Patent Application
20240352522
The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 647144).
This invention relates to methods for detecting the presence or absence of target nucleic acid in a sample.
Nucleic acid detection is central to a wide variety of scientific techniques and underpins various detection methods, such as detection of pathogens, genetic conditions and gene expression levels.
Traditional methods for nucleic acid detection typically require amplification of target nucleic acid prior to detection, and so are inherently susceptible to errors induced by enzyme variety, nonspecific amplification of target, amplification biases and limited multiplexing capacity.
The nucleic acid ‘RNA’ is one of four major classes of biomacromolecules in the cell and is a core element of gene expression and gene expression regulation. In addition to its importance for cellular physiology, double and single-stranded RNA forms the genomic material of numerous viruses, including major human pathogenic viruses such as Influenza, Zika, Ebola and SARS-COV-2 which have single-stranded RNA genomes.
Methods traditionally used for detection of RNA include RNA sequencing, Northern blot, and quantitative polymerase chain reaction (qPCR). qPCR is a gold standard method for RNA detection that relies on the real-time fluorescence monitoring of amplified target RNA. In addition to above-mentioned problems associated with target nucleic acid amplification prior to detection, qPCR also requires intensive sample preparation and labour intensive optimization.
As highlighted by the Covid-19 pandemic, there is an urgent need for faster and more reliable nucleic acid detection methods which are less reliant on skilled users and are better-suited to multiplexing, such as for detection of viral RNA and RNA transcripts.
The inventors have overcome the above problems by identifying a novel method for detecting the presence or absence of target nucleic acid(s) in a sample. In more detail, the inventors discovered that the presence or absence of target nucleic acids can be determined efficiently and with a high degree of specificity and sensitivity by excising and detecting specific target probe(s) from the target nucleic acid. Advantageously, the method of the invention avoids the need for intensive sample preparation and/or amplification of target nucleic acid(s).
The method of the invention is rapid and can be readily multiplexed allowing the detection of multiple target nucleic acids in a single reaction. The high level of sensitivity and specificity achieved by the invention enables target nucleic acid detection, and optionally quantification, even when present at low concentrations.
The invention provides a method for detecting the presence or absence of a target nucleic acid in a sample, the method comprising the steps of: (a) contacting the sample with a cutting reagent for excising a target probe from the target nucleic acid to provide an excise mixture; (b) contacting the excise mixture with a nucleic acid carrier comprising a capture oligonucleotide that is complementary to the target probe; and (c) detecting binding of the target probe to the capture oligonucleotide; wherein binding of the target probe to the capture oligonucleotide indicates presence of the target nucleic acid in the sample, and the absence of binding of the target probe to the capture oligonucleotide indicates absence of the target nucleic acid in the sample.
In one embodiment, the cutting reagent comprises: (a) cutting oligonucleotides which are complementary to target nucleic acid sequences immediately upstream and immediately downstream of the target probe, and (b) an enzyme suitable for cutting the target nucleic acid at sites hybridised to cutting oligonucleotides.
In one embodiment, the target nucleic acid is RNA, optionally wherein the target nucleic acid is selected from single-stranded RNA, double-stranded RNA, mRNA, miRNA, and non-coding RNA.
In one embodiment, the target nucleic acid is single-stranded RNA and the cutting oligonucleotides comprise single-stranded DNA. In one embodiment, the enzyme suitable for cutting the target nucleic acid at sites hybridised to cutting oligonucleotides is ribonuclease H (RNase H).
In one embodiment, the nucleic acid carrier is a single stranded DNA (ssDNA) carrier. In one embodiment, the nucleic acid carrier comprises one or more reference labels that allow the identity of the nucleic acid carrier, the location of the capture oligonucleotides, and/or the identity of the capture oligonucleotides to be determined.
In one embodiment, the nucleic acid carrier comprises more than one capture oligonucleotide complementary to different target probes, optionally wherein the different target probes are derived from different target nucleic acids.
In one embodiment, the capture oligonucleotide binds to a signalling oligonucleotide in the absence of target probe. In one embodiment, the signalling oligonucleotide is displaced from the capture oligonucleotide in the presence of target probe. In one embodiment, the capture oligonucleotide comprises an overhang that is complementary to the target probe but is not complementary to the signalling oligonucleotide, and wherein in the presence of target probe, the target probe binds to the overhang and displaces the signalling oligonucleotide from the capture oligonucleotide.
In one embodiment, detecting binding of the target probe to the capture oligonucleotide comprises detecting binding of the capture oligonucleotide to the signalling oligonucleotide.
In one embodiment, the signalling oligonucleotide comprises a structural, chemical and/or fluorescent label. In one embodiment, the signalling oligonucleotide comprises a ligand label, and optionally wherein the method further comprises contacting the nucleic acid carrier with a receptor that interacts with the ligand.
In one embodiment, the ligand is biotin and the receptor is avidin, neutravidin, traptavidin or streptavidin, and wherein detecting binding of the capture oligonucleotide to the signalling oligonucleotide comprises detecting the presence of biotin, avidin, neutravidin, traptavidin, streptavidin and/or biotin/avidin, biotin/neutravidin, biotin/traptavidin or biotin/streptavidin complexes.
In one embodiment, the ligand is an antigen and the receptor is an antibody, and wherein detecting binding of the capture oligonucleotide to the signalling oligonucleotide comprises detecting the presence of antigen and/or antigen/antibody complexes.
In one embodiment, the capture oligonucleotide comprises a fluorescent label and the signalling oligonucleotide comprises a quencher and wherein detecting binding of the target probe to the capture oligonucleotide comprises detecting the presence or absence fluorescence.
In one embodiment, binding of the target probe to the capture oligonucleotide is detected using nanopore-based detection methods.
In one embodiment, binding of the target probe to the capture oligonucleotide is detected by spectroscopic-based detection methods.
In one embodiment, the method further comprises quantifying the level of target nucleic acid in the sample by quantifying the level of binding of the target probe to the capture oligonucleotide and/or by quantifying the level of binding of the signalling oligonucleotide to the capture oligonucleotide.
In one embodiment, the method comprises detecting the presence or absence of more than one target nucleic acid in the sample.
In one embodiment, the method comprises excising more than one target probe from the target nucleic acid, optionally from more than one target nucleic acid.
In one embodiment, the target probe has a GC content of 40-60%. In one embodiment, the target probe comprises a terminal region that has a GC content of 40-60%, optionally wherein the terminal region of the target probe is 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 30 nt, 40 nt, or 50 nt starting from the 3′ and/or the 5′ end of the target probe.
In one embodiment, the target probe has less than 80% sequence identity to other sequences that may be present in the sample, such as other regions of the target nucleic acid.
In one embodiment, the target probe is located in an unhybridized region of the target nucleic acid.
In one embodiment, the target nucleic acid is derived from a virus, optionally wherein the virus is selected from a coronavirus, Influenza virus, Zika virus, Ebola virus, Dengue virus, Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, Flavivirus, and Alphavirus. In one embodiment, the target nucleic acid is a coronavirus genome, optionally the SARS-COV-2 genome.
In one embodiment, the target nucleic acid is derived from a microorganism, optionally wherein the target nucleic acid is derived from a bacteria or a fungi.
In one embodiment, the target nucleic acid is derived from a pathogen, optionally wherein the pathogen is a viral pathogen, bacterial pathogen or a fungal pathogen.
