WO2022150283A1 - Détection de sras-cov-2 - Google Patents

Détection de sras-cov-2 Download PDF

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WO2022150283A1
WO2022150283A1 PCT/US2022/011087 US2022011087W WO2022150283A1 WO 2022150283 A1 WO2022150283 A1 WO 2022150283A1 US 2022011087 W US2022011087 W US 2022011087W WO 2022150283 A1 WO2022150283 A1 WO 2022150283A1
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tcep
cov
sars
phosphine
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Nodar Makharashvili
James O'Connell MCNAMARA
Steve Pemberton
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Nuclease Probe Technologies, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

  • Rapid and inexpensive assays that detect the SARS-CoV-2 coronavirus in human and other sample types are needed to identify infected individuals and monitor infection prevalence in communities. Detection of infections enables the selective isolation of infected subjects from uninfected individuals which can greatly reduce disease transmission. Efficient infection detection can also enable treatment early in the course of illness, improving treatment outcomes. Additionally, viral detection methods can be used to monitor the prevalence of infections in communities, providing information that can be used to guide behavioral adjustments at the community level to limit the impact of outbreaks.
  • PCR-based assays that detect viral RNA
  • antigen assays that detect SARS-CoV-2 proteins (usually proteins of the viral capsid)
  • assays that detect SARS-CoV-2 infection signatures in the cells and molecules of the human immune system i.e., host-response tests such as antibody tests.
  • host-response tests such as antibody tests.
  • the PCR and antigen tests are the assay types that are most useful for identifying individuals with active infections. Both PCR and antigen tests are known to have limited sensitivity for SARS-CoV-2 infections; of the two, PCR tests are more sensitive and are considered the gold-standard tests.
  • PCR assays for SARS-CoV-2 is likely due in part to the high stability of the SARS-CoV-2 viral capsid in human specimens.
  • Studies that evaluate the stability of the viral capsid in human nasopharyngeal swab samples (via RT -PCR-based detection of the RNA it encapsulates) found little if any degradation with refrigerated sample storage over the course of several days.
  • An important limitation of PCR tests is that, in principle, they cannot differentiate between patients who are shedding live virus and those who have recovered from an infection and are shedding fragments of virus; positive results may be found in both cases. This is due to a molecular idiosyncratic property of PCR.
  • antigen tests detect viral proteins, usually via antibodies that bind with high affinity and specificity to target viral proteins. While less sensitive than PCR assays, antigen tests that detect viral proteins could provide a more accurate means of identifying individuals who are shedding live virus. In particular, tests that detect viral proteins that are subject to rapid degradation in vivo (i.e., viral proteins that exhibit short half-lives) may provide results that show a better correlation with the presence and abundance of live virus. However, current antigen tests that are commercially available detect proteins that make up SARS-CoV-2 viral particles. The components of the viral particle, especially the proteins, have evolved to be stable in the extracellular environments in which they encounter their cellular targets.
  • tests that detect these proteins can serve as robust tools for detecting viral material in individuals who may have been infected with SARS-CoV-2, but they can also be expected to yield positive results for some time after an individual is no longer shedding substantial amounts of infective viral particles.
  • Antigen tests that detect viral proteins that are not part of the viral particles themselves may provide a means of detecting the presence of the SARS-CoV-2 virus, in addition to a means of determining the presence, absence, or relative amount of viable virus. While antigen tests have exhibited lower sensitivity than PCR, they have the potential to provide superior sensitivity because proteins (the molecular targets of antigen tests) are roughly ⁇ 2, 000-fold more abundant than nucleic acids (Marguerat et al., 2012).
  • the disclosure provides compositions and methods that enable rapid and specific detection of SARS-CoV-2 in human samples via detection of the enzymatic activity of the SARS-CoV-2-derived NendoU nuclease. In other aspects and embodiments, this disclosure provides compositions and methods that protect nucleic acids from degradation in human samples.
  • compositions and methods involve the use of Tris(2- carboxyethyl)phosphine, or “TCEP” and related compounds, which in some embodiments are combined with RNase inhibitors such as nickel, or manganese, or zinc (or other divalent cations) to stabilize RNA against degradation by RNases present in human samples such as blood and saliva, while permitting degradation by NendoU nuclease of SARS-CoV-2 (if present).
  • RNase inhibitors such as nickel, or manganese, or zinc (or other divalent cations)
  • these compositions promote selective digestion of the substrates by NendoU, thus enabling detection of SARS-CoV-2 in the samples via increases in fluorescence.
  • the specific and sensitive detection of the SARS- CoV-2 NendoU nuclease was also found to be facilitated by a DEAE-matrix-based ion exchange protein purification method that selectively captures and concentrates this nuclease.
  • the stabilizing effects of TCEP and related compounds find use for other applications, such as stabilizing RNA and/or DNA (e.g., extracellular DNA) in various mammalian (e.g., human) sample types, such as blood, serum, and plasma, with utility that includes assays that measure nucleic acid biomarkers.
  • the stabilizing effects of TCEP and related compounds find use for stabilizing protein biomarkers in samples, for example, from protease degradation.
