WO2022136023A1 - Solution permettant de détecter une séquence d'arn viral - Google Patents

Solution permettant de détecter une séquence d'arn viral Download PDF

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WO2022136023A1
WO2022136023A1 PCT/EP2021/085677 EP2021085677W WO2022136023A1 WO 2022136023 A1 WO2022136023 A1 WO 2022136023A1 EP 2021085677 W EP2021085677 W EP 2021085677W WO 2022136023 A1 WO2022136023 A1 WO 2022136023A1
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domain
probe
strand
sequence
group
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PCT/EP2021/085677
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Yi Sun
Mohsen MOHAMMADNIAEI
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Danmarks Tekniske Universitet
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the invention relates to a solution for detection of a viral RNA sequence, the solution comprising a double stranded (duplex) initiation helix, at least one signal system, and at least two probes A and B having hairpin structures.
  • Nucleic acid-based diagnosis is well known within the field for e.g. screening nasopharyngeal swab samples for viral RNA. This is commonly used today e.g. to identify those who have an active coronavirus infection.
  • the ‘gold standard’ for RNA detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR).
  • RNA is firstly transcribed into complementary DNA (cDNA) by reverse transcriptase, and then the cDNA is amplified exponentially with the help of Taq polymerase.
  • cDNA complementary DNA
  • Taq polymerase The qRT-PCR is extremely powerful due to its high sensitivity and specificity. The method has been widely established in many countries, playing key roles in controlling the pandemic. However, the qRT-PCR requires a dedicated machine to accurately cycle through different temperatures, and the whole reaction can take up to 3 hr due to time-consuming heating and cooling steps.
  • RPA recombinase polymerase amplification
  • CRISPR-Cas13-mediated enzymatic signal amplification for detection of SARS-CoV-2.
  • the present invention solves many of the above problems by using a solution comprising a double stranded (duplex) initiation helix, at least one signal system, and at least two probes A and B having hairpin structures.
  • the double stranded initiation helix as disclosed herein may also be called “reporter”. It is comprised of a template strand and a recognition strand, wherein the recognition strand has a toehold sequence.
  • the present invention is based on toehold-mediated strand displacement (TMSD), which is used as an alternative isothermal amplification technique.
  • TMSD are competitive hybridization reactions, where an incoming nucleic acid strand outcompetes the other strand from a DNA or RNA duplex to form a better matched duplex.
  • the kinetics of strand displacement is modulated by the toehold - the short single-stranded DNA segment overhanging on the original duplex.
  • the process is controlled by the Gibbs free energy of hybridization, which is non-enzymatic and purely entropy driven.
  • the presence of the incoming strand can trigger cascade branch migration reactions for signal amplification.
  • the present invention discloses a modification to this termed an isothermal strand migration and amplification (NISMA) assay, which is an ingenious one-pot, enzyme-free, isothermal assay that can rapidly detect a viral RNA sequence in clinical samples.
  • NISMA isothermal strand migration and amplification
  • the present invention exchanges the long viral RNA into a short DNA template using a DNA duplex containing a toehold overhang (reporter).
  • the DNA template then initiated the cascade unfolding of two DNA molecular beacon structures (probes), leading to dramatic enhancement in e.g. fluorescence intensity.
  • the present invention is able to detect e.g. SARS- CoV-2 RNA in 30 min at 42 °C with a limit of detection of 10 Copies/pL (see table 2 bellow in the examples) of the viral RNA sequence.
  • a solution comprising: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a’, and c domain; wherein the first group of the at least one signal system is
  • colorimetric detection as disclosed herein is included all detections which results in a coloured output, even if said output is not recognized by the human eye as coloured.
  • Colorimetric assays result in a coloured reaction product that absorbs light in the visible range. The optical density of the reaction product is typically proportional to the amount of analyte being measured.
  • Fluorescent assays are simply a variation of colorimetric assay.
  • the reaction product emits fluorescence when excited by light of a particular wavelength.
  • the relative fluorescence units (emitted photons of light) that are detected are typically proportional to the amount of analyte being measured.