In one embodiment, the target nucleic acid is an RNA transcript.
In one embodiment, the target nucleic acid is a therapeutic nucleic acid, optionally wherein the therapeutic nucleic acid is selected from siRNA, shRNA, miRNA, RNA aptamer, DNA aptamer, mRNA, splice-switching oligonucleotides, antisense oligonucleotides, RNA decoys and peptide nucleic acids.
In one embodiment, the target nucleic acid is a genetic biomarker, optionally wherein the target nucleic acid is selected from a gene, an RNA transcript or a region thereof. In one embodiment, the genetic biomarker is associated with a disease or condition, optionally wherein the disease or condition is cancer or an increased risk thereof, or a hereditary disease or condition.
In one embodiment, the target nucleic acid comprises a single nucleotide variant when compared to a reference nucleic acid.
In one embodiment, the sample is obtained from a subject that has been treated with a therapeutic. In one embodiment, the method comprises comparing the level of target nucleic acid in the sample to the level present in a sample from a subject who has not been treated with the therapeutic. In one embodiment, the target nucleic acid is an RNA transcript.
In one embodiment, the sample is obtained from a subject, optionally wherein the subject is a human. In one embodiment, the sample is selected from blood, serum, plasma, saliva, sputum, urine, faeces, cerebrospinal fluid, a lung tissue sample, a bronchoalveolar lavage sample, a nose and/or throat swab sample, or a biopsy sample.
The invention provides a method for detecting the presence or absence of target nucleic acid(s) in a sample. The methods of the invention are rapid and exhibit a high degree of sensitivity and specificity. Advantageously, the methods of the invention can be readily multiplexed allowing the simultaneous detection and quantification of multiple target nucleic acids in a single reaction. Moreover, the methods of the invention can advantageously differentiate between closely related sequences in a single sample, e.g. viral or genetic variants such as single-nucleotide variants.
Nucleic acid detection methods known in the art often rely on amplification of the target nucleic acid, or a region thereof. Amplification, e.g. by polymerase chain reaction (PCR), typically requires intensive sample preparation and may exhibit amplification biases, whereby some targets are more efficiently amplified than others. As a result, the specificity and sensitivity of the method varies depending on the target. Amplification biases also reduce the reliability of quantitative nucleic acid detection because targets that are more efficiently amplified appear to be more abundant relative to targets with lower amplification efficiency.
The inventors have overcome these problems by developing a nucleic acid detection method that relies on the detection of specific probes that are excised from the target nucleic acid, without requiring amplification. The target nucleic acid can be detected, and optionally quantified, rapidly and with a high degree of specificity and sensitivity. Advantageously, avoiding amplification of the target nucleic acid avoids the need for intensive sample preparation and reduces the potential for errors introduced by nonspecific amplification and amplification biases.
Existing nucleic acid detection methods may also rely on random fragmentation of nucleic acids to produce shorter fragments which are then detected. During random fragmentation, e.g. by chemical fragmentation or non-specific enzymatic cleavage, all nucleic acids present in a sample (i.e. target and non-target nucleic acids) are non-specifically fragmented, resulting in an abundance of nucleic acid fragments. An abundance of nucleic acid fragments is problematic because, e.g.: (i) it leads to an increased risk of off-target binding between non-target fragments and detection reagents, thereby reducing the specificity of the method and increasing the potential for false-positive results; and (ii) it increases the potential for hybridisation between fragments, thereby preventing or reducing interaction of target nucleic acid(s) with detection reagents, in turn reducing the sensitivity of the method and increasing the potential for false-negative results. Non-specific fragmentation also risks fragmentation of target nucleic acids, thereby reducing the sensitivity and reproducibility of the method, and further increasing the potential for false-negative results.
In overcoming problems with existing methods, the inventors designed a method for excising specific probes (herein “target probe(s)”) from target nucleic acids prior to detection. By limiting the fragmentation of nucleic acids present in a sample, the present invention advantageously reduces the potential for off-target binding with detection reagents and significantly improves the specificity and sensitivity of detection. Excision of specific probes ensures that nucleic acid fragments are generated only in the presence of target nucleic acid. Limiting the generation of non-target probes allows the methods of the invention to retain a high level of sensitivity, even in the presence of total DNA and/or RNA isolated from a patient sample. The methods of the invention also ensure that the target probes have a well-defined sequence allowing detection reagents (nucleic acid carriers and capture oligonucleotides) to be designed with a high level of specificity and reproducibility.
The target probes are designed to maximise the efficiency of: (i) excision of the target probe from the target nucleic acid by the cutting reagents; and (ii) detection of the target probe by capture oligonucleotides. Although existing nucleic acid detection methods often rely on the detection of well-conserved regions of target sequences, the inventors found that probes located in well-conserved regions can exhibit low excision efficiency, thereby reducing the sensitivity of the method. The present invention enables considerable flexibility in designing and optimising selection of target probes and is not restricted to the detection of well-conserved regions of target sequences. Target probes for use in the invention are typically designed for efficient excision from the target nucleic acid and/or for efficient recognition by detection reagents, thereby ensuring a high level of sensitivity and enabling quantification of target nucleic acid(s). A high level of sensitivity ensures that the methods of the invention can reliably detect target nucleic acids at low abundance.
The inventors identified several criteria that may be used to identify target probes that are efficiently excised and detected by the methods of the invention. In some embodiments, target probes have a guanine-cytosine (GC) content of 40-60%. In some embodiments, target probes comprise a terminal region that has a GC content of 40-60%. In some embodiments, target probes have a GC content of 40-60% and a terminal region that has a GC content of 40-60%. A GC content of 40-60% was identified by the inventors as being advantageous because it helps ensure that the probe and the corresponding capture oligonucleotide establish a stable interaction.
In some embodiments, target probes have a high specificity, i.e. low similarity to other nucleic acids that may be present in the sample. Selecting target probes that have high specificity limits the potential for cross-hybridization between the target probe and other nucleic acids. Limiting cross-hybridisation advantageously increases the sensitivity of the methods of the invention because target probes that are excised from the target nucleic acid remain unhybridized and free to interact with detection reagents. Low similarity to other nucleic acids typically means that the target probe has a low level of sequence identity relative to other nucleic acids that may be in the sample, including other regions of the target nucleic acid.
In some embodiments, target probes are located in (or excised from) regions of the target nucleic acid that are unstructured, e.g. regions of the target nucleic acid that are, or are predicted to be, unhybridized. Typically, target probes are located in regions of the target nucleic acid that have the highest likelihood of being unhybridized regions based on published or predicted 3D structures of a target nucleic acid. For single stranded RNA (ssRNA) targets, target probes are typically located in unstructured regions including RNA loops, RNA bulges, and unpaired RNA segments.
The invention provides a method for detecting the presence or absence of a target nucleic acid in a sample. The method comprises contacting the sample with a cutting reagent for excising a target probe from the target nucleic acid to provide an excise mixture.
In some embodiments, the cutting reagent comprises: (i) cutting oligonucleotides which are complementary to regions of the target nucleic acid that are directly upstream and downstream of the target probe sequence; and (ii) an enzyme suitable for cutting target nucleic acid hybridised to the cutting oligonucleotides. Cutting oligonucleotides rely on specific base pairing interactions with complementary regions of the target nucleic acid to ‘flank’ the target probe sequence. Enzymatic cutting of the target nucleic acid-cutting oligonucleotide hybrid results in excision of the target probe from the target nucleic acid.