  • Reactions also contained PolyU fluorogenic RNA probe (1 lmer poly-uridine flanked with fluorophore and quencher; 0.1 pM concentration). Fluorescence was measured with a Synergy HI Biotek plate-reader after incubation for 30 minutes at 37 °C.
  • FIG. 2 demonstrates that TCEP protects RNA from degradation by human saliva nucleases.
  • FIG. 3 demonstrates that TCEP protects RNA from degradation by human saliva nucleases.
  • PolyU a poly-uridine (l lmer) fluorogenic RNA probe, was combined (at 1 mM final concentration) with 25% saliva or water (see labels) in solutions with 50 mM Tris pH 7.0, 250 mM KC1, 20 mM MnCh and either: 1) 20 mM DTT and 5 mM TCEP; 2) 20 mM DTT; 3) 5 mM TCEP or 4) No DTT or TCEP.
  • FIG. 4 demonstrates that TCEP protects RNA from degradation by human plasma nucleases.
  • PolyU a poly-uridine (1 lmer) fluorogenic RNA probe (at 0.1 pM concentration), was combined with human plasma (10% final concentration; anti-coagulant is lithium heparin) or water (see labels), in reactions with 250 mM KC1, and either: 1) 20 mM DTT and 5 mM TCEP; 2) 5 mM TCEP; or 3) 20 mM DTT.
  • FIG. 5 demonstrates that TCEP protects RNA from degradation by human plasma nucleases in ⁇ 24 hour incubation.
  • RNaseAlert ® IDT was incubated for ⁇ 24 hours at room temperature with human plasma (lithium heparin) or water (upper x-axis labels), plus either: nothing additional (i.e., water), 10 mM TCEP, or with a lx concentration of the Streck RNA Complete BCT solution (lower x-axis labels).
  • FIG. 6 shows nuclease assay specificity for SARS-CoV-2 demonstrated with nuclease-spiked human saliva samples.
  • NendoU orthologues of each of the indicated human coronaviruses were spiked into 50% human saliva (provided by an asymptomatic donor) at a final concentration of 50 pM.
  • the spiked samples were then processed with a NendoU- detection protocol that was engineered to specifically detect the SARS-CoV-2 NendoU orthologue. Briefly, each 500 pL sample was combined with 500 pL H2O, 50 pL 1 M Tris- HCl, pH 8.0 and 15 pL 5 M NaCl. The samples were then combined with 25 pL DEAE magnetic beads and incubated on ice for 10 minutes.
  • FIG. 7 evaluates performance of a nuclease-based SARS-CoV-2 detection assay with patient nasopharyngeal swab samples.
  • 750 pL of each of 10 patient nasopharyngeal samples collected in saline (that were previously evaluated with a PCR assay for SARS-CoV-2- see labels) were processed with a SARS-CoV-2 NendoU-detection protocol. Briefly, each sample (and control, see below) was combined with 750 pL of 50 mM Tris-HCl, pH 8.0, 0.05% triton X-100 and then with 50 pL DEAE magnetic beads.
  • Beads suspensions were incubated on ice for 10 minutes and then, using a magnet for liquid/beads separations, beads were washed once with 1 mL of NaOAc pH 5.2, 75 mM KC1, 0.05% triton X-100 and then with 1 mL of 50 mM Tris-HCl pH 7.0, 75 mM KC1, 0.005% triton X-100. Proteins were eluted from the beads by resuspending with 25 pL 2x reaction buffer (100 mM Tris-HCl, 500 mM KC1, 40 mM MnCh, 10 mM TCEP, 40 mM DTT).
  • Controls include saliva from an asymptomatic individual, unspiked (25% Saliva Only), or spiked with 5 pM NendoU (25% Saliva +NendoU); note the controls have 25% saliva after the 750 pL volumes of input saliva are combined with 750 pL buffer in the first step detailed above. Control samples were also supplemented with 75 mM NaCl prior to processing to match the saline in the clinical samples. Dotted line is retrospectively drawn to indicate elevated signal in samples that were positive for SARS-CoV-2 by PCR.
  • the present invention in certain aspects and embodiments relates to methods and compositions that enable detection of microbial pathogens via the detection of the enzymatic activity of their nucleases.
  • Nucleic acid probes for the detection of pathogen-related nucleases are described in WO 2015/120406 and WO 2013/033436, which are hereby incorporated by reference in their entireties.
  • inexpensive, rapid and sensitive molecular assays can detect nucleases of microbial pathogens via their unique enzymatic and structural properties.
  • This approach entails the engineering of assays that use quenched fluorescent oligonucleotide substrates for nucleases and the reaction buffers in which they are presented to specifically detect nucleases of target pathogens, such as viral, bacterial, and fungal pathogens.
  • Digestion of a fluorogenic oligonucleotide substrate yields an increase in fluorescence which can be detected with various established fluorescence measuring devices such as fluorescence plate-readers.
  • each target nuclease serves both as biomarker for the pathogen and also as the signal-amplifying component of the assay.
  • This approach exploits the highly diverse and ubiquitous presence of nucleases in living cells.
  • the nucleases are encoded in viral genomes and are produced within host cells that are infected by the viruses.
