., see above, or EP 1 759 012 A2).

[0133] In some embodiments, at least one recombinase cofactor is λOrf disclosed in the first aspect, or a fusion protein comprising the second aspect.

[0134] Another recombinase coenzyme of interest could be an enzyme that promotes efficient dissociation of the recombinase / dsDNA complex after DNA synthesis has commenced. These coenzymes include those capable of stimulating 3' to 5' dissociation and those capable of supporting 5' to 3' dissociation. Additional recombinase coenzymes could include a variety of polymerases that can replace RecA or equivalent, preferably thermostable, recombinases in the 3' to 5' direction and can stimulate 3' to 5' dissociation of the recombinase-dsDNA complex. These DNA polymerases preferably include thermostable equivalents of *E. coli* PolV and homologous polymerases from other species. Other recombinase coenzymes include a class of enzymes called helicases, which can be used to promote the dissociation of the recombinase from dsDNA. This promotes dissociation in both the 5' to 3' and 3' to 5' directions. Helicases are essential components of the in vivo recombination process, used to move the branching point of the recombination intermediate from one place to another, separate the strand, and dissociate and recover components bound to DNA. Additional recombinase coenzymes include *E. coli* RecG homologs, preferably thermostable enzymes. RecG can stimulate the dissociation of branched structures. In nature, this enzyme acts to reverse the replication fork at DNA damage sites: by unwinding the leading and lagging strands, it drives the replication fork back to produce four-way connections.

[0135] When the enzyme and the reaction targeted by these suitable reaction components are known, those skilled in the art can readily determine the suitable reaction components for the enzymatic reaction, as well as their concentrations and mixing methods. Several aspects particularly relevant to the method of the present invention will be disclosed in detail below.

[0136] Nucleic acid amplification reactions will require dNTPs (nucleotide triphosphates), such as dATP, dGTP, dCTP, and dTTP. In leading and lagging strand amplification, ATP, GTP, CTP, and UTP may also be included to synthesize RNA primers. Additionally, ddNTPs (ddATP, ddTTP, ddGTP, and ddGTP) can be used to generate fragment ladders. The concentration of dNTPs used can be from 1 µM to 200 mM for each NTP. A mixture of dNTPs and ddNTPs can be used, where the concentration of ddNTPs is 1 / 100 to 1 / 1000 of the concentration of dNTPs (1 µM to 200 mM). The use of UTPs may be particularly suitable when using a carry-over prevention kit (e.g., AmpErase, as called by Thermo Fisher). In these embodiments, a suitable, commonly used recombinant uracil N-glycosylation enzyme (UNG) can be used to prevent the amplified products from being re-amplified in subsequent reactions. This allows the reaction to proceed more quickly and avoids false positive results.

[0137] In some embodiments, the method may further include at least one reducing agent, preferably dithiothreitol (DTT). The concentration of DTT may be from 1 mM to 50 mM.

[0138] In some embodiments, the method may further include at least one RNase inhibitor.

[0139] The buffer solution used in the isothermal method of this invention can be a Tris-HCl buffer, a Tris-acetate buffer, or a combination thereof, but other buffer systems are also suitable. The concentration of the buffer can be from about 10 nm to 500 nm, depending on the buffer system used and the pH of the reaction to be performed. As those skilled in the art know, the latter is affected by the optimal activity of the enzyme of interest.

[0140] Suitable standard reaction conditions and reagent concentrations (e.g., enzymes, primers, probes, dNTPs, ATP, buffers, pH, etc.) for the isothermal amplification methods disclosed herein are known to those skilled in the art and can be selected from, for example, Zanoli and Spoto (see above); Li et al. (see above); Hoser et al. (see above); Van Ness et al. (see above); and specific reaction conditions considered for particular embodiments of the invention are disclosed herein. Generally, the concentrations of recombinases, SBB proteins, and / or recombinase cofactors used in the art for isothermal amplification, particularly in the context of RPA reactions, can also be used for the recombinases, SBB proteins, and / or recombinase cofactors of the present invention. Example 5 illustrates a non-limiting exemplary composition and procedure for isothermal amplification using the proteins of the present invention.