  • the probe A comprising the hairpin structure M1 is labeled with e.g. a fluorophore, such as FAM and e.g. a quencher, such as a black hole quencher (BHQ) at the end of the stem, and when viral RNA is present, the hairpin structure of M1 is opened, and the b' sequence is accessible to function as a toehold for the subsequent hybridization with the b sequence on probe B, wherein due to the strand displacement process, the M2 displaces the initial template strand by hybridization with probe A to form a more stable duplex structure, and wherein the displaced template strand acts as a fuel to open another probe A and initiating a new hybridization cycle, and wherein unfolding of probe A results in a distinct change in the fluorescence intensity, as the fluorophore and quencher are brought further apart, and hereby based on the intensity shift, the positive and negative test results can be easily distinguished by an optical reader.
  • a fluorophore such as FAM
  • a solution for colorimetric detection of a viral RNA sequence comprising: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a’, and c
  • RNA sequence is meant that the system can test different types of samples.
  • the pathogens can come from e.g., clinical samples, food samples, or environmental samples.
  • genome RNA sequence it should also be able to detect genome DNA and short RNAs e.g. microRNA.
  • a viral RNA sequence is the partial/complete nucleotide sequence of a viral RNA- genome.
  • a genome is all genetic material of an organism. The genome includes both the genes (the coding regions) and the noncoding nucleotide regions.
  • the invention also provides a general solution for detection of long nucleic acid sequence, such as mRNA, genome DNA, etc.
  • the viral RNA sequence is a long nucleic acid sequence (above 100 nt), an mRNA sequence, or a genome DNA sequence.
  • the transcription part it is necessary to have a balanced design on the double stranded (duplex) initiation helix, which is composed of the template strand and the recognition strand with enhanced affinity for the viral RNA sequence (by e.g. including an LNA/INA sequence in the recognition strand).
  • the critical issue in the design of the recognition strand is to have (i) high affinity to efficiently and specifically bind to the whole RNA genome, and (ii) sufficient stability to easily undergo the strand displacement.
  • One possibility of obtaining such excellent conditions is to introduce higher affinity only at the toehold sequence of the recognition strand. Further, the toehold length could be varied.
  • One advantage of the present invention is that in the template recycling and signal amplification part, the design of the M1 enables modification of fluorophore and quencher at the 3’ and 5’ends of the nucleotide to lower the production cost and provide very low background noise to significantly increase the sensitivity, dynamic range and readout speed of the assay.
  • Another advantage of the present invention is the use of a double stranded (duplex) initiation helix comprising two strands, a template strand and a recognition strand having a toehold sequence.
  • the present invention is able to detect a viral RNA/DNA sequence in a whole or substantially whole/full viral RNA/DNA genome without the use of any enzyme and the need to reverse transcribe the RNA into DNA and/or cut the viral nucleic acid into smaller pieces prior to incubation with the reaction mixture of the present invention.
  • a Limit of Detection (LOD) of at least 1 nM such as at least 100 fM, such as at least 50 fM, such as at least 5 fM, or such as at least 0.2 fM can be obtained.
  • LOD Limit of Detection
  • the present invention allows for a very simple signal readout with high throughput using e.g. 96 well or 384 well plate reader device.
  • Another advantage of the present invention is that the reaction is controlled by the Gibbs free energy of hybridization, which is non-enzymatic and purely entropy driven.
  • the present invention uses at least four different domains in the nucleotide sequences, a, b, c, and d domain, in addition to three complimentary nucleotide sequences, a’, b’, and c’ domain, where a’ is complimentary to a, b’ is complimentary to b, and c’ is complimentary to c.
  • a double stranded (duplex) initiation helix is a short double-stranded DNA complex, with an overhanging toehold on one of the strand.
  • a template strand is a piece of DNA oligonucleotide, such as e.g. 22 nucleotides (nt) sharing the same sequence of the target gene.
  • a domain is a fragment of a DNA sequence.
  • a recognition strand is a piece of DNA oligonucleotide (e.g. 30-40 nt) that has the complementary sequence of the target gene. It is partially complementary to the template strand with a toehold sequence overhanging at its end region invigorated to have a higher affinity to recognize and bind with the target RNA genome.