  • This technical platform depends on the discovery of a combination of substrate and reaction conditions that yield substrate digestion selectively by the target nuclease and not by the off-target nucleases that are found within a particular sample type or extract. For instance, determination of substrate and reaction conditions that enable selective detection of a nuclease of a bacterial pathogen within a mammalian (e.g., human) blood sample that contains many off-target (e.g., mammalian or human) nucleases can be used as a molecular diagnostic approach for bacterial bloodstream infections (Menzies, 1977).
  • Sample processing methods such as methods that specifically purify a target nuclease from a sample prior to incubation of the purified nuclease with the optimal substrate in the optimal reaction conditions can further enhance both specificity (by eliminating off-target nucleases) and sensitivity (by concentrating the target nuclease).
  • coronavirus NendoU efficiently digests RNA substrates, with preferential cleavage 3’- of uridine nucleotides (Bhardwaj et al., 2006).
  • the detection of SARS-CoV-2 with quenched fluorescent RNA substrates that include uridines is a promising approach for SARS-CoV-2 detection.
  • using unmodified RNA especially RNA that includes unmodified pyrimidine nucleotides as a reporter for a pathogen-derived nuclease in human samples presents a substantial challenge due to the ubiquitous presence of high activity levels of endogenous nucleases, such as RNase A that efficiently digest such RNA oligos (Behlke, 2008; Yang, 2011).
  • this disclosure provides methods that are both highly sensitive for the SARS-CoV-2 NendoU orthologue and exhibit low background with respect to endogenous human nucleases and off-target NendoU orthologues.
  • these methods include an ion exchange purification step with a weak anion exchange matrix, such as DEAE (diethyl ami noethyl), which selectively captures and concentrates the SARS- CoV-2 NendoU orthologue.
  • DEAE diethyl ami noethyl
  • the invention provides reaction mixtures that provide further specificity for SARS-CoV-2 NendoU vs.
  • the reaction buffer comprises TCEP-HC1 (Tris(2-carboxyethyl)phosphine) (“TCEP”), a disulfide bond-reducing agent (Bums, 1991; Getz et al., 1999), which exhibited an unexpected degree of inhibition of endogenous human RNases, but did not impede NendoU digestion of RNA substrates.
  • TCEP-HC1 Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP is more stable than other reducing agents commonly used in protein biochemistry procedures such as DTT (Getz et al., 1999), a property that provides an advantage for some applications.
  • DTT Trimet al., 1999
  • An additional, previously unappreciated mechanism by which TCEP protects nucleic acids from degradation may underlie the profound impact on RNA stability.
  • nuclease substrates do not need to be fluorogenic; they include distinct affinity tags (or modifications enabling conjugation to surfaces, macromolecules, etc.) flanking an oligonucleotide substrate.
  • FAM fluorescein amidite
  • biotin examples include FAM (fluorescein amidite) and biotin.
  • FAM fluorescein amidite
  • Biotin can be bound by various proteins with high affinity, such as streptavidin.
  • Colorimetric methods for detecting enzymatic activity of target nucleases such as methods that use nanoparticles to yield a visible color change upon nuclease-based digestion of oligonucleotide substrates are an alternative nuclease-detection format that can be interfaced with the present invention to enable detection of SARS-CoV-2 via detection of the SARS-Cov-2 NendoU nuclease.
  • An example of this approach is described in Tiet, P, et al., 2017, which is hereby incorporated by reference.
  • NendoU (also known as non- structural protein 15, or “Nspl5”) is a potential diagnostic target of the SARS-CoV-2 virus that does not form part of the viral capsid. NendoU is encoded in the SARS-CoV-2 genome and is expressed intracellularly in host cells that are infected with the virus. Its role in viral pathogenesis is thought to be carried out entirely within the host cells (Deng and Baker, 2018). As NendoU has apparently not evolved to play a role in the extracellular milieu, it is likely more susceptible to degradation in this environment than the proteins that make up the viral capsid. In any case, NendoU represents a different category of targets from the antigen tests currently available.
  • the SARS-CoV-2 NendoU protein shares 39 to 45% amino acid sequence identity with its orthologues in the alternative human coronaviruses that are in common circulation (which includes the 229E, NL63, OC43 and HKU1 human coronaviruses).
  • a common immunoassay format entails an initial binding event in which a primary antibody binds directly to the antigen (if it is present in a sample), followed by incubation with a secondary antibody, which is tagged with a reporter moiety (e.g., a fluorophore, or a signal-generating enzyme). The secondary antibody then binds the primary antibody, thereby forming a complex that is coupled to one or more reporter molecules, which enable detection.
  • a reporter moiety e.g., a fluorophore, or a signal-generating enzyme
  • immunoassays include detection of proteins that exhibit enzymatic activity, such as beta-galactosidase, proteases, and nucleases, which can be detected via enzymatic substrates that yield a change in color, fluorescence or other detectable signal upon enzymatic processing.