[0141] For the purposes of this invention, a short amplicon length is generally preferred because it improves amplification efficiency. Furthermore, the shorter the target to be amplified (and therefore the shorter the amplicon), the faster the corresponding detection results. This, of course, represents a significant advantage for rapid testing, such as at-home testing, enabling the rapid identification of potential infections.

[0142] When using the methods disclosed herein to provide reliable diagnostic results, the design of primers and optional probes is particularly important. In preferred embodiments of the methods of the invention, especially if used for diagnostic purposes, the conventional length of the amplicon, influenced by the corresponding primer design, is typically from about 50 to about 400 base pairs (bp), preferably from 100 bp to about 200 bp, and most preferably from about 120 bp to about 150 bp. Amplicon lengths below 200 bp are considered advantageous for diagnostic purposes because the length of the amplicon affects the efficiency of amplicon amplification and is therefore directly related to the analytical sensitivity of the reaction.

[0143] Unlike PCR, primers used in this invention are typically relatively long (approximately 30 to 35 nucleotides). Shorter primers (18 to 25 nucleotides) can also be used in this invention, but this may reduce reaction speed and sensitivity, which should be avoided in diagnostic settings.

[0144] In one embodiment of the method of the present invention, at least one forward primer and at least one reverse primer are provided as target sequence-specific primers, wherein at least one of these primers, preferably a reverse primer, is provided at a higher concentration relative to the other primer, wherein the concentration may be, for example, about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 to 2.5, 2.6 to 2.9, 3 to 4, 5 to 6, 7 to 8, or 9 to 10 times or more. Typically, primers for multiplex analysis, or a set of primers or several sets of primers, are of interest and can be provided according to the methods disclosed herein, wherein the primers are typically provided in equimolar ratios. However, it has been found that for certain purposes, such as for the diagnosis of RNA viruses, it may be suitable to provide a higher concentration of one primer (depending on whether it is a (+) or (-) ssRNA virus) to obtain more cDNA for subsequent amplification steps, thereby improving the performance of the reaction.

[0145] Isothermal amplification was initially thought to be a method for nucleic acid amplification of DNA. Subsequent studies showed that RNA amplification was also suitable if additional enzymes, such as reverse transcriptases (e.g., murine leukemia virus (MuLV) reverse transcriptase), were added to the same reaction chamber. In the context of viral diagnostics, RNA amplification and thus the detection of viral RNA is crucial. This allows not only the detection of the genetic material of RNA viruses but also the detection of viral transcripts, which also originate from DNA viruses. The latter can be particularly important, for example, in situations where viral transcripts are released into the bloodstream, while viral particles and viral DNA largely remain in tissues where biological samples are difficult to obtain.

[0146] In embodiments involving RNA amplification, the method may include reverse transcriptase. The method may include a protein containing the functions of both DNA polymerase and reverse transcriptase, thereby enabling a single protein to perform both functions.

[0147] In some embodiments, the kits and methods of the present invention can be used as target sequences for single-stranded or double-stranded DNA, cDNA, and / or RNA sequences. Furthermore, the kits and methods can be used for various amplification reactions on various nucleic acid materials.

[0148] In some implementations, DNA and RNA nucleic acid sequences are provided as target sequences. For the detection of DNA viruses or retroviruses, combined detection of viral DNA and viral RNA (via reverse transcription in the same procedure) offers significant advantages because all possible targets that may be present in the sample can be detected together, such as the viral DNA itself and the RNA transcripts of the viral DNA, thus greatly improving sensitivity.

[0149] In one embodiment, the method is carried out at a temperature of about 20°C to about 50°C, preferably about 22°C to about 45°C, and most preferably about 25°C to about 42°C.