  • Complimentary refers to the complementary nature of nucleic acid bases. When two complementary strands of DNA or RNA are alongside each other, the bases match up with their complement, that is, thymine (or uracil) with adenine, and guanine with cytosine. When a strand is fully complementary means that all bases in said sequence are complementary.
  • a toehold sequence is a sequence on the recognition strand such that when the two strands (the template and recognition strand) are hybridizes to each other due to complementary of their nucleotide sequences, the recognition strand has an overhanging region, called the toehold sequence, which is complementary to the target sequence but not to the template strand sequence. This means that when the template and recognition strands form the double stranded initiation helix the toehold sequence will not be hybridized with the template strand and therefore will be found as a single strand domain as an overhang on the duplex structure.
  • a signal system refers to the reaction process that produces an output optical signal in response to an input viral RNA.
  • a hairpin structure is a structure comprising a loop, which is an unpaired loop of nucleotides that is created when the strand folds and forms base pairs with another section of the same strand, i.e. it occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop.
  • the resulting structure looks like a loop or a U-shape.
  • a hairpin structure may also be called a stem-loop. Hairpins are a common type of secondary structure in RNA molecules.
  • covalently attached is meant that the probes are attached on the substrate through covalent bond, which is a chemical bond that involves the sharing of electron pairs between atoms.
  • kits for colorimetric detection of a viral RNA sequence comprising a solution and a sample collector for collecting sample from a patient, wherein the solution comprises: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having
  • a solution for detecting a viral RNA sequence in a sample comprises: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a
  • a solution for use in a method of diagnosis comprising: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a’, and c domain; wherein
  • a method for colorimetric detection of a viral RNA sequence comprises the steps of: providing a sample to be analysed for the viral RNA sequence; lysing the provided sample and extracting the total viral RNA; adding the extracted total viral RNA to a solution for colorimetric/fluorescent detection of the viral RNA sequence, wherein the solution comprises: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein
  • Lysing the provided sample and extracting the total viral RNA is e.g. performed by adding a lysis buffer to break the cell membrane, such that total RNA of all sizes is released to the solution. The total RNA is then isolated and purified from the solution.
  • Adding the extracted total viral RNA to a solution for colorimetric detection means that one is detecting the presence of the viral RNA based on the change of colour intensity/wavelength of the solution.
  • An optical reader based on an intensity shift is a device that can record the change of the light intensity.
  • the predetermined temperature is a constant isothermal temperature. This is especially advantageous as it would then be a isothermal detection system, not requiring thermal cycles, such as is the case for e.g. PCR amplification.
  • the constant isothermal temperature is selected from between 25 and 50 °C.
  • the sample is a cell lysate.
  • the predetermined time is between 20 and 60 minutes, such as between 25 and 40 minutes, such as between 25 and 35 minutes.
  • the sample to be analysed for the viral RNA sequence comprises less than 1 nM of total viral RNA, such as less than 100 fM of total viral RNA, such as less than 50 fM of total viral RNA, or such as less than 10 fM of total viral RNA.
  • the recognition strand d domain is a toehold sequence composed partially or fully of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • LNA/INA enables the recognition strand to a have higher affinity to efficiently and specifically bind to the whole RNA genome.
  • the toehold sequence may be reduced in length.
  • Locked Nucleic Acid is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon.
  • the structure of LNA has improved specificity and affinity as a monomer or a constituent of an oligonucleotide.
  • Intercalating Nucleic Acids refers to the insertion of a new intercalating pseudo-nucleotide (IPN) into a DNA strand.
  • IPN is the phosphoramidite of (S)-1-0-(4,4'-dimethoxytriphenylmethyl)-3- 0-(1-pyrenylmethyl)glycerol.
  • INAs oligodeoxyribonucleotide
  • the recognition strand is an LNA strand, an INA strand, or combinations hereof.
  • the design of the recognition strand have to have a high binding affinity (to the target sequence) to efficiently and specifically bind to the whole RNA genome, while also have sufficient stability to facilitate the strand displacement.