In some embodiments, the target nucleic acid is a ssRNA target and the cutting reagent comprises single stranded DNA (ssDNA) cutting oligonucleotides and ribonuclease H (RNase H). In the presence of ssRNA target, ssDNA cutting oligonucleotides bind to complementary regions of the ssRNA upstream and downstream of the target probe resulting in the formation of RNA:DNA hybrid regions upstream and downstream of the target probe sequence. RNase H cuts RNA in these RNA:DNA hybrid regions by hydrolysing RNA phosphodiester bonds, thereby excising the target probe from the ssRNA target.
Advantageously, the specificity of the cutting reagents allows pre-determined, specific probes to be excised from the target nucleic acid while reducing or avoiding the generation of non-specific nucleic acid fragments which can otherwise interfere with target probes and/or capture oligonucleotides and reduce the sensitivity and/or specificity of the method.
When target nucleic acid is present in the sample, excision of the target probe(s) can occur and so the excise mixture contains excised target probe(s). In the absence of target nucleic acid, the excise mixture does not contain target probe.
The excise mixture may be treated prior to being contacted with the nucleic acid carrier, e.g. to remove or denature enzyme and/or to separate the target probe from longer nucleic acids, such as nucleic acids that are more than 100 nucleotides (nt), more than 200 nt, more than 500 nt, more than 1000 nt, or more than 2000 nt in length. Typically, the target probes are not purified in the excise mixture.
The sample is contacted with cutting reagent under conditions that allow: (i) the cutting oligonucleotides to bind to complementary regions of the target nucleic acid; and (ii) the enzyme to cut the target nucleic acid where the cutting oligonucleotides are bound. The conditions may comprise different phases, e.g. a first phase that allows the cutting oligonucleotides to bind to complementary regions of the target nucleic acid and a second phase that allows the enzyme to cut the target nucleic acid where the cutting oligonucleotides are bound. The cutting oligonucleotide binding phase may comprise incubating the sample with cutting oligonucleotides at a temperature that is optimal for cutting oligonucleotides to anneal to the target nucleic acid. The temperature will vary depending on the nature of the target nucleic acid and cutting oligonucleotides used. The enzymatic cutting phase may comprise incubating at a temperature that is within the optimal activity range for that enzyme, but which does not result in dissociation of the cutting oligonucleotides from the target nucleic acid.
For example, when the enzyme is RNase H, the mixture may be incubated at a temperature in the range of 20-95° C., e.g. at 25-85° C., 25-80° C., 25-75° C., 25-70° C., 25-65° C., 25-60° C., 25-55° C., 25-50° C., 25-45° C., 25-40° C., 30-85° C., 30-80° C., 30-75° C., 30-70° C., 30-65° C., 30-60° C., 30-55° C., 30-50° C., 30-45° C., 30-40° C., 35-40° C. or 36-38° C. In one embodiment, the mixture is incubated at 37° C. Following excision of the target probe, the conditions may further comprise an inactivation phase comprising incubation under conditions that result in the enzyme being inactivated, e.g. to prevent unwanted enzymatic activity during subsequent method steps.
The terms “probe” and “target probe” are used interchangeably herein to refer to a specific region of the target nucleic acid that is excised from the target nucleic acid and subsequently detected. Reference to “probe” and “probes” should be understood to encompass both a singular probe and multiple (i.e. more than one) probes, unless otherwise indicated.
In some embodiments, the target probe(s) are 10-100 nucleotides (nt) in length, for example 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-50 nt, 10-40 nt, 10-35 nt, 10-30 nt, 10-25 nt, 10-20 nt, 15-100 nt, 15-90 nt, 15-80 nt, 15-70 nt, 15-60 nt, 15-50 nt 15-40 nt, 15-35 nt, 15-30 nt, 15-25 nt, 15-20 nt, 20-100 nt, 20-90 nt, 20-80 nt, 20-70 nt, 20-60 nt, 20-50 nt 20-40 nt, 20-35 nt, 20-30 nt, 20-25 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt in length. In some embodiments, the target probe(s) are 20 nt in length.
The methods of the invention may comprise excising and detecting one probe from a single target nucleic acid. The methods of the invention may comprise excising and detecting one probe from more than one target nucleic acid, e.g. one probe from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 different target nucleic acids. For example, the methods of the invention may comprise excising and detecting one probe from more than one target nucleic acid, e.g. one probe from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 different target nucleic acids.
The methods of the invention may comprise excising and detecting more than one probe from a single target nucleic acid. For example, the methods of the invention may comprise excising and detecting 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 probes from a single target nucleic acid. For example, the methods of the invention may comprise excising and detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 probes from a single target nucleic acid.
The method of the invention may comprise excising and detecting more than one probe from more than one target nucleic acid. For example, the methods of the invention may comprise excising and detecting 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 probes from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 different target nucleic acids. For example, the methods of the invention may comprise excising and detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 probes from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 different target nucleic acids.
To optimise sensitivity, the inventors identified several criteria for designing probes that are susceptible to optimal excision and detection. In some embodiments, target probes: (a) have a guanine-cytosine (GC) content of 40-60%; (b) comprise a terminal region that has a GC content of 40-60%; (c) have a high specificity, i.e. low similarity to other nucleic acids that may be present in the sample to limit cross-hybridization; and/or (d) are located in unstructured (unhybridized) regions of the target nucleic acid.
The invention provides a method of identifying target probes for use in the method of the invention, comprising identifying regions of the target nucleic acid sequence that: (a) have a GC content of 40-60%; (b) comprise a terminal region that has a GC content of 40-60%; (c) have a high specificity, i.e. low similarity to other nucleic acids that may be present in the sample to limit cross-hybridization; and/or (d) are located in unstructured, i.e. unhybridized, regions of the target nucleic acid.
The GC content is the percentage of bases in a nucleic acid molecule that are either guanine (G) or cytosine (C). A probe with a GC content of 40-60% indicates that 40-60% of the bases forming the probe are guanine or cytosine. In some embodiments, probes have a GC content of 40-60%. In some embodiments, probes have a GC content of 40-55%, 40-50% 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, 50-55%, 40%, 45%, 50%, 55% or 60%. In some embodiments, probes have a GC content of less than 40%. In some embodiments, probes have a GC content of more than 60%.
A probe comprising a terminal region that has a GC content of 40-60% means one or both terminal regions of the probe have a GC content of 40-60%. In some embodiments, probes comprise a terminal region with a GC content of 40-55%, 40-50% 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, 50-55%, 40%, 45%, 50%, 55% or 60%. In some embodiments, probes comprise a terminal region with a GC content of less than 40%. In some embodiments, probes comprise a terminal region with a GC content of more than 60%.
In some embodiments, the probe terminal region comprises 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 30 nt, 40 nt, or 50 nt starting from either the 3′ and/or the 5′ end of the probe. In some embodiments, the probe terminal region comprises 4 nt starting from either the 3′ or the 5′ end of the probe. In some embodiments, the probe terminal region comprises 6 nt starting from the 5′ end of the probe.
In some embodiments, the probe has less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 20% sequence identity to other nucleic acids that may be present in the sample, such as other regions of the target nucleic acid.
In some embodiments, the target nucleic acid is RNA and the target probe is located in an RNA loop, RNA bulge, or an unpaired RNA segment.