[0150] The reaction of the present invention can be incubated for 5 minutes to 16 hours, for example 15 minutes to 3 hours, or 30 minutes to 2 hours. Incubation can be carried out until the desired degree of amplification is achieved (see EP 2 336 361 A2). In some embodiments, the reaction can be carried out under stirring to increase the contact rate of the various components, thereby improving the efficiency of the method.

[0151] In some embodiments, probes may be used in the kits and methods disclosed herein, wherein at least two different probes are preferably used: one associated with the target nucleic acid to be amplified and one associated with a positive control. The use of a reliable positive control is very helpful, especially in the diagnostic field, and more particularly in the diagnosis of infectious agents (such as SARS-CoV-2), for which currently available test kits do not include such a positive control, as a positive control allows for the conclusion of whether the reaction is effective. This is especially important and offers significant advantages when the test is not performed by laboratory professionals but in a home setting or at a testing site.

[0152] In the context of this invention, any luminescence detection system can be used to rapidly and reliably detect amplification results of interest. Luminescence is a general term for any kind of non-thermal spontaneous emission of light from a substance. Luminescence includes various types of luminescence, among which chemiluminescence (including bioluminescence and electroluminescence) and photoluminescence (including fluorescence and phosphorescence) are most relevant to the purposes of this invention.

[0153] Probes that can be incorporated into the kits and methods of this invention include, for example, commercially available TwistAmp™ exo probes (typically about 46 to 52 nucleotides in length) and TwistAmp® fpg probes (typically 32 to 35 nucleotides in length). These probes are used for real-time fluorescence detection. Both types of probes are typically labeled with a fluorophore, a quencher (e.g., Black HoleQuencher), wherein the quencher is closely adjacent to the fluorophore to transiently block the fluorescence signal; and a blocking group at the 3' end (e.g., C3-spacer, phosphate group, biotin-TEG, or amino group) to prevent polymerase from extending from the 3' end (see also Li et al., Analyst, 2019, see above, particularly...). Figure 3 A and Figure 3B). Real-time detection is based on cleavage of a fluorescent probe at a base-free site (also known as a depurinyl / depyrimidine site, located in DNA (rarely in RNA), which contains neither purine nor pyrimidine bases), with cleavage occurring between the fluorophore and the quencher. The base-free site can be tetrahydrofuran (THF), dSpacer (a derivative of THF), or a dR group (the deoxyribose of a base-free site linked by a COC linker). For example, *E. coli* exonuclease III cleaves the TwistAmp™ exo probe at the THF or dSpacer site, while *E. coli* glycosylase / lyase fpg (formamidopyrimidine-DNA glycosylase) cleaves the TwistAmp™ fpg probe at the dR site. After enzyme cleavage, the TwistAmp® exo probe can be used as a forward primer. However, due to the different catalytic mechanism (β-elimination) of the *E. coli* glycosylase / lyase fpg protein, this catalytic mode does not produce an extendable 3'-OH group, but rather a 3'-phosphate group. Therefore, the TwistAmp™ fpg probe cannot be used as a primer. It is conceivable to use various probes or labels to label target nucleic acid bases or nucleotides with detectable tags, including probes that do not require the addition of at least one additional activating enzyme, provided that at least one suitable light source and a corresponding detection device specifically detects the presence of the probe and / or label by generating a detectable signal are present.

[0154] In another embodiment, alternative luminescent dyes can be used (see Roy et al., Biosens Bioelectron. December 15, 2016; doi:10.1016 / j.bios.2016.06.065).

[0155] As is known in the field of molecular detection technology (including methods using PCR and isothermal methods), there are various ways to visualize and analyze the results of amplification reactions. These techniques are known to those skilled in the art and can be used in the kits and methods disclosed herein.