  • a DNA toehold harboring different toehold length has been tested with and without strand modifications in the form of LNA or INA.
  • the INA® technology Proprietor: PentaBase A/S
  • PentaBase A/S is on the basis of insertion of a base unite (intercalator) that is intercalated into nucleobases without disrupting or substituting any nucleotide in the nucleic acid sequence.
  • the second group of the at least one signal system is covalently attached to probe A.
  • the solution does not comprise enzymes, i.e. the solution is an enzyme-free solution.
  • An enzyme is a substance produced by a living organism, which acts as a catalyst to bring about a specific biochemical reaction. That the solution is enzyme-free means that no such catalyst is used in the transcription and amplification steps as described herein.
  • the at least one signal system formed from the first group and the second group is a quencher-fluorophore signalling system, a Forster resonance energy transfer (FRET) signalling system, a gold-nanoparticle signalling system, or a florescent nanoparticle signalling system.
  • FRET Forster resonance energy transfer
  • a quencher refers to a substance that decreases the fluorescence intensity of a given substance, while a fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
  • Forster resonance energy transfer refers to a mechanism describing energy transfer between two light-sensitive molecules.
  • Gold-nanoparticles are small gold particles with a diameter of 1 to 100 nm, while florescent nanoparticles are small particles with a diameter of 1 to 100 nm that emit light upon light excitation.
  • the at least one signal system formed from the first group and the second group is a quencher-fluorophore signalling system or a fluorophore-quencher signalling system.
  • the quencher is a selected from a black hole quencher, a dabcyl quencher, Iowa black Dark Quencher; and the fluorophore is selected from a fluorescein, a rhodamine, a cyanine, a coumarin, Alexa fluor (488), Alexa fluor (532), Alexa fluor (546), Alexa fluor (594), Alexa fluor (647), Alexa fluor (660), Alexa fluor (750), ATTO (488), ATTO (532), ATTO (550), ATTO (565), ATTO (590), ATTO (633), ATTO (647N), HEX, ROX, TAMRA, or Texas Red.
  • a black hole quencher is a substance that absorbs excitation energy from a fluorophore.
  • Dabcyls' (a dabcyl quencher is) has an absorbance maximum of 479 nm, and an effective absorbance range of 346-489 nm. It is the preferred quencher for pairing with fluorescent dyes that emit in the blue to green part of the visible range (442-506 nm). The emission spectra of this set of dyes sufficiently overlaps the absorbance spectrum of Dabcyl to allow the latter to quench the fluorescence of the former with a high degree of efficiency.
  • Fluorescein is a fluorophore that has an absorption maximum at 494 nm and emission maximum of 512 nm (in water).
  • Rhodamine is a family of related dyes, a subset of the triarylmethane dyes.
  • Coumarin or 2H-chromen-2-one is an aromatic organic chemical compound with formula C9H6O2. Its molecule can be described as a benzene molecule with two adjacent hydrogen atoms replaced by a lactone-like chain, forming a second sixmembered heterocycle that shares two carbons with the benzene ring.
  • the at least one signal system formed from the first group and the second group is a gold-nanoparticle signalling system.
  • AuNPs gold-nanoparticles
  • the gold nanoparticles are at least 10 nm, such as at least 20 nm, such as at least 30 nm, such as 40 nm, such as 50 nm.
  • the toehold sequence is at least 7 nucleotides, such as at least 8 nucleotides, such as at least 9 nucleotides, such as at least 10 nucleotides, such as at least 11 nucleotides, such as at least 12 nucleotides, such as at least 13 nucleotides, such as at least 14 nucleotides, or such as at least 15 nucleotides.
  • the toehold sequence is at least 9 nucleotides. In one or more embodiments, the toehold sequence is at least 15 nucleotides.
  • the toehold sequence is no more than 25 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, or such as no more than 16 nucleotides.
  • the toehold sequence is between 7 and 25 nucleotides, such as between 10 and 20 nucleotides, such as between 14 and 16 nucleotides.