Target probes are excised from the target nucleic acid by the cutting reagent. In some embodiments, the cutting reagent comprises: (i) cutting oligonucleotides which are complementary to regions of the target nucleic acid that are directly upstream and downstream of the target probe; and (ii) an enzyme suitable for cutting target nucleic acid hybridised to the cutting oligonucleotides. The cutting reagent may comprise multiple cutting oligonucleotides that are complementary to regions upstream and downstream of multiple probes. The cutting reagent may comprise cutting oligonucleotides that are complementary to the full length of the target nucleic acid except the target probe(s).
The terms “cutting oligos” or “cutting oligonucleotides” are used interchangeably herein to refer to short, single stranded nucleic acids which are complementary to regions of the target nucleic acid directly upstream and downstream of the probe. Cutting oligonucleotides base-pair (anneal) with complementary sequences of the target nucleic acid and render these annealed regions susceptible to enzymatic cutting.
In some embodiments, the cutting oligonucleotides are 10-50 nucleotides (nt) in length. In some embodiments, the cutting oligonucleotides are 10-40 nt, 10-35 nt, 10-30 nt, 10-25 nt, 10-20 nt, 15-40 nt, 15-35 nt, 15-30 nt, 15-25 nt, 15-20 nt, 20-40 nt, 20-35 nt, 20-30 nt, 20-25 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt in length. In some embodiments, the cutting oligonucleotides are 20 nt in length. In some embodiments, the cutting oligonucleotides are more than 50 nt in length, e.g. more than 100 nt, more than 500 nt, more than 1000 nt, more than 1500 nt, or more than 2000 nt in length.
In some embodiments, the cutting oligonucleotides are complementary to the 10-50 nt of the target nucleic acid directly upstream of the target probe. In some embodiments, the cutting oligonucleotides are complementary to the 10-50 nt of the target nucleic acid directly downstream of the target probe. In some embodiments, the cutting reagent comprises cutting oligonucleotides complementary to the 10-50 nt of the target nucleic acid directly upstream of the target probe and cutting oligonucleotides complementary to the 10-50 nt of the target nucleic acid directly downstream of the target probe. In some embodiments, the cutting oligonucleotides are complementary to the 10-40 nt, 10-35 nt, 10-30 nt, 10-25 nt, 10-20 nt, 15-40 nt, 15-35 nt, 15-30 nt, 15-25 nt, 15-20 nt, 20-40 nt, 20-35 nt, 20-30 nt, 20-25 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt of the target nucleic acid directly upstream and/or directly downstream of the target probe. In some embodiments, the cutting oligonucleotides are complementary to the 20 nt of the target nucleic acid directly upstream and directly downstream of the target probe. In some embodiments, the cutting oligonucleotides are complementary to the full length of the target nucleic acid with the exception of the target probe. The cutting oligonucleotides are not complementary to the target probe.
In some embodiments, the enzyme suitable for cutting target nucleic acid hybridised to the cutting oligonucleotides is a nuclease. In some embodiments, annealing of cutting oligonucleotides to the target nucleic acid forms a double stranded region that is susceptible to cutting by the nuclease. In some embodiments, the target nucleic acid comprises ssRNA, the cutting oligonucleotides comprise ssDNA and the enzyme comprises ribonuclease, e.g. ribonuclease H. When annealed to ssRNA, ssDNA cutting oligonucleotides form RNA:DNA hybrid regions which are recognised and cut by RNase H. RNase H nucleases are non-sequence specific endonucleases which hydrolyse RNA phosphodiester bonds in RNA:DNA hybrids. Cutting of such RNA:DNA hybrid regions by RNase H results in excision of specific target probes from the target nucleic acid.
In some embodiments, cutting oligonucleotides are linked to a nuclease (any suitable linkage may be employed). In such an embodiment, binding of the cutting oligonucleotides to the complementary regions of the target nucleic acid brings the nuclease into close proximity with the target nucleic acid which then excises the probe. In some embodiments, the nuclease is a CRISPR associated (Cas) endonuclease. In some embodiments, the target probe is cut from that target nucleic acid using double specific cleavage using CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) systems. In this embodiment, the cutting reagent comprises cutting oligos (guide nucleic acids) complexed with Cas nucleases.
In some embodiments, the cutting reagent comprises DNAzymes (deoxyribozymes or DNA enzymes). DNAzymes are single-stranded DNA molecules with catalytic capabilities which can be employed as site-specific RNA cutting enzymes. DNAzymes have two binding arms and a middle catalytic domain that cuts at a specific RNA position. Binding arms can be designed that are specific to regions downstream and upstream of the target probe.
The method comprises contacting the excise mixture with a nucleic acid carrier. The nucleic acid carrier is typically a single stranded nucleic acid to which capture oligonucleotide(s) are bound. In some embodiments, the nucleic acid carrier is a DNA carrier. In some embodiments, the nucleic acid carrier is an RNA carrier. The nucleic acid carrier may be a single stranded DNA (ssDNA) carrier. Nucleic acid carriers may be 100 nucleotides (nt), 200 nt, 500 nt, 1000 nt, 2000 nt, 3000 nt, 4000 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10,000 nt or more in length.
The capture oligonucleotide(s) are complementary to the target probe(s). In some embodiments, the capture oligonucleotide(s) comprises a nucleotide that is not complementary to the target probe (i.e. there is a mismatch). In some embodiments, the capture oligonucleotide(s) comprises two, three, four, five, or more mismatches to the target probe.
In some embodiments, the nucleic acid carrier comprises one capture oligonucleotide. In some embodiments, the nucleic acid carrier comprises more than one capture oligonucleotides that are complementary to the same or different target probes. Typically, each capture oligonucleotide is complementary to a single target probe.
In some embodiments, the nucleic acid carrier comprises a single capture oligonucleotide that is complementary to a single target probe. In some embodiments, the nucleic acid carrier comprises more than one capture oligonucleotide complementary to a single target probe e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 capture oligonucleotides which are complementary to a single target probe. In some embodiments, the nucleic acid carrier comprises more than one capture oligonucleotide complementary to a single target probe e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 capture oligonucleotides which are complementary to a single target probe.
In some embodiments, the nucleic acid carrier comprises capture oligonucleotides that are complementary to single target probes from more than one target nucleic acid, e.g. single target probes from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500 or 1000 target nucleic acids. In some embodiments, the nucleic acid carrier comprises more than one capture oligonucleotide complementary to different probes from a single target nucleic acid, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 different probes from a single target nucleic acid. The nucleic acid carrier may comprise more than one capture oligonucleotides that are complementary to more than one probe from more than one target nucleic acid, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 different probes from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 target nucleic acids.
In some embodiments, the nucleic acid carrier comprises capture oligonucleotides that are complementary to single target probes from more than one target nucleic acid, e.g. single target probes from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500 or at least 1000 target nucleic acids. In some embodiments, the nucleic acid carrier comprises more than one capture oligonucleotide complementary to different probes from a single target nucleic acid, e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 different probes from a single target nucleic acid. The nucleic acid carrier may comprise more than one capture oligonucleotides that are complementary to more than one probe from more than one target nucleic acid, e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 different probes from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, or at least 1000 target nucleic acids.
The nucleic acid carrier may comprise one or more reference label(s), e.g. structural, chemical or fluorescent label(s). The reference label(s) may be used to identify the nucleic acid carrier and/or to identify the location and/or the identity of capture oligonucleotides.