[0156] In some embodiments of the sixth or eighth aspect, at least one target-specific primer (including single-stranded and partially double-stranded primers) is itself labeled with a detectable marker. It should be noted that fluorescence quenchers are also considered detectable markers. For example, a fluorescence quencher may come into contact with a fluorescent dye, and the amount of quenching it produces will also be detected. To avoid interfering with the reaction, the detectable marker should not interfere with the amplification reaction. In the case of partially double-stranded primers having both an invasive and a non-invasive strand, the manner in which the detectable marker is attached should not interfere with the amplification reaction of the invasive strand. The non-invasive strand of a partially double-stranded primer does not extend, therefore there are no restrictions on labeling the non-invasive strand, the only exception being that the label on the non-invasive strand must not interfere with the extension reaction of the invasive strand. Labeled primers offer the advantage of faster detection of amplification products. Furthermore, the detection of unincorporated markers (i.e., labeled oligonucleotides that have not yet extended) will allow for monitoring of the reaction status.

[0157] In another embodiment, the double-stranded primers can be labeled to detect the separation of the two strands. As described above, after multiple rounds of amplification, the invasive and non-invasive strands of some double-stranded primers will separate. After this separation, the non-invasive strand does not participate in the amplification reaction, thus allowing for real-time detection and monitoring of the amplification reaction results.

[0158] Furthermore, the detectable label can be a fluorescent label or an enzyme, and the label quencher (also known as a label inhibitor) can be a fluorescence quencher or an enzyme inhibitor. In these cases, the label is detected by fluorescence or enzyme inhibition. If a fluorescent label is used, the detectability of the label will be fluorescence, or if an enzyme is used, the detectability of the label will be enzyme activity.

[0159] In some embodiments of the sixth or eighth aspect, a separate probe is provided, which may be target sequence specific. Furthermore, the probe can be used to provide a detectable or visible signal, thereby facilitating monitoring of the results of the amplification reaction. The probe may carry an activatable label or signaling component, which can be activated, for example, by adding an enzyme, such as exonuclease III (Exo III, exo herein) used in the Twist-RPA reaction. In some embodiments, the label may also bind to at least one primer. Although the detectable portion may be selected from the group consisting of fluorescent dyes, enzymes, fluorescence quenchers, enzyme inhibitors, radioactive labels, chromogenic agents, and combinations thereof, the use of radioactive materials and / or commonly used (chemical, biological, or other hazardous substances) should be avoided for non-laboratory applications of the methods of the present invention to allow for rapid adjustment and to ensure the safety of the system end-user, particularly when used in private environments. Furthermore, this also facilitates waste management, as the system can be easily discarded after use.

[0160] In some embodiments of the sixth or eighth aspect, at least one probe and / or marker is provided, wherein the at least one probe and / or at least one marker comprises a directly or indirectly detectable portion.

[0161] All primers or probes used in the kits and methods disclosed herein may be naturally occurring DNA and / or RNA sequences, or synthetic primers or probes including naturally occurring bases and / or backbones, or synthetic elements including covalently and / or non-covalently linked tags to primers or probes, or including at least one phosphate thioester backbone used to increase the stability of the primer or probe, or to improve the amplification of DNA sequences by means of a DNA polymerase with proofreading activity (Skerra, Nucleic Acids Res 20, 3551-3554, doi:10.1093 / nar / 20.14.3551 (1992)), and any combination thereof.

[0162] The concentration of at least one SSB protein used in any embodiment of the present invention may be from 50 ng / μl to 1500 ng / μl, preferably from 100 ng / μl to 800 ng / μl, more preferably from 200 ng / μl to 600 ng / μl, and most preferably from 300 ng / μl to 500 ng / μl.

[0163] According to various embodiments of the present invention, each protein may include at least one tag, which may be located at the N-terminus, C-terminus, or within the protein sequence (without interfering with the function of the protein, preferably between two separate domains), or any combination thereof (e.g., typically different N-terminal and C-terminal tags, such as two different affinity tags, or a combination of a solubility tag and a visualization tag, etc.).

[0164] Optionally or additionally, nanostructured elements can be used in the reaction mixture of the sixth or eighth aspect of the present invention to further optimize the amplification results, for example, ZnO nanostructures (Ma et al., Virus Res., 2017, 232:34-40. doi: 10.1016 / j.virusres.2017.01.021).