  • the recognition strand d domain is a toehold sequence composed partially of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • the toehold sequence is at least 9 nucleotides.
  • the recognition strand d domain is a toehold sequence composed partially or fully of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof, and the toehold sequence is at least 9 nucleotides, such as 15 nucleotides.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • the recognition strand d domain is a toehold sequence composed partially of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof, and the toehold sequence is at least 9 nucleotides.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • the a and a’ domain sequences are both between 4 and 10 nucleotides, such as between 5 and 9 nucleotides, such as between 6 and 8 nucleotides.
  • the b and b’ domain sequences are both between 5 and 11 nucleotides, such as between 6 and 10 nucleotides, such as between 7 and 9 nucleotides.
  • the c and c’ domain sequences are both between 10 and 20 nucleotides, such as between 12 and 18 nucleotides, such as between 14 and 16 nucleotides.
  • the sample collector is a nasopharyngeal swab sample collector for collecting a nasopharyngeal swab sample from a patient.
  • the kit further comprises lysis and extraction solutions for lysing and extracting the total viral RNA from the sample, such as the nasopharyngeal swab sample.
  • the detection is colorimetric detection of the viral RNA sequence in the sample.
  • the detection is enzyme-free colorimetric detection of the viral RNA sequence.
  • the detection is isothermal colorimetric detection of the viral RNA sequence, such as colorimetric detection at isothermal conditions selected from between 25 and 50 °C.
  • Isothermal conditions is conditions wherein.
  • An example of such as an isothermal process which is a thermodynamic process in which the temperature of a system remains constant. The transfer of heat into or out of the system happens so slowly that thermal equilibrium is maintained.
  • a detection time from contact between the solution and the viral RNA sequence to a readout is between 20 and 60 minutes, such as between 25 and 40 minutes, such as between 25 and 35 minutes.
  • contact between the solution and the viral RNA sequence to a readout is meant that from the time the solution is mixed with a sample to be analysed for the viral RNA sequence until the time the solution is able to give a readout.
  • the viral RNA sequence is extracted from swab specimens.
  • the viral RNA sequence is comprised in a cell lysate.
  • the viral RNA sequence is composed of at least 85 nucleotides, such as at least 100 nucleotides, such as at least 150 nucleotides, such as at least 200 nucleotides. If a short target RNA like miRNA (19-23 nt) is targeted then the first part, the transcription, is excluded and the assay would be faster (20 min) and cheaper (no need for reporter T/INA). This is advantageous compared to PCR, as the PCR has challenges to detect short nucleic acids due to the low melting temperature of short nucleic acids making it difficult to precisely design f/w primers.
  • the sample comprises less than 1 nM of the viral RNA sequence, such as less than 100 fM of the viral RNA sequence, such as less than 50 fM of the viral RNA sequence, or such as less than 10 fM of the viral RNA sequence.
  • the solution is for use in the in vitro diagnosis of SARS-CoV-2.
  • Figure 1 shows a schematic diagram of an isothermal strand migration and amplification (NISMA) assay as disclosed according to some embodiments.
  • NISMA isothermal strand migration and amplification
  • Figure 2 shows a schematic diagram of another NISMA assay as disclosed according to some embodiments.
  • Figure 3 shows a PAGE analysis illustrating the amplification part of the NISMA assay.
  • Figure 5 shows a) typical curves illustrating the kinetics of the fluorescence signal of the assay, designed for N gene, over time for serial dilutions of the template from 300 nM down to 300 aM. Scr denotes for the scrambled sequence, b) Plotting the linear response of the assay (signal readout at 20 min) over different template concentrations ranging from 3 fM to 300 nM for b) N gene and c) RdRP gene.
  • Figure 6 shows a PAGE analysis showing the transcription of mimic SARS-2 DNA into the template.
  • Figure 7 shows a plotting of the FAM fluorescence signal of the NISMA assay on RdRP and N genes comprising different reporters.
  • FIG. 1 shows a schematic diagram of an isothermal strand migration and amplification (NISMA) assay as disclosed according to some embodiments as disclosed herein.