Capture oligonucleotides typically comprise a region that is complementary to the nucleic acid carrier and a region that is complementary to the target probe. When the region complementary to the nucleic acid carrier is bound thereto, the region complementary to the target probe forms an overhang (i.e. unhybridized) region.
In some embodiments, capture oligonucleotides are 10-100 nucleotides (nt) in length. In some embodiments, capture oligonucleotides are 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-50 nt, 10-40 nt, 10-35 nt, 10-30 nt, 10-25 nt, 10-20 nt, 15-100 nt, 15-90 nt, 15-80 nt, 15-70 nt, 15-60 nt, 15-50 nt, 15-40 nt, 15-35 nt, 15-30 nt, 15-25 nt, 15-20 nt, 20-100 nt, 20-90 nt, 20-80 nt, 20-70 nt, 20-60 nt, 20-50 nt, 20-40 nt, 20-35 nt, 20-30 nt, 20-25 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt in length. Typically, the region of the capture oligonucleotide that is complementary to the target probe is the same length as the corresponding target probe.
In some embodiments, capture oligonucleotides are bound to signalling oligonucleotides that are complementary thereto to form capture-signalling hybrids. In some embodiments, signalling oligonucleotides are 10-100 nt in length. In some embodiments, signalling oligonucleotides are 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-50 nt, 10-40 nt, 10-35 nt, 10-30 nt, 10-25 nt, 10-20 nt, 15-100 nt, 15-90 nt, 15-80 nt, 15-70 nt, 15-60 nt, 15-50 nt, 15-40 nt, 15-35 nt, 15-30 nt, 15-25 nt, 15-20 nt, 20-100 nt, 20-90 nt, 20-80 nt, 20-70 nt, 20-60 nt, 20-50 nt, 20-40 nt, 20-35 nt, 20-30 nt, 20-25 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt in length. In some embodiments, signalling oligonucleotides are 14 nt in length.
The capture oligonucleotide typically has a higher affinity for the target probe than for the signalling oligonucleotide. The affinity between two oligonucleotides may be determined e.g. by the dissociation constant (Kd) wherein a lower dissociation constant indicates a higher binding affinity. Two sequences that are fully complementary have a higher binding affinity than two sequences that contain mismatches (i.e. non-complementary nucleotides). Thus, in some embodiments, the capture-signalling hybrid contains at least one mismatch. When there is a mismatch between the target probe and the capture oligonucleotide, particularly the toehold region of the capture oligonucleotide, the strand displacement reaction is slowed because the formation of strand displacement (i.e. the interaction between the probe and the capture-signalling duplex) is destabilised. As a result, probes comprising mismatches can be distinguished from probes that are fully complementary based on the efficiency of strand displacement.
Wherein signalling oligonucleotide is present, the presence of target probe causes displacement of the signalling oligonucleotide from the capture oligonucleotide and/or prevents binding of the signalling oligonucleotide to the capture oligonucleotide. In such embodiments, capture oligonucleotides preferentially interact with target probes via base pairing interactions to form capture-probe hybrids.
Wherein signalling oligonucleotide is present, the absence of target probe (e.g. when the sample does not contain target nucleic acid) typically results in hybridisation of signalling oligonucleotide to the capture oligonucleotide to form capture-signalling hybrids.
Wherein signalling oligonucleotide is not present, the presence of target probe typically results in formation of capture-probe hybrids.
Wherein signalling oligonucleotide is not present, the absence of target probe (e.g. when the sample does not contain target nucleic acid) typically results in unhybridized capture oligonucleotides.
In some embodiments, the capture oligonucleotide in the capture-signalling hybrid comprises an overhang that is complementary to a region (typically a terminal region) of the target probe. An “overhang” refers to at least one unpaired nucleotide. The overhang may be referred to as a toehold region herein. In the presence of target probe, the target probe interacts with the overhang region of the capture oligonucleotide in the capture-signalling hybrid and subsequent base-pairing between complementary bases of the target probe and capture oligonucleotide result in displacement of the signalling oligonucleotide (referred to herein as strand displacement). Displacement of the signalling oligonucleotide results in the formation of capture-probe hybrids. In some embodiments, the capture oligonucleotide comprises a 5′ overhang. In some embodiments, the capture oligonucleotide comprises a 3′ overhang. In some embodiments, the capture oligonucleotide comprises a 5′ overhang and a 3′ overhang. In some embodiments, the overhang is 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 30 nt, 40 nt, or 50 nt in length. In some embodiments, the overhang is 4 nt in length.
In some embodiments, capture oligonucleotides are contacted with signalling oligonucleotides after being contacted with the excise mixture. In such embodiments, signalling oligonucleotides may bind to capture oligonucleotides that are not already bound to complementary target probes, to form capture-signalling hybrids.
Signalling oligonucleotides comprise labels that can be detected using suitable methods known in the art, e.g. spectroscopic, photochemical, or microscopic methods. In some embodiments, the signalling oligonucleotide comprises a fluorescent label, a chemical label and/or a structural label. In some embodiments, the signalling oligonucleotide comprises a label selected from a nucleic acid nanostructure (e.g. nucleic acid nanostructure, nucleic acid origami structure, hairpin structure or multi-hairpin structure), a ligand (e.g. biotin or an antigen), an enzyme, a radioactive tag and/or a fluorescent tag.
The method of the invention comprises detecting binding of the target probe to the capture oligonucleotide; wherein binding of the target probe to the capture oligonucleotide indicates presence of the target nucleic acid in the sample, and the absence of binding of the target probe to the capture oligonucleotide indicates absence of the target nucleic acid in the sample.
The method may comprise detecting binding between the probe and capture oligonucleotide directly, e.g. by detecting the presence or absence of unhybridized (i.e. single stranded) and/or hybridized (i.e. double stranded) capture oligonucleotide(s). In such embodiments, the presence of unhybridized capture oligonucleotide(s) indicates the absence of target probe (and thus absence of the target nucleic acid in the sample), and the presence of hybridized capture oligonucleotides (i.e. capture-probe hybrids) indicates the presence of target probe (and thus presence of the target nucleic acid in the sample).
The method may comprise detecting binding between the probe and capture oligonucleotide indirectly, e.g. by detecting the presence or absence of capture-signalling hybrids. Wherein signalling oligonucleotide is present, absence of capture-signalling hybrids indicates presence of the target probe (and thus presence of the target nucleic acid in the sample). Presence of capture-signalling hybrids indicates absence of the target probe (and thus absence of the target nucleic acid in the sample). The skilled person will understand that the method used to detect the presence or absence of capture-signalling hybrids is dependent on the signalling oligonucleotide label.
In some embodiments, the signalling oligonucleotide is conjugated to a ligand. In this embodiment, presence or absence of capture-signalling hybrids may be determined by detecting the presence or absence of the ligand. In some embodiments, the amount of ligand is quantified and correlated to the amount of target probe and/or the amount of target nucleic acid in the sample.
In some embodiments, the signalling oligonucleotide is conjugated to a ligand and the method further comprises contacting the nucleic acid carrier with a receptor that is specific for said ligand. In this embodiment, presence or absence of capture-signalling hybrids may be determined by detecting the presence or absence of ligand/receptor complexes. In some embodiments, the amount of ligand/receptor complex is quantified and correlated to the amount of target probe and/or the amount of target nucleic acid in the sample.