[0165] The invention will now be further described with reference to the following non-limiting embodiments.

[0166] Example

[0167] Example 1: Identification of novel recombinase and SSB protein

[0168] To identify novel recombinases and SSB proteins, gene and protein sequences with moderate or high sequence similarity to known recombinases and SSB proteins are searched, as these have the highest probability of identifying candidates of interest.

[0169] Bioinformatic identification of novel recombinases

[0170] In the first step, candidate genes were searched across 98 different genomes. Surprisingly, no recombinases (e.g., Helicobacter pharmacophagus and Aeromonas pharmacophagus) were annotated in most genomes. Instead, some genomes encoded more than one recombinase (Escherichia coli, Salmonella pharmacophagus, Shigella pharmacophagus, and Burkholderia pharmacophagus). The encoded recombinases were primarily annotated as NinB7NinG, RecT / E, tyrosine recombinases, or YqaJ recombinases. Several other recombinases were not annotated to any family and were described as putative recombinases. RecA-like recombinases were annotated only in two Arthrobiosis pharmacophagus species. However, these showed very low sequence identity with the known recombinase T6 UvsX, suggesting they are less likely to be good candidates. A phylogenetic tree was constructed based on the available sequence data. This analysis proposed two candidates: the Pseudomonas virus H66 recombinant-associated protein RdgC and the Bacillus virus pony ReRF recombinase protein. However, sequence alignment with T6 UvsX again showed very low sequence identity.

[0171] Based on phylogenetic analysis, a BLAST (NCBI) amino acid sequence search was performed, using T6 UvsX as a reference sequence for evaluation. From a large number of hits, five distinct candidates with high or moderate sequence identity were selected. Among these five proteins, the DNA recombination / repair protein RecA [E. f. EGI strain EGI FJ00015] was considered the most promising candidate. Therefore, available sequence data from different E. f. strains were analyzed and compared. Interestingly, strain EGI FJ00015 encodes two different RecA sequences – referred to in this paper as RecA1 and RecA2. RecA2 is a classic RecA homolog found in all available E. f. genomes, while RecA1 is similar to the UvsX enzyme. Furthermore, RecA1 and RecA2 show high sequence similarity to each other.

[0172] Genome analysis of different *Staphylococcus* strains suggested that bacterial UvsX-like recombinases might be promising candidates. Therefore, a novel BLAST search was performed, limited to bacterial taxa. Of the hits, RecA1 from *Staphylococcus* strain EGI FJ00015 (highly similar to the known T6 UvsX) and RecA from *Shigella flexneri* (moderately similar to T6 UvsX) were selected for structural analysis.

[0173] Structural analysis of novel recombinase variants

[0174] Since no structural information was available for the novel protein, 2D elements were predicted using the Psi Pred program, and 2D structures were created using the 2dss software for alignment. Finally, 3D structures were created using AlphaFold. The resulting structures were compared with those obtained by X-ray crystallography for T4 UvsX and T4 Gp2. For both newly identified recombinase variants, 2D and 3D structural analyses showed high similarity up to approximately amino acid 340, after which the predicted structures showed significant differences.

[0175] Identification of novel SSB proteins

[0176] BLAST was used to search for bacterial SSB proteins, with RB69 GP32 as a reference sequence. Among a group of highly similar proteins, the following proteins were identified: one labeled as a hypothetical protein from the *Staphylococcus* strain EGI FJ00015, and another labeled as a single-stranded DNA-binding protein from *Shigella flexneri*. Both showed high sequence similarity to RB69Gp32, and both candidates were selected for further structural analysis.

[0177] Structural analysis of novel SSB variants

[0178] 2D structural elements showed very high similarity to Rb69 and Gp32 up to approximately amino acid 220, with more pronounced structural differences thereafter. Small deviations were also identified between amino acids 30 and 80. Analysis of the 3D structure confirmed these results and showed high similarity up to approximately amino acid 235.