  • proteins that exhibit enzymatic activity such as beta-galactosidase, proteases, and nucleases
  • Nucleases are enzymes that cleave nucleic acids, and nucleases can be detected via their enzymatic activities, for instance, with fluorogenic oligonucleotide substrates. Due to their high structural and enzymatic diversity and the fact that they are essential for life (i.e., they are ubiquitous), nucleases have the potential to provide a category of enzymatic biomarkers with wide applicability in molecular diagnostics. In principle, the presence of a distinct nuclease in a “diseased” sample and its absence in a normal sample can be used as the basis for a screening or diagnostic assay for the disease. Specificity for a target nuclease can be achieved by engineering assays to selectively enable detection of the target nuclease, while minimizing or eliminating the enzymatic activity of off-target nucleases that may be present.
  • Nucleases have diverse substrate preferences, including some that are selective for single-stranded RNA, single-stranded DNA, double-stranded RNA, double-stranded DNA, or sequence-specific substrates (i.e., restriction endonucleases) (Yang, 2011). Some nucleases can readily digest chemically modified nucleic acid substrates, such as substrates with 2'-0-methyl and 2'-fluoro modified nucleotides, whereas others cannot. Fluorogenic oligonucleotide substrates can be engineered to match the substrate preferences of target nucleases while minimizing or eliminating the susceptibility to digestions by off-target nucleases. Similarly, the composition of the reaction buffer in which a nuclease is detected can have profound effects on the abilities of target and off-target nucleases to digest a substrate.
  • Oligos with the following attributes are expected to yield sensitive and specific detection of NendoU (e.g., as the oligo component of fluorogenic oligonucleotides).
  • Oligos that include unmodified uridine nucleotides that also include alternative nucleotides, such as chemically modified nucleotides may enable sensitive NendoU detection, but with limited specificity.
  • Alternative nucleotides/modifications of such oligos could include sugar modifications, such as modifications of the 2'-position of the ribose moiety, phosphorothioate modifications, base modifications, locked nucleic acid nucleotides, unlocked nucleic acid nucleotides, DNA nucleotides, unmodified RNA purine nucleotides.
  • Oligos that include such nucleotides/modifications might be engineered to include several nucleotides that are non-cleavable by NendoU or other nucleases, (such as modified nucleotides, unmodified purine RNA nucleotides, DNA nucleotides) adjacent to or flanking a NendoU-cleavable nucleotide(s) (e.g., unmodified uridines, or uridines that have modifications of the C5 and/or C6 positions of the uracil base). These uracil modifications may provide specificity for NendoU vs. off-target nucleases such as RNase A.
  • Oligos that consist of several nucleotides (such as 2-100 nucleotides) of unmodified uridines or of uridines modified to be selectively cleavable by NendoU (modified at positions C5 and/or C6 of the base) may yield greater sensitivity as many cleavage sites would be present.
  • Substrates with unmodified uridines are expected to be most useful when used in concert with NendoU purification methods and/or reagents that block or inhibit off-target nuclease digestion of these substrates (e.g., TCEP).
  • Oligos that include unmodified cytosine nucleotides may also be sensitive to NendoU (Bhardwaj et al., 2008).
  • the reaction buffer can have a profound impact on the activity of nucleases.
  • optimal salt concentrations and buffer compositions can be highly nuclease- dependent.
  • divalent cations are known to have a profound impact on the activities of nucleases (Yang, 2011).
  • the activities of some nucleases is dependent on magnesium (e.g., Endonuclease I of E. coli ⁇ calcium (e.g., micrococcal nuclease of S. aureus ), manganese, or zinc, and others exhibit cation-independent enzymatic activity (e.g., bovine pancreatic RNase A) (Cuatrecasas et al., 1967; Lehman et al., 1962; Yang, 2011).
  • divalent cations are known to inhibit the enzymatic activities of some nucleases.
  • manganese, zinc and mercury are potent inhibitors of micrococcal nuclease (Cuatrecasas et al., 1967). This contrasts with other nucleases that depend on some of these same divalent cations for their activity (e.g., manganese (Mrel l) and zinc (nuclease PI)) (Yang, 2011).
  • Incorporation in the reaction buffer of divalent cations that selectively inhibit off-target nucleases is one means of promoting specificity for target nuclease detection.
  • Disulfide bond-reducing agents can be used to eliminate disulfide bonds in proteins. In cases where a target nuclease exhibits enzymatic activity in the presence of disulfide bond-reducing agents (i.e., activity is independent of disulfide bonds), the inclusion of such agents in the reaction buffer can be used to reduce the activity of off-target nucleases, many of which require disulfide bonds for enzymatic activity. Disulfide bond-reducing agents in common use include dithiothreitol (DTT) and beta-mercaptoethanol (BME).
  • DTT dithiothreitol
  • BME beta-mercaptoethanol
  • a target nuclease is present in very low concentrations or is present in samples that comprise highly diverse mixtures of off-target nucleases, such as stool samples
  • the sensitivity and specificity of target nuclease detection can be greatly enhanced by capturing and concentrating the target nuclease prior to incubating it with the engineered probe/reaction condition combination.
  • a monoclonal antibody can be coupled to magnetic beads to capture the target nuclease from the sample. After washing the nuclease-coupled beads, they can be resuspended in a small volume (e.g., ⁇ 60 pL), effectively concentrating the nuclease for detection using a fluorogenic oligonucleotide probe.