  • the NISMA assay is a one-pot assay with all the reagents added simultaneously in a single tube, which simplifies the operation procedures and avoids contamination.
  • the key components of the assay include a “reporter” (the duplex initiation helix) and two “probes” (probe M1 and probe M2) that consist of molecular beacon structures.
  • the reporter is a dsDNA composed of a nucleic acid strand with a toehold, such as a locked nucleic acid (LNA) strand with a toehold, partially complementary to the “template” DNA strand, and completely complementary to the viral RNA/DNA.
  • the probe M1 is labeled with a signaling system, such as a fluorophore and a quencher, at the end of the stem. In the absence of the viral RNA, the spontaneous interactions between the reporter and probes are kinetically blocked, and there is no fluorescence signal as the fluorophore and quencher are in close vicinity.
  • the workflow of the NISMA assay is as following. Firstly, e.g. a nasopharyngeal swab sample is collected from the patient. Next, the sample is lysed and total RNA is extracted. When viral RNA is present (Fig. 1 - “Transcription”, left panel), it hybridizes with the LNA (the recognition strand) through the strand displacement process initiated by the toehold sequence, a step called toehold- mediated exchange or toehold medicated strand displacement. It results in the release of the template strand, such that we are able to transcribe the long viral RNA into the short DNA template. This step is critical, as the subsequent signal amplification is much more efficient with the short template.
  • the template then hybridizes with the molecular beacon structure of probe A (hairpin structure M1) (Fig. 1 “Amplification”, right panel).
  • the hairpin structure of M1 is opened, and the b' sequence is accessible to function as a toehold for the subsequent hybridization with the b sequence on probe B (hairpin structure M2).
  • the M2 displaces the initial template by hybridization with M1 to form a more stable duplex structure.
  • the displaced template acts as a fuel to open another M1 hairpin and initiating a new hybridization cycle. The process is termed as toehold-mediated signal amplification.
  • Unfolding probe A results in a distinct change in the fluorescence intensity, as the fluorophore and quencher are brought further apart. After a 30 minute reaction at 42 °C, more M1/M2 pairs are formed, and the fluorescence intensity of the solution increases significantly (see the examples, e.g. figure 4). Based on the intensity shift, the positive and negative test results can be easily distinguished by an optical reader.
  • FIG 2 shows a schematic diagram of another NISMA assay, where colorimetric detection is by using probe A and probe B functionalized with gold nanoparticles (AuNPs), i.e., in this setup, the two probes (probe A and probe B) that consist of molecular beacon structures (M1 and M2) are immobilized on the AuNPs.
  • AuNPs gold nanoparticles
  • the M2 displaces the initial template by hybridization with M1 to form a more stable duplex structure.
  • LSPR localized surface plasma resonance
  • N isothermal strand migration and amplification
  • the NISMA mechanism comprises two parts of “transcription” and “amplification”. At first, it was made sure that the amplification part on a short template sequence would work properly. Optimum, 22 nt DNA sequence identical to the RdRP and N genes were chosen for the template and M1 and M2 were subsequently designed. The rational design of the M1 and M2 was prerequisite to eradicate their interaction in the absence of the template. As shown in the PAGE analysis of figure 3, from the left panel lane 5, it can be clearly seen that M1 and M2 did not interact/hybridize with each other before the template invasion. Lane 4 shows that the M1 was easily opened and hybridized with template. Interestingly, template could not also open the M2 in the absence of M1 (lane 6).
  • the probe M1 was labeled with FAM/BHQ-1 (M1_fam/bhq) while the probe M2 was unmodified.
  • 24 pl of reaction mixtures composed of M1_fam/bhq (9 ng/pL), M2 (35 ng/pL) and the reporter (T/INA 1 15 ) (150 ng/pL) were prepared and incubated with 15 pl of SARS- CoV-2 RNA extracted from patient oropharyngeal (OP) swab samples.
  • the reaction temperature was fixed at the optimum 42 ⁇ 1 °C and the fluorescence signal arising from FAM was recorded over time.