In some embodiments, the signalling oligonucleotide comprises a biotinylated nucleotide. In some embodiments, the signalling oligonucleotide is covalently attached to biotin. In these embodiments, the method may comprise contacting the nucleic acid carrier with avidin, neutravidin, traptavidin or streptavidin. In this embodiment, the presence or absence of capture-signalling hybrids is determined by detecting the presence or absence of biotin/avidin, biotin/neutravidin, biotin/traptavidin or biotin/streptavidin complexes. Biotin/avidin, biotin/neutravidin, biotin/traptavidin or biotin/streptavidin complexes may be detected using nanopore based methods as described herein.
As used herein, references to avidin encompass neutravidin, traptavidin and streptavidin, and vice versa. Avidin, neutravidin, traptavidin and streptavidin for use in the methods of the invention are typically monomeric or monovalent, although multimeric forms (e.g. divalent trivalent or tetravalent) may also be employed.
In some embodiments, the signalling oligonucleotide is conjugated to an antigen and the method comprises contacting the nucleic acid carrier with an antibody specific for the antigen. In this embodiment, presence or absence of capture-signalling hybrids is determined by detecting the presence or absence of antigen/antibody complexes. In some embodiments, the presence or absence of antigen/antibody complexes is determined by nanopore-based methods.
In some embodiments, the signalling oligonucleotide is conjugated to an enzyme and the method comprises contacting the nucleic acid carrier with a substrate specific for the enzyme. In this embodiment, presence or absence of capture-signalling hybrids is determined by detecting the presence or absence of enzymatic reaction product(s). In some embodiments, the enzyme is horseradish peroxidase (HRP) and substrate is selected from 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5,5′-Tetramethylbenzidine (TMB), and 3,3′-Diaminobenzidine (DAB). Suitable methods for identifying and quantifying the products of HRP enzyme reactions are known in the art. For example, HRP converts chromogenic substrates, including TMB, DAB and ABTS, into coloured products which can be detected using spectrophotometric methods. The intensity of signal produced by the enzyme substrate may be correlated to the abundance of target probe, and therefore the abundance of target nucleic acid in the sample.
In some embodiments, the capture oligonucleotide is conjugated to a fluorophore and the signalling oligonucleotide is conjugated to a quencher. When the capture oligonucleotide is bound to the signalling oligonucleotide, the fluorophore and quencher are in close proximity and the fluorophore is quenched, i.e. does not fluoresce. In the presence of target probe, the signalling oligonucleotide is displaced and the fluorophore and quencher are separated resulting in the fluorophore fluorescing. In some embodiments, the fluorophore is a fluorescein, such as 6-carboxyfluorescein (6-FAM), and the quencher is Dabcyl or tetramethylrhodamine. The presence or absence of fluorescence can be detected by methods known in the art, e.g. fluorescence spectroscopy. The presence of fluorescence indicates that the signalling oligonucleotide has been displaced or prevented from binding to the capture oligonucleotide, thereby indicating the presence of capture-probe hybrids. The intensity of fluorescence may be quantified and correlated to the abundance of probe, and therefore to the abundance of target nucleic acid in the sample.
The presence or absence of capture-signalling hybrids may be detected using nanopore-based methods. In nanopore-based detection methods, an ionic current passes through the nanopore due to an applied potential. When nucleic acid carriers translocate through a nanopore, a current signature or current trace is produced which corresponds to the current level detected over time as the nucleic acid carrier translocates through the nanopore. The current signature may be compared to a negative control (e.g. a current trace produced by the nucleic acid carrier in the absence of target probe and/or presence of signalling oligonucleotide); and/or to a positive control (e.g. a current trace produced by the nucleic acid carrier in the presence of target probe and/or absence of signalling oligonucleotide). A current signature or current trace may also be referred to herein as an event or a nanopore event.
The nucleic acid carrier may comprise one or more reference labels or identification labels that produce an identifiable signal in the current trace. Reference labels, and the current signals they produce, may be used to locate and/or identify capture oligonucleotides. This is advantageous because it allows capture-signalling hybrids to be differentiated, even when the same signalling labels are used. Identification labels, and the current signals they produce, may be used to identify the nucleic acid carrier. Advantageously, identification labels allow nucleic acid carriers from multiple reactions to be combined in a single nanopore-based detection assay.
Wherein a nanopore is used for detecting binding of the target probe to the capture oligonucleotide, the type of nanopore used will depend on whether the binding is being detected directly (e.g. by identifying single stranded capture oligonucleotides and double stranded capture-probe hybrids) or indirectly (e.g. by detecting capture-signalling hybrids). The nanopore may be a solid state or a biological nanopore. In some embodiments, the nanopore is a glass nanopore. For direct detection of capture-probe binding, nanopores with a diameter of about 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm are typically used. For example, direct detection of capture-probe binding, nanopores with a diameter of about 3 nm-about 10 nm, about 3 nm-about 9 nm, about 3 nm-about 8 nm, about 3 nm-about 7 nm, about 3 nm-about 6 nm, about 3 nm-about 5 nm, about 3 nm-about 4 nm, about 4 nm-about 10 nm, about 4 nm-about 9 nm, about 4 nm-about 8 nm, about 4 nm-about 7 nm, about 4 nm-about 6 nm, about 4 nm-about 5 nm, about 5 nm-about 10 nm, about 5 nm-about 9 nm, about 5 nm-about 8 nm, about 5 nm-about 7 nm, about 5 nm-about 6 nm, about 6 nm-about 10 nm, about 6 nm-about 9 nm, about 6 nm-about 8 nm, about 6 nm-about 7 nm, about 7 nm-about 10 nm, about 7 nm-about 9 nm, about 7 nm-about 8 nm, about 8 nm-about 10 nm, about 8 nm-about 9 nm, or about 9 nm-about 10 nm, are typically used. For indirect detection of capture-probe binding, nanopores with a diameter of about 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm are typically used. For example, for indirect detection of capture-probe binding, nanopores with a diameter of about 10 nm-about 20 nm, about 10 nm-about 19 nm, about 10 nm-about 18 nm, about 10 nm-about 17 nm, about 10 nm-about 16 nm, about 10 nm-about 15 nm, about 10 nm-about 14 nm, about 10 nm-about 13 nm, about 10 nm-about 12 nm, about 10 nm-about 11 nm, about 11 nm-about 20 nm, about 11 nm-about 19 nm, about 11 nm-about 18 nm, about 11 nm-about 17 nm, about 11 nm-about 16 nm, about 11 nm-about 15 nm, about 11 nm-about 14 nm, about 11 nm-about 13 nm, about 11 nm-about 12 nm, about 12 nm-about 20 nm, about 12 nm-about 19 nm, about 12 nm-about 18 nm, about 12 nm-about 17 nm, about 12 nm-about 16 nm, about 12 nm-about 15 nm, about 12 nm-about 14 nm, about 12 nm-about 13 nm, about 13 nm-about 20 nm, about 13 nm-about 19 nm, about 13 nm-about 18 nm, about 13 nm-about 17 nm, about 13 nm-about 16 nm, about 13 nm-about 15 nm, about 13 nm-about 14 nm, about 14 nm-about 20 nm, about 14 nm-about 19 nm, about 14 nm-about 18 nm, about 14 nm-about 17 nm, about 14 nm-about 16 nm, about 14 nm-about 15 nm, about 15 nm-about 20 nm, about 15 nm-about 19 nm, about 15 nm-about 18 nm, about 15 nm-about 17 nm, about 15 nm-about 16 nm, about 16 nm-about 20 nm, about 16 nm-about 19 nm, about 16 nm-about 18 nm, about 16 nm-about 17 nm, about 17 nm-about 20 nm, about 17 nm-about 19 nm, about 17 nm-about 18 nm, about 18 nm-about 20 nm, about 18 nm-about 19 nm, or about 19 nm-about 20 nm are typically used. The skilled person will readily understand that the diameter of the nanopore used will depend on the size of the label that is associated with the signalling oligonucleotide.