  • This nuclease capture/concentration procedure leverages the high specificity of monoclonal antibodies for the target nuclease to provide an orthogonal means of increasing target-specificity, and also increases the sensitivity by concentrating the nuclease in a smaller volume.
  • the methods may purify or enrich target nuclease from complex preparations using chromatography, prior to incubating them with fluorogenic oligonucleotide probes and buffer.
  • chromatography e.g., ion exchange chromatography (or ion exchange matrices), size-exclusion chromatography, and alternative affinity chromatography/affmity matrices such as lectin- coupled matrices, carbohydrate-coupled matrices, RNA-coupled matrices, and DNA- coupled matrices (e.g., DNA-sepharose).
  • Alternative nucleic acid-coupled matrices that could be useful include chemically-modified RNA- or DNA- coupled matrices.
  • ion-exchange chromatography include ion exchange spin columns, many of which are commercially available, and ion exchange resins, such as surface-modified Sepharose, agarose, and magnetic beads.
  • the matrices can be used in various formats.
  • the ion exchange spin columns include charged resins that bind proteins based on charge-charge interactions. As nucleases have diverse charges on their surfaces, a subset of nucleases in a sample will interact with a particular ion exchange matrix in particular buffer conditions. Upon applying a sample to the upper chamber of a spin column and centrifuging the column, the sample passes through the matrix (i.e., resin). Some proteins will bind the matrix and others will flow through to the lower chamber.
  • Elution buffers can (likewise) be used to selectively elute a subset of the bound proteins from the matrix.
  • Ion exchange matrices include cation-exchange and anion-exchange matrices. Identification of a matrix that binds off-target nucleases (e.g., host nucleases found in saliva, nasopharyngeal samples, blood, sewage), but not on-target nucleases, such as NendoU of the SARS-CoV-2 human coronavirus, can be used to isolate a sample extract (the flow through) that is enriched for the target nuclease. Procedures like this can be used to pre- process samples to increase specificity for detection of the target nuclease.
  • off-target nucleases e.g., host nucleases found in saliva, nasopharyngeal samples, blood, sewage
  • on-target nucleases such as NendoU of the SARS-CoV-2 human coronavirus
  • heparin-coupled surfaces such as heparin-coupled Sepharose beads.
  • Heparin is a carbohydrate that is negatively charged at neutral pH and will interact with positively charged molecules, including proteins with positively charged patches on their surfaces.
  • TCEP buffers with millimolar concentrations of TCEP provide superior stabilization of RNA in the presence of plasma nucleases versus a commercial product (Streck) that is commonly used to stabilize extracellular RNA in blood samples.
  • TCEP was previously shown to protect RNA from degradation that otherwise occurs when RNA is heated in the presence of magnesium (Rhee and Burke, 2004). This study did not demonstrate the utility of TCEP in protecting RNA from enzymatic degradation or at room temperature or 37 °C.
  • RNA from degradation by non-enzymatic degradation processes at elevated temperatures and also the stabilization against RNases such as those found in blood and saliva (according to this disclosure) that is in excess to the stabilization provided by other reducing agents, suggest a mechanism of action distinct from the inactivation of RNases via reduction of disulfide bonds.
  • TCEP-RNA and TCEP-DNA adducts may be reversibly forming in solutions comprising both RNA and TCEP, and DNA and TCEP, respectively.
  • Adducts of TCEP with proteins is also anticipated with similar stabilization effects vs. proteases.
  • Such adducts could in principle, prevent the digestion of nucleic acids by some nucleases, but not others, depending on the manner in which the nucleases interact with their substrates. For instance, phosphine adducts to the pyrimidine rings (e.g., to the 5 th and/or 6 th carbons of the rings), could selectively block some nucleases, but not others.
  • nucleic acid stabilization reagents e.g., for stabilization of extracellular RNA and extracellular DNA
  • clinical sample types including blood, plasma, serum, saliva, stool, bronchoalveolar lavage, sputum, urine, cerebrospinal fluid, rectal or anal swab samples, nasopharyngeal swab samples, throat swab samples, skin and soft tissue swab samples, vaginal swab samples.
  • phosphine-based compounds that may be used to protect nucleic acids or proteins from degradation by nucleases or proteases, respectively, that are found in human and animal specimens and fluids include tris(3-hydroxypropyl)phosphine (THPP), tris(hydroxymethyl)phosphine, mmTCEP, dmTCEP and tmTCEP, and derivatives of these compounds (Cline et al., Biochemistry, 2004; Patel et al., Biochemistry, 2020).
  • THPP tris(3-hydroxypropyl)phosphine
  • mmTCEP tris(hydroxymethyl)phosphine
  • dmTCEP dmTCEP
  • tmTCEP dmTCEP
  • derivatives of these compounds Cline et al., Biochemistry, 2004; Patel et al., Biochemistry, 2020.
  • the phosphine compound is TCEP or salt thereof.
  • the phosphine compound may be TCEP-HCl.