  • FIG. 6 demonstrates a typical PAGE analysis on the transcription of mimic DNA into the template using T/INA 1 - 15 (harboring high affinity 15 nt toehold sequence). It is clear that the reporter (T/INA 1 - 15 ) is well purified, as there is no band corresponding to the template or INA 1 - 15 in the lane 3. However, incubation of T/INA 1 - 15 with the mimic DNA resulted in a higher band in the gel (lane 5) to demonstrate the formation of mimic DNA/INA 1 - 15 following by the successful displacement of the template.
  • the NISMA assay on N gene showed faster and more sensitive performance, which might be due to the presence of the N gene at the far 3’ end region of the SARS-CoV-2 RNA genome to facilitate the access of reporter to its sequence of interest.
  • a solution comprising: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a’, and c domain; wherein the first group of the at least one signal system is covalently attached to probe A and the second
  • a solution for colorimetric detection of a viral RNA sequence comprising: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an a, b, c, b’, and a’ domain and M2 is a nucleotide sequence having a, b, c’, b’, a’, and c domain; wherein the first group of the at least one
  • the recognition strand d domain is a toehold sequence composed partially or fully of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof. 4.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • the at least one signal system formed from the first group and the second group is a quencher-fluorophore signalling system, a Forster resonance energy transfer (FRET) signalling system, a gold-nanoparticle signalling system, or a florescent nanoparticle signalling system.
  • FRET Forster resonance energy transfer
  • the quencher is a selected from a black hole quencher, a dabcyl quencher, or an Iowa black Dark Quencher; and the fluorophore is selected from a fluorescein, a rhodamine, a cyanine, a coumarin, Alexa fluor (488), Alexa fluor (532), Alexa fluor (546), Alexa fluor (594), Alexa fluor (647), Alexa fluor (660), Alexa fluor (750), ATTO (488), ATTO (532), ATTO (550), ATTO (565), ATTO (590), ATTO (633), ATTO (647N), HEX, ROX, TAMRA, or Texas Red.
  • the quencher is a selected from a black hole quencher, a dabcyl quencher, or an Iowa black Dark Quencher
  • the fluorophore is selected from a fluorescein, a rhodamine, a cyanine, a coumarin, Alexa
  • the gold nanoparticles are at least 10 nm, such as at least 20 nm, such as at least 30 nm, such as 40 nm, such as 50 nm.
  • toehold sequence is at least 7 nucleotides, such as at least 8 nucleotides, or such as at least 9 nucleotides.
  • the toehold sequence is no more than 25 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, or such as no more than 16 nucleotides. 14. The solution according to any of items 1-13, wherein the toehold sequence is between 7 and 25 nucleotides, such as between 10 and 20 nucleotides, such as between 14 and 16 nucleotides.
  • kits for colorimetric detection of a viral RNA sequence comprising the solution according to any of items 1-17 and a sample collector for collecting sample from a patient.
  • sample collector is a nasopharyngeal swab sample collector for collecting a nasopharyngeal swab sample from a patient.
  • kit according to any of items 18-19, wherein the kit further comprises lysis and extraction solutions for lysing and extracting the total viral RNA from the sample, such as the nasopharyngeal swab sample.
  • any of items 21-23 wherein the detection is isothermal colorimetric detection of the viral RNA sequence, such as colorimetric detection at isothermal conditions selected from between 25 and 50 °C. 25.
  • a detection time from contact between the solution and the viral RNA sequence to a readout is between 20 and 60 minutes, such as between 25 and 40 minutes, such as between 25 and 35 minutes.
  • the viral RNA sequence is composed of at least 85 nucleotides, such as at least 100 nucleotides, such as at least 150 nucleotides, such as at least 200 nucleotides.
  • the sample comprises less than 1 nM of the viral RNA sequence, such as less than 100 fM of the viral RNA sequence, such as less than 50 fM of the viral RNA sequence, or such as less than 10 fM of the viral RNA sequence.
  • a detection time from contact between the solution and the viral RNA sequence to a readout is between 20 and 60 minutes, such as between 25 and 40 minutes, such as between 25 and 35 minutes.
  • the viral RNA sequence is comprised in a cell lysate.