[0179] Example 2: Oligonucleotide Binding Analysis

[0180] Figures 1A to 1C Examples of purification of LuSSB1, LuSSB1, and SfRecA are shown. To test the oligonucleotide binding activity of the purified proteins, electrophoretic mobility shift analysis (EMSA) was performed on several newly identified variants (see [link to documentation]). Figures 2A to 2F (and Table 1).

[0181] This assay is based on the detection of gel migration of FAM-labeled oligonucleotides after protein binding. Limits for oligonucleotide migration were determined using polyacrylamide gel electrophoresis. RPA conditions were applied for the binding reaction. Reaction conditions for oligonucleotide binding in a 20 μL reaction were: 20 mM tris-acetate pH 7.4, 90 mM KOAc, 10 mM MgOAc, 2 mM DTT, 1.23 μM oligonucleotides, and varying amounts of protein (serially diluted in 20 mM tris-acetate pH 7.4). The reaction was initiated by adding protein to the incubation mixture and run at 42 °C for 15 min. After 15 min, the reaction was stopped by adding 20 μL of pre-chilled 40% glycerol.

[0182] After incubation, the samples were electrophoresed in a 12% TBE gel, and the FAM labeling of oligonucleotides was detected.

[0183] Table 1 EMSA Results

[0184]

[0185] Example 3: Activity assay of recombinase and recombinase cofactor

[0186] The recombinase activity of purified proteins was detected based on the recombinase-mediated ATP-to-ADP conversion.

[0187] The 20 μl reaction mixture contained 50 mM tris-acetate / KOAc (pH 7.9), 150 mM KOAc, 14 mM MgOAc, 5 mM DTT, 2 mM ATP, and varying amounts of ssDNA and recombinase protein. The reaction was initiated at 42 °C by adding MgOAc, incubated for 2 min, 4 min, and 8 min, and then immediately frozen after a 1:36 dilution in water. The negative control contained the same components but was not initiated by adding MgOAc. Samples and controls were then analyzed using the Kinase-Glo® Max assay, in which the luminescent signal generated by luciferase was linearly correlated with the ATP concentration in the sample.

[0188] Exemplary results of activity measurement are as follows:

[0189] The activity of the recombinase cofactor was analyzed in a similar manner. The recombinase cofactor does not convert ATP to ADP; however, under specific conditions (potassium acetate concentration >150 mM), it can enhance the activity of the recombinase. In this assay, the concentration of the recombinase cofactor that doubled the recombinase activity was determined. The reaction mixture was the same as described above, except that the recombinase cofactor was added.

[0190] Example 5: Isothermal amplification using a novel protein

[0191] Various combinations and conditions were established to test newly identified recombinases and SSB proteins in isothermal amplification reactions. Figure 3 An exemplary result for LuSSB1 is shown in the figure.

[0192] Establish an RPA reaction in a 50 μl sample with the following protein composition:

[0193] The RPA reaction was carried out in an Axxin T8 isotherm.

[0194] Figure 4 Further exemplary RPA results are shown using 330 ng / μl LuSSB1 or 580 ng / μl Rb69 GP32 in the above reaction mixture.

[0195] Additional reaction mixtures containing 400 ng / μl LuSSB1 or 600 ng / μl Rb69 Gp32 were tested according to the method described above.

[0196] Interestingly, in otherwise identical RPA reactions, LuSSB1 exhibited a significant performance increase compared to RB69 Gp32. Therefore, LuSSB1 has a considerable enhancing effect on standard RPA reactions. Different amplification settings have been performed on LuSSB1 and other novel variants disclosed herein, and further experiments are underway.

[0197] Example 6: Asymmetric primer concentration

[0198] To obtain optimal amplification results, the amplified protein can be used in an amplification mixture containing asymmetric primer concentrations. Setting one primer (preferably the reverse primer) at a higher concentration in the primer pair can promote the amplification reaction, especially when the amount of the target nucleic acid to be detected is low.