A biological nanopore may be a transmembrane protein nanopore. Examples of transmembrane protein pores include β-barrel pores and α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-strands. β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin (α-HL), anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) and other pores, such as lysenin. α-helix bundle pores comprise a barrel or channel that is formed from α-helices. α-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as WZA and ClyA toxin. A biological nanopore may be a transmembrane pore derived from or based on MspA, α-HL, lysenin, CsgG, ClyA, or haemolytic protein fragaceatoxin C (FraC).
Examples of transmembrane pores derived from or based on MspA are described in WO 2012/107778. Examples of transmembrane pores derived from or based on α-hemolysin are described in WO 2010/109197. Examples of transmembrane pores derived from or based on lysenin are described in WO 2013/153359. Examples of transmembrane pores derived from or based on CsgG are described in WO 2016/034591 and WO 2019/002893. Examples of transmembrane pores derived from or based on ClyA are described in WO 2017/098322. Examples of transmembrane pores derived from or based on FraC are described in WO 2020/055246.
The nanopore may be a DNA origami pore. Examples of DNA origami pores are described in WO 2013/083983, WO 2018/011603, and WO 2020/025974.
The nanopore may be a solid state nanopore. Examples of solid state nanopores are described in WO 2016/127007.
In nanopore-based detection methods, signalling oligonucleotides typically comprise a structural label that produces an identifiable current signal, i.e. reduction in current, when translocated through the nanopore. The number of capture-signalling hybrids that are detected may be quantified and correlated to the amount of target probe and/or target nucleic acid in the sample.
In some embodiments, signalling oligonucleotides comprise a biotin label and capture-signalling hybrids are detected by detecting the presence or absence of biotin using nanopore-based detection methods. In some embodiments, the capture oligonucleotides are further contacted with streptavidin, neutravidin, traptavidin or avidin and capture-signalling hybrids are detected by detecting the presence or absence of biotin/streptavidin, biotin/neutravidin, biotin/traptavidin or biotin/avidin complexes using nanopore-based detection methods.
In some embodiments, signalling oligonucleotides comprise an avidin, neutravidin, traptavidin or streptavidin label and capture-signalling hybrids are detected by detecting the presence or absence of avidin, neutravidin, traptavidin or streptavidin using nanopore-based detection methods. In some embodiments, the capture oligonucleotides are further contacted with biotin and capture-signalling hybrids are detected by detecting the presence or absence of biotin/streptavidin, biotin/neutravidin, biotin/traptavidin or biotin/avidin complexes using nanopore-based detection methods.
The size and duration of current blockages (as represented by reduction(s) in current) can be used to differentiate between different signalling labels because the size of the current blockage is typically relative to the size of the label (larger labels typically produce larger peaks/greater reductions in current, and vice versa). Multiple labels corresponding to multiple different capture-signalling hybrids can therefore be identified in a single reaction.
The superior sensitivity of the method of the invention advantageously allows for quantitative detection of target nucleic acids. As described in detail above, the methods of the invention comprise detecting the presence or absence of target probes which (if present) are derived directly from the target nucleic acid. Advantageously, the abundance of target probes is not altered, (e.g. by prior amplification) in the method of the invention and so the abundance of target probe corresponds directly to the abundance of target nucleic acid.
The methods of the invention may comprise calibrating the level of capture-probe or capture-signalling hybrids with the abundance of target probe present in the sample. The sample may be contacted with a known amount of capture oligonucleotides and the difference between the level of capture-signalling hybrids present in the sample and the level present in a negative control (e.g. in the absence of target probe) may be used to determine the abundance of target probe, and therefore the abundance of target nucleic acid in the sample. For example, a 50% reduction in the level of capture-signalling hybrids relative to the negative control indicates that 50% of capture oligonucleotides have interacted with probe to form capture-probe hybrids. The number of capture-probe hybrids detected corresponds to the abundance of target nucleic acid present in the sample.
Advantageously, the method of the invention can be readily adapted to allow detection of target nucleic acids in a wide variety of concentration ranges. Probes can be designed to optimise the dynamic range (the range of target nucleic acid concentration that can be detected) by altering: (i) the toehold length wherein a shorter toehold length typically increases the dynamic range; (ii) the GC content wherein a lower GC content increases the dynamic range; (iii) the length of the probe wherein shorter probes increase the dynamic range; and/or (iv) alternating the position of the toehold wherein a 5′ toehold exhibits faster displacement than a 3′ toehold and therefore decreases the dynamic range. Decreasing the efficiency of strand displacement typically increases the dynamic range of the assay because a more concentrated sample is required to achieve equivalent strand displacement rates.
The methods of the invention can be readily multiplexed by employing signalling oligonucleotides with distinguishable labels or by differentiating between different capture oligonucleotides using nucleic acid carrier reference labels. In these embodiments, the relative and/or absolute abundance of multiple target nucleic acid(s) can be measured in a single reaction.
As used herein, the term “target nucleic acid” encompasses a single target nucleic acid and multiple (i.e. more than one) target nucleic acids. The target nucleic acid may comprise RNA, e.g. single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), or DNA, e.g. single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), or combinations thereof. The target nucleic acid may be messenger RNA (mRNA), microRNA (miRNA), non-coding RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) or ribosomal RNA (rRNA). The target nucleic acid may be autosomal DNA, or mitochondrial DNA. The target nucleic acid may be a naturally occurring or synthetic nucleic acid.
When detecting the presence or absence of double-stranded target nucleic acid, the method may further comprise denaturing the target nucleic acid to produce single-stranded nucleic acid prior to contacting the target nucleic acid with cutting reagents.
The method of the invention may be used to detect the presence or absence of more than one target nucleic acid in a sample. For example, the method of the invention may be used to detect the presence or absence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1000 target nucleic acids in a sample.
In some embodiments, the sample contains the target nucleic acid. In some embodiments, the sample does not contain target nucleic acid. In some embodiments, the sample comprises non-target nucleic acid(s). The sample may be obtained from a cell culture. The sample may be obtained from a subject.
The subject may be selected from a human or a non-human animal, such as a murine, bovine, equine, ovine, canine, or feline animal. The sample may be selected from the group consisting of, but not limited to, blood, serum, plasma, saliva, sputum, urine, faeces, cerebrospinal fluid, a lung tissue sample, a bronchoalveolar lavage sample, a nose and/or throat swab sample, or a biopsy sample.
The sample may be treated prior to use in the method of the invention. For example, the sample may be treated to lyse cells, remove and/or denature proteins. Nucleic acid extraction may be performed on the sample prior to use in the method of the invention. Suitable nucleic acid extraction methods are known in the art and include methods that extract total DNA and/or RNA from samples.