  • the viral RNA sequence is composed of at least 85 nucleotides, such as at least 100 nucleotides, such as at least 150 nucleotides, such as at least 200 nucleotides.
  • the sample comprises less than 1 nM of the viral RNA sequence, such as less than 100 fM of the viral RNA sequence, such as less than 50 fM of the viral RNA sequence, or such as less than 10 fM of the viral RNA sequence.
  • a method for colorimetric detection of a viral RNA sequence comprises the steps of: providing a sample to be analysed for the viral RNA sequence; lysing the provided sample and extracting the total viral RNA; adding the extracted total viral RNA to a solution for colorimetric detection of the viral RNA sequence, wherein the solution comprises: a double stranded (duplex) initiation helix, wherein the initiation helix comprises two strands, a template strand having an a’, b’, and c’ domain and a recognition strand having an a, b, c, and d domain, wherein the template strand is fully complimentary to the recognition strand, and wherein the recognition strand d domain is a toehold sequence; at least one signal system formed from a first group and a second group; at least two probes A and B, wherein probe A is a hairpin structure M1 and probe B is a hairpin structure M2, wherein M1 is a nucleotide sequence having an
  • the sample to be analysed for the viral RNA sequence comprises less than 1 nM of total viral RNA, such as less than 100 fM of total viral RNA, such as less than 50 fM of total viral RNA, or such as less than 10 fM of total viral RNA.
  • the recognition strand d domain is a toehold sequence composed partially or fully of Locked Nucleic Acid (LNA), Intercalating Nucleic Acids (INA), or combinations hereof.
  • LNA Locked Nucleic Acid
  • INA Intercalating Nucleic Acids
  • the solution does not comprise enzymes, i.e. the solution is an enzyme-free solution.
  • the at least one signal system formed from the first group and the second group is a quencher-fluorophore signalling system, a Forster resonance energy transfer (FRET) signalling system, a gold- nanoparticle signalling system, or a florescent nanoparticle signalling system.
  • FRET Forster resonance energy transfer
  • the quencher is a selected from a black hole quencher, a dabcyl quencher, or an Iowa black Dark Quencher; and the fluorophore is selected from a fluorescein, a rhodamine, a cyanine, a coumarin, Alexa fluor (488), Alexa fluor (532), Alexa fluor (546), Alexa fluor (594), Alexa fluor (647), Alexa fluor (660), Alexa fluor (750), ATTO (488), ATTO (532), ATTO (550), ATTO (565), ATTO (590), ATTO (633), ATTO (647N), HEX, ROX, TAMRA, or Texas Red.
  • the quencher is a selected from a black hole quencher, a dabcyl quencher, or an Iowa black Dark Quencher
  • the fluorophore is selected from a fluorescein, a rhodamine, a cyanine, a coumarin, Alexa
  • the gold nanoparticles are at least 10 nm, such as at least 20 nm, such as at least 30 nm, such as 40 nm, such as 50 nm.
  • toehold sequence is at least 7 nucleotides, such as at least 8 nucleotides, or such as at least 9 nucleotides.
  • toehold sequence is no more than 25 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, or such as no more than 16 nucleotides.
  • toehold sequence is between 7 and 25 nucleotides, such as between 10 and 20 nucleotides, such as between 14 and 16 nucleotides.

Abstract

L'invention concerne une solution permettant de détecter une séquence d'ARN viral, la solution comprenant une hélice d'initiation double brin (duplex), au moins un système de signaux et au moins deux sondes A et B ayant des structures en épingle à cheveux. L'invention concerne en outre un kit comprenant ladite solution, et l'utilisation de ladite solution pour par exemple détecter de l'ARN viral dans un échantillon.
PCT/EP2021/085677 2020-12-21 2021-12-14 Solution permettant de détecter une séquence d'arn viral WO2022136023A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112029910A (zh) * 2020-09-30 2020-12-04 东南大学 SARS-CoV-2病毒核酸检测方法

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN112029910A (zh) * 2020-09-30 2020-12-04 东南大学 SARS-CoV-2病毒核酸检测方法

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