  • the invention employs reaction compositions and mixtures that comprise phosphine-based nucleic acid-protecting compounds and also include other reagents that are known to inhibit nucleases.
  • the phosphine-based nucleic acid-protecting compounds and the additional nuclease inhibitors would be useful in combination via additive or synergistic effects on nucleic acid stabilization.
  • Nuclease inhibitors that are expected to be useful in such buffers include some divalent cations, including Ca++, Mg++, Mn++, Zn++, Cu++, Co++, Ni++, Sr++, and divalent cation chelating agents including EDTA and EGTA.
  • nuclease inhibitors that may be useful in combination with phosphine-based nucleic acid-protecting compounds include other reducing agents including DTT, BME, and glutathione; antibody -based nuclease inhibitors (i.e., inhibitory antibodies), protein-based nuclease inhibitors, nucleic acid-based nuclease inhibitors, including nucleic acid-based nuclease-inhibiting aptamers, nuclease-inhibiting nucleotide analogues, proteases including proteinase K, chemicals that denature proteins including SDS, and chaotropic salts including guanidinium salts.
  • antibody -based nuclease inhibitors i.e., inhibitory antibodies
  • protein-based nuclease inhibitors i.e., inhibitory antibodies
  • protein-based nuclease inhibitors i.e., inhibitory antibodies
  • protein-based nuclease inhibitors i.e., protein-based nu
  • reactions are performed at pH 2 to about pH 9, such as in the range of about pH 3 to about pH 8.
  • the reaction can be performed near physiological pH, such as in the range of about pH 6 to about 8.
  • the selectivity of the reaction is enhanced by a low pH, such as a pH of about 2 to about pH 5.5 (e.g., a pH of from about 3 to about 5).
  • Extracellular RNA is found in human blood, saliva, urine, cerebrospinal fluid, and other specimen types (Murillo et al., 2019). Because RNA can be efficiently digested by RNases, which are also present in these specimens, the accuracy and reproducibility of RNA- detection methods depends on minimizing the impact of RNA degradation on the assay results. Practical implementation of diagnostics that measure RNA biomarkers is facilitated by compositions that preserve RNA integrity in samples for hours or days, reducing the need for immediate on-site isolation of RNA from the samples. Clinical samples such as blood have many different types of RNases present and these enzymes have diverse properties.
  • RNA present in clinical samples include chemical reagents that denature proteins (e.g., SDS, and chaotropic salts such as guanidinium thiocyanate or guanidium chloride), formaldehyde releasing agents that inactivate RNases by forming covalent bonds with them, various RNase-specific macromolecular inhibitors including anti-RNase antibodies and inhibitory proteins ((Bender et al., 2020; Chomczynski and Sacchi, 1987); M. Rohan Fernando, US 9,926,590 B2, 2018).
  • denature proteins e.g., SDS, and chaotropic salts such as guanidinium thiocyanate or guanidium chloride
  • formaldehyde releasing agents that inactivate RNases by forming covalent bonds with them
  • various RNase-specific macromolecular inhibitors including anti-RNase antibodies and inhibitory proteins
  • Divalent cation chelators such as EDTA, and EGTA will inhibit RNases that require divalent cations such as calcium and magnesium as cofactors; however, some RNases, such as RNase A, do not require divalent cations for enzymatic activity and are thus not inhibited by cation chelators (Yang, 2011).
  • reducing agents such as DTT and BME inhibit RNases such as RNase A which require disulfide bonds between select amino acids to exhibit enzymatic activity (Sela et al., 1957); other RNases do not require disulfide bonds for their activity and are unaffected by reducing agents.
  • Proteases such as proteinase K have been used to digest RNases, with substantial, though incomplete effects (Bender et al., 2020).
  • the use of formaldehyde to cross-link amino acids of RNases has been found to result in poor nucleic acid quality or recovery, making this approach of limited value (Vander Plaetsen et al., 2017).
  • an assay that detects SARS-CoV-2 in biological samples via the ability of its NendoU RNase to digest nucleic acid substrates with unmodified RNA nucleotides must have selectivity for this enzyme versus other RNases that are commonly found in the relevant sample types.
  • These “off-target” nucleases include the orthologues of NendoU that are produced by distinct human coronaviruses in common circulation, including the 229E, NL63, OC43, and HKU1 human coronaviruses.
  • Other important off-target RNases include mammalian RNases that are found in clinical samples such as blood, saliva and nasopharyngeal swab samples. These include various forms of RNase A.
  • This disclosure provides assay conditions that enable the specific detection of SARS- CoV-2 NendoU via its digestion of substrates such as poly-uridine (RNA) oligonucleotides, as well as RNaseAlert ® fluorogenic substrate (IDT).
  • substrates such as poly-uridine (RNA) oligonucleotides, as well as RNaseAlert ® fluorogenic substrate (IDT).
  • RNA poly-uridine
  • IDTT RNaseAlert ® fluorogenic substrate
  • reaction buffers were prepared that enable preferential detection of the SARS-CoV-2 NendoU orthologue with a fluorogenic RNA substrate consisting of 11 uridine nucleotides flanked with a fluorophore and quencher (FIG. 1).