[0199] Figure 5 An exemplary amplification reaction for amplifying monkeypox virus DNA using the following reaction mixture at symmetrical and asymmetrical primer concentrations is shown:

[0200] The primer concentrations are either symmetrical (210 nM for both primers) or asymmetrical (forward primer: 210 mM; reverse primer: 840 mM).

[0201] Analysis using the protein of the present invention in a reaction mixture with asymmetric primer pairs is ongoing, and the analysis shows that such methods using the novel enzyme of the present invention combined with asymmetric primer concentrations can achieve significant improvements over currently available amplification methods.

[0202] Example 7: LuSSB1 mutants and variants

[0203] To increase the solubility of LuSSB1 or LuSSB2 enzymes and thus their versatility, mutants with increased folding and solubility can be created. As shown in SEQ ID NOs 21 to 23, positions T246, A247, and A2448 can be mutated individually or in combination to HIS to increase the solubility of LuSSB1. LuSSB2 can be modified in a similar manner.

[0204] Additionally, the N-terminus or C-terminus of LuSSB1 can carry a penta-HIS or hexa-HIS tag to optimize purification. An exemplary variant (double hexa-HIS tag) is shown in SEQ ID NO: 24. This tag (N-terminus, C-terminus, or both ends) can also be combined with any of the LuSSB1 variants of SEQ ID NO: 21 to 23 or with any other enzyme disclosed herein.

[0205] Example 7: LuSSB1 mutants and variants

[0206] Enzyme stability and performance are extremely important for optimizing point-of-care RPA assays. Therefore, the granule mixture was optimized, and different LuSSB1 mutants were tested. Figure 6 As shown, LuSSB1 performs excellently under various conditions.

[0207] The enzyme mixture used and tested was:

[0208] Notably, LuSSB1 and its variants, as well as tagged mutants, all exhibited excellent performance. Figure 5 The data were generated from variants with dual HIS tags (SEQ ID NO: 24). SEQ ID NO: 21, 22, and 23 were demonstrated to be more reliable, and achieving the same performance rate required less enzyme (~50 ng / µl to 200 ng / µl), resulting in additional improvements due to the enzymes potentially being more stable and thus requiring even less enzyme.

Claims

1. A protein or a functional fragment thereof, preferably supporting isothermal amplification, wherein, The protein comprises or is composed of the following: a) *Seminobacterium* ( Lutimaribacter SSB1, comprising or consisting of: the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it, preferably wherein the amino acid sequence has a serine residue at position 64 of SEQ ID NO 1 or 2; or SSB1 of the genus *Synthobacterium*, comprising or consisting of: the amino acid sequence of SEQ ID NO: 21, 22 or 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO: 21, 22 or 23, respectively.

2. A group of at least two proteins or functional fragments thereof, preferably supporting isothermal amplification, wherein, One of the proteins is the protein of claim 1 or a functional fragment thereof, and the group comprising at least one additional protein, which further comprises or consists of the following: b) *S. spp.* SSB2, comprising or consisting of: the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or c) Shigella flexneri ( Shigella flexneri SSB, comprising or consisting of: the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or d) λ phage β protein, comprising or consisting of: the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or e) *RecA1*, comprising or consisting of: the amino acid sequence of SEQ ID NO: 5 or 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or f) *RecA2*, comprising or consisting of: the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or g) Shigella flexneri RecA, comprising or consisting of: the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or h) Escherichia coli ( Escherichia coli RecT, comprising or consisting of: the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with it; or i) λ phage Orf, which comprises or consists of the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with it.

3. A fusion protein comprising at least one protein as described in claim 1 or 2 and at least one additional functional moiety, said at least two moiety being optionally separated by at least one linker; and / or said fusion protein comprising at least one affinity tag, preferably at the C-terminus and / or N-terminus.

4. The fusion protein according to claim 1 or 2, wherein, The fusion protein contains at least one GST tag, MBP tag, Strep tag and / or HIS tag, preferably containing at least one HIS tag.

5. A nucleic acid molecule encoding at least one protein as described in claim 1 or 2; and / or at least one fusion protein as described in any one of claims 3 to 4.