  • Both buffers tested in the experiment shown in FIG. 1 include divalent cations that can serve as inhibitors of alternative nucleases, but permit the activity of NendoU of SARS-CoV-2.
  • the activity observed for SARS-CoV-2 in this experiment occurred in the presence of the disulfide-bond reducing reagents, beta- mercaptoethanol (MnCh buffer) and TCEP (NiCh buffer).
  • disulfide bond reducing reagents may therefore be used to promote assay specificity for SARS-CoV-2 NendoU.
  • Buffer components were evaluated for compatibility with SARS-CoV-2 NendoU enzymatic activity, and for ability to suppress mammalian RNases that are present in clinical sample types such as human saliva and plasma.
  • TCEP a disulfide bond reducing agent
  • a reaction buffer that includes 5 mM TCEP yielded only modest activation of the PolyU probe when incubated with human saliva (FIG. 2).
  • the PolyU probe contains 11 unmodified RNA uridines, providing a highly sensitive substrate for RNases present in human saliva, such as RNase A.
  • RNaseAlert ® is a fluorogenic RNA substrate used to detect RNase activity; it is commercially available from Integrated DNA Technologies, Inc. (Coralville, IA). As RNaseAlert ® has been widely used to detect RNase activity in many studies, it provides a familiar means of detecting RNase activity. TCEP exhibited a similar degree of RNase inhibition, when measured with RNaseAlert ® , to that observed with PolyU probe-based RNase detection (FIG. 2).
  • Disulfide bond reducing agents such as DTT and BME, are known to inhibit enzymes whose activity depends on disulfide bonds, such as RNase A.
  • TCEP is an alternative disulfide bond reducing agent that is not commonly used. The results shown in FIG. 2 were unexpected for two reasons. First, the impact of disulfide bond reducing agents on RNase activity of mammalian fluids has been described as modest, whereas TCEP provided robust RNase inhibition. Second, since DTT is present in the buffer (in addition to TCEP) and in the buffer that does not include TCEP (FIG.
  • TCEP may have value in stabilizing RNA in blood, plasma, or serum samples.
  • the practical utility of TCEP in this context would depend on its ability to stabilize RNA in such fluids for much longer time periods (e.g., 12-24 hours).
  • a leading product currently available for stabilizing RNA in blood samples is the Streck Complete RNA BCT, a blood collection tube that contains an RNA stabilizing liquid composition. RNA present in blood samples collected in these tubes is stabilized from degradation by RNases present in the blood.
  • TCEP While serving as an inhibitor of mammalian RNases, TCEP is compatible with the nuclease activity of SARS-CoV-2 NendoU (see FIG. 1, NiCh buffer). Inclusion of TCEP in the reaction buffer of an assay for this nuclease could thus promote specificity for the NendoU nuclease in preparations where mammalian RNases may be present. TCEP was incorporated in the reaction buffer for a SARS-CoV-2 NendoU detection assay protocol that also includes a SARS-CoV-2 NendoU DEAE ion exchange matrix purification step as a means of providing further specificity for this target enzyme.
  • This assay exhibited highly specific detection for SARS-CoV-2 NendoU in the context of human saliva (FIG. 6). This result demonstrates that the combination of a target-tailored purification protocol with reaction conditions that enable selective enzymatic activity of the target enzyme can yield specificity vs. off-target enzymes of both viral and human origin.
  • the fluorescence levels generated when fluorogenic oligonucleotide probes are incubated with samples that contain nucleases capable of digesting the probe are dependent on the concentration of the nuclease; higher concentrations of nucleases generate higher fluorescence levels. It is anticipated that the concentration of the NendoU nuclease in a sample is dependent on the concentration of virus in the sample. It is thus anticipated that the fluorescence signal strength of the NendoU detection assays of this invention will provide a semi-quantitative measure of the viral concentration in a sample.
  • the NendoU detection assays may thus have utility in determining the concentration of SARS-CoV-2 in a sample.
  • US Patent 9,926,560 describes compositions for preservation of cell-free nucleic acids, which is hereby incorporated by reference in its entirety, particularly with regard to the use of compounds as nucleic acid stabilizing reagents in blood collection tubes.
  • TCEP In addition to its use in the SARS-CoV-2 detection assay and as a component of blood collection tubes (i.e., protection of extracellular nucleic acids), TCEP (and related compounds) may also be useful as component s) of collection tubes for additional clinical sample types, including collection tubes for nasopharyngeal swabs, anal or rectal swabs, saliva, bronchoalveolar lavage samples, vaginal swabs, cerebrospinal fluid, skin and soft tissue infection samples. TCEP (and related compounds) may also be useful as components of RNase-inhibitor cocktails and/or protease-inhibitor cocktails, or as a standalone RNase inhibitor or a standalone protease inhibitor.
  • TCEP millimolar (final) concentrations of TCEP (or related compounds) in lysis buffers that also include lytic reagents such as detergents or denaturants (e.g., chaotropic agents) that disrupt cellular membranes
  • lytic reagents such as detergents or denaturants (e.g., chaotropic agents) that disrupt cellular membranes
  • TCEP (or related compounds) could be included as reagent(s) in kits for protein or nucleic acid isolation as a separate reagent that could be combined by the end-user with other kit components to yield buffers or solutions that protect proteins and/or nucleic acids from enzymatic degradation.
  • NendoU NendoU orthologues of the following human coronaviruses were obtained from the Uniprot.org database: SARS- CoV2; HCoV-229E; HCoV-NL63; HCoV-OC43; HCoV-HKUl .
  • Protein accession numbers and amino acid numbers used (in brackets) are as follows: SARS-CoV2 (accession number: P0DTD1[6453 - 6798]); HCoV-229E (accession number: P0C6X1[6111 - 6458]); HCoV- NL63 (accession number: P0C6X5[6086 - 6429]); HCoV-OC43 (accession number: P0C6X6, [6422 - 6796]); HCoV-HKUl (accession number: P0C6X3, [6480 - 6853]).
  • DNA sequences encoding each protein were codon-optimized for bacterial expression, synthesized, and cloned into the pET28a expression plasmid as custom orders by GenScript Biotech.
  • the protein expression plasmids were transformed into BL21- pLys expression cells according to the manufacturer’s recommendations (Thermo Scientific). Transformed bacterial clones were expanded in LB supplemented with kanamycin at 37 °C, with shaking. After an initial outgrowth period, expression was induced with addition of Isopropyl -b-D-thiogalactopyranoside (IPTG) (Fisher Scientific) and the cultures were grown for an additional 20 hours at 18 °C. Protein purification. The His-tagged nucleases were purified with standard methods.
  • the cells were harvested by centrifugation, lysed in the presence of protease inhibitors, the lysate was clarified with centrifugation and the proteins were captured with Ni-NTA agarose (Invitrogen). The agarose was washed and then proteins were eluted with imidazole-containing buffer. Protein products were analyzed with polyacrylamide gel electrophoresis (PAGE). Purified proteins were flash frozen in liquid nitrogen and stored at -80 °C.
  • NiCh was acquired from Alfa Aesar; KC1 was acquired from Invitrogen; PVSA (poly (vinyl sulfonic acid) was acquired from Polysciences; human plasma (lithium heparin is anticoagulant) was acquired from BioIVT ; DEAE magnetic beads were acquired from Bioclone, Inc..
  • the Streck RNA Complete BCT tubes were acquired from Streck.
  • RNaseAlert ® was acquired from Integrated DNA Technologies, Inc. (IDT) of
  • RNA oligo flanked with a fluorophore and quenchers (5’-FAM- rUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrU-IAbRQSp-3’, where FAM is fluorescein amidite, rU is a uridine nucleotide (RNA), and IAbRQSp is the Iowa Black RQ quencher connected with a spacer).
  • FAM fluorescein amidite
  • rU is a uridine nucleotide (RNA)
  • IAbRQSp is the Iowa Black RQ quencher connected with a spacer
  • Tris(2-carboxyethvDphosphine stabilization of RNA comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry. Anal Biochem 325, 137-143.
  • the human cytomegalovirus UL98 gene encodes the conserved herpesvirus alkaline nuclease. J Gen Virol 78 ( Pt 11), 2953-2961.

Abstract

L'invention concerne des compositions pour stabiliser l'ARN et/ou des protéines, comprenant des composés de phosphine, et, facultativement, des réactifs supplémentaires qui réduisent l'activité de la ribonucléase ou de la protéase mammifère. L'invention concerne également des mélanges réactionnels comprenant les compositions pour détecter le SRAS-CoV-2. L'invention concerne en outre des tubes de sang à prise directe comprenant les compositions.
PCT/US2022/011087 2021-01-05 2022-01-04 Détection de sras-cov-2 WO2022150283A1 (fr)

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US20100209930A1 (en) * 2009-02-18 2010-08-19 Streck, Inc. Preservation of cell-free nucleic acids
US20160338342A1 (en) * 2010-07-26 2016-11-24 Biomatrica, Inc. Compositions for stabilizing dna, rna and proteins in blood and other biological samples during shipping and storage at ambient temperatures
WO2017214338A1 (fr) * 2016-06-07 2017-12-14 Drawbridge Health, Inc. Procédés et dispositifs pour des molécules fortes ou stabilisantes
US20190127729A1 (en) * 2016-02-11 2019-05-02 Sarstedt Ag & Co. Kg Device and method for isolating nucleic acids from whole blood

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Publication number Priority date Publication date Assignee Title
US20100209930A1 (en) * 2009-02-18 2010-08-19 Streck, Inc. Preservation of cell-free nucleic acids
US20160338342A1 (en) * 2010-07-26 2016-11-24 Biomatrica, Inc. Compositions for stabilizing dna, rna and proteins in blood and other biological samples during shipping and storage at ambient temperatures
US20190127729A1 (en) * 2016-02-11 2019-05-02 Sarstedt Ag & Co. Kg Device and method for isolating nucleic acids from whole blood
WO2017214338A1 (fr) * 2016-06-07 2017-12-14 Drawbridge Health, Inc. Procédés et dispositifs pour des molécules fortes ou stabilisantes

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