6. A vector or expression construct comprising at least one nucleic acid molecule as described in claim 5.

7. A cell comprising at least one protein according to claim 1 or 2; and / or at least one fusion protein according to any one of claims 3 to 4; and / or at least one nucleic acid molecule according to claim 5; and / or at least one vector or expression construct according to claim 6.

8. A kit comprising at least one protein according to claim 1 or 2; and / or at least one fusion protein according to any one of claims 3 to 4; and / or at least one nucleic acid molecule according to claim 5; and / or at least one vector or expression construct according to claim 6; and / or at least one cell according to claim 7.

9. The kit according to claim 8, wherein, The kit includes: (i) at least one single-chain binding protein, optionally the single-chain binding protein of claim 1 or 2 (a), (b), (c) and / or (d), preferably the single-chain binding protein of claim 1 (a), and / or at least one fusion protein comprising the same as claimed in any one of claims 3 to 4; (ii) at least one recombinase, optionally the recombinase of claim 2 (e), (f), (g) and / or (h), and / or at least the fusion protein comprising any one of claims 3 to 4; (iii) Optionally at least one recombinase cofactor, optionally the recombinase cofactor of claim 2 (i), and / or at least the fusion protein comprising the recombinase cofactor of any one of claims 3 to 4; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) Optional at least one reverse transcriptase; (vii) A suitable reaction component, including at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analogue, and further optional crowding agent. (viii) Optionally, at least one probe and further optionally, at least one enzyme that activates the probe.

10. Use of the protein of claim 1 or 2; and / or the fusion protein of any one of claim 3 or 4; and / or the nucleic acid molecule of claim 5; and / or the vector or expression construct of claim 6; the cell of claim 7; and / or the kit of claim 8 or 9 for isothermal amplification of at least one target nucleic acid molecule, optionally for the detection of viral RNA and / or DNA, and / or for sample amplification for nucleic acid sequencing, preferably wherein the amplification is performed in vitro, and / or preferably wherein the amplification is performed with a field-available test, preferably using the kit of claim 8 or 9.

11. A method for isothermal amplification of at least one target sequence, preferably in a biological sample, the method comprising the following steps: (a) Provide (i) at least one single-chain binding protein, optionally the single-chain binding protein of claim 1 or 2 (a), (b), (c) and / or (d), preferably the single-chain binding protein of claim 1 (a), and / or at least one fusion protein comprising the same as any one of claims 3 to 4; (ii) at least one recombinase, optionally the recombinase of claim 2 (e), (f), (g) and / or (h), and / or at least the fusion protein comprising any one of claims 3 to 4; (iii) Optionally at least one recombinase cofactor, optionally the recombinase cofactor of claim 2 (i), and / or at least the fusion protein comprising the recombinase cofactor of any one of claims 3 to 4; (iv) at least one, preferably at least two, target sequence-specific primers; (v) at least one DNA polymerase; (vi) Optional at least one reverse transcriptase; (vii) A suitable reaction component, including at least one suitable buffer, a mixture of dNTPs and ddNTPs or dNTPs, optionally ATP or an ATP analogue, and further optional crowding agent. (b) Provide at least one biological sample to be analyzed to detect the presence of at least one target sequence, and optionally provide a preferred sterile extraction kit to obtain the biological sample; (c) Optionally: providing at least one probe and optionally providing at least one enzyme for activating said probe, thereby generating a detectable signal; (d) Adding starting reagents to initiate the amplification reaction; and (e) Obtaining at least one amplified target sequence and / or obtaining at least one detectable signal, each signal indicating successful amplification of the corresponding target sequence. Step (a) includes providing at least one protein as described in claim 1 or 2 and / or at least one fusion protein as described in any one of claims 3 to 4 in sub-steps (i), (ii) and / or (ii).

12. The method according to claim 11, wherein, The method does not include the use of ATP or ATP analogues and / or energy regeneration systems.

13. The method according to claim 11 or 12, wherein, The method is performed in the absence of recombinase cofactor.

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