Methods of the invention may be used to detect the presence or absence of target nucleic acids derived from a pathogen. Presence of a target nucleic acid derived from a pathogen in the sample typically indicates presence of the pathogen in the sample, whereas absence of the target nucleic acid in the sample typically indicates absence of the pathogen in the sample. Methods of the invention may be used to detect target nucleic acid derived from a viral pathogen, a bacterial pathogen, or a fungal pathogen.
The target nucleic acid may be viral nucleic acid, e.g. a viral genome, such as a ssRNA viral genome. The ssRNA viral genome may be derived from a virus selected from an Influenza virus, Zika virus, Ebola virus, coronavirus, Dengue virus, Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, Flavivirus, and Alphavirus. In some embodiments, the target nucleic acid is derived from a coronavirus, such as SARS-CoV-2.
The nucleic acid carrier may comprise multiple capture oligonucleotides that are specific for different probes derived from the same pathogen. Alternatively, the nucleic acid carrier may comprise multiple capture oligonucleotides that are specific for probe(s) derived from different pathogens.
In some embodiments, target probes are designed to detect the presence or absence of a particular pathogen, and/or to detect the presence or absence of particular pathogen variants. For example, probes that are common to a group of pathogens (e.g. SARS-COV-2) may be used to detect the presence or absence of a pathogen within that group; and probes that are specific to a particular variant may be used to detect the presence or absence of different variants.
In some embodiments, methods of the invention are used to quantify the relative abundance of multiple pathogens in the sample. Advantageously, the methods of the invention may be used to identify the predominant pathogen, or pathogen variant, in the sample.
Methods of the invention may be used to detect the presence or absence of a genetic biomarker, e.g. a genetic variant. The genetic biomarker may be associated with a particular disease or condition, or an increased risk thereof. For example, the genetic biomarker may be associated with cancer, or an increased risk thereof. The genetic biomarker may be associated with a hereditary disease or condition. The presence of the genetic biomarker may indicate the presence of a disease or condition, or increased risk of a disease or condition, that is associated with that biomarker. The absence of the genetic biomarker may indicate the absence of a disease or condition, or reduced risk of a disease or condition, that is associated with that biomarker.
In methods for detecting the presence or absence of a genetic biomarker, the target nucleic acid may be the genome of a subject, or a region thereof, a gene or a region thereof, or may be an RNA transcript produced by a gene of interest. The method may comprise detecting the presence or absence, and optionally the relative abundance, of multiple variants of a particular genetic marker in a single reaction.
Nucleic acid carriers may comprise capture oligonucleotides that are specific for all known variants of a genetic marker.
Detection of target nucleic acid(s) relies on specific base-pairing between target probes and capture oligonucleotides which are complementary thereto. Target probes and capture oligonucleotides that are fully complementary establish a more stable interaction than capture-probe pairs containing mismatches. Methods of the invention can therefore be used to differentiate between highly similar sequences, e.g. target nucleic acids containing single nucleotide variants (SNVs). This is particularly advantageous because the method of the invention can be used as a screening method for the detection of SNVs, and SNV-based biomarkers. The region of the capture oligonucleotide(s) that is specific to a particular SNV may be positioned in the toehold overhang region of capture-signalling hybrids.
In some embodiments, the target nucleic acid comprises a SNV when compared to a reference nucleic acid. The reference nucleic acid may be the wild type form of the target nucleic acid. In some embodiments, the target nucleic acid is a gene or a region thereof comprising a SNV when compared to the wild type form of the gene.
Methods of the invention may be used to detect the presence or absence, and optionally the abundance, of RNA transcript(s). Thus, methods of the invention may be used in transcriptomics, i.e. to detect the presence or absence, and optionally the abundance, of RNA transcripts in a sample. The method of the invention may be used to measure the presence or absence, and optionally the abundance, of transcripts derived from a single cell (e.g. single cell transcriptomics).
Methods of the invention may be used to detect the presence or absence, and optionally the abundance, of therapeutic nucleic acids in a sample. Therapeutic nucleic acids are typically administered to a subject to treat a disease or condition. For example, the therapeutic nucleic acid may be selected from siRNA, shRNA, miRNA, RNA or DNA aptamers, mRNA, splice-switching oligonucleotides, antisense oligonucleotides, RNA decoys and peptide nucleic acids. Methods of the invention may be used to detect the presence or absence of therapeutic nucleic acids in samples obtained at different time points and/or from different tissues or locations allowing the pharmacokinetics of the therapeutic nucleic acid to be tracked.
Methods of the invention may also be used to detect the presence or absence, and optionally the abundance, of native nucleic acids in the presence or absence of treatment with a drug candidate designed to target the native nucleic acid. For example, a patient may be administered a drug (e.g. a therapeutic nucleic acid) that is designed to lower the expression of a particular gene or to reduce the abundance of the corresponding gene transcript. The methods of the invention may be used to detect the presence or absence, and optionally the abundance, of the particular gene transcript in samples obtained in the presence or absence of the drug candidate to determine the activity of the drug on the gene transcript. Samples may be obtained from the subject at different time points and/or from different tissues or locations to determine the pharmacokinetics of the drug candidate.
Methods of the invention may be used to screen drug candidates for their ability to reduce or increase the abundance of gene transcripts in a sample. For example, various cell cultures may be treated with putative drug candidates. Following treatment, the methods of the invention may be used to detect the abundance of the gene transcript of interest in the various cell cultures.
The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are in no way limiting.
The inventors developed a method for detecting the presence of SARS-COV-2 and Escherichia virus MS2 target nucleic acids. As shown herein, the inventors designed DNA carriers for detecting the presence or absence of short RNA probes that are recognised and excised from SARS-COV-2 and MS2. The DNA carrier was assembled by annealing capture oligonucleotides that are complementary to regions of SARS-COV-2 and MS2 to single-stranded DNA. The inventors found that multiple RNA probes derived from SARS-COV-2 and MS2 could be detected in-parallel, even at ultralow concentrations. In the present example, ultrasensitive nanopore based detection methods were used to identify the presence or absence of capture-signalling hybrids. The design of DNA carrier structures was verified with AFM imaging and gel electrophoresis.
An overview of the experimental design for viral RNA detection is shown in
The SARS-COV-2 probes and MS2 probes used in the present example are provided in Tables 1 and 2, respectively.
To excise the probes from SARS-COV-2 and MS2 RNA, two complementary cutting oligonucleotides are hybridised upstream and downstream of the probe of interest. Cutting oligonucleotides for the MS2 probes in Table 2 are provided in Table 3, respectively.
To make the DNA carrier, a long linear single-stranded DNA scaffold was annealed with short complementary oligonucleotides (purchased from Integrated DNA Technologies) (Table 22). To prepare the linear scaffold, single-stranded circular M13mp18 DNA (7249 nt, Guild Biosciences, USA) was cleaved by restriction enzymes after binding to a short oligonucleotide (39 nt) which created double-stranded restriction sites (see the protocol details provided in Bell, N. A. W. & Keyser, U. F. Nat. Nanotechnol. 11, 645-651 (2016)). The oligonucleotide set for making a specific design was prepared by mixing the required oligonucleotides (with 200 nM final concentration of each in the mixture).
To produce DNA carriers for the detection of the SARS-COV-2 and MS2, several fully complementary oligonucleotides (i.e. those listed in Table 22) were replaced with capture oligonucleotides which comprise a region complementary to the DNA scaffold and an overhang region complementary to the target probe. Capture oligonucleotides for the SARS-COV-2 and MS2 virus probes and the fully complementary oligonucleotides which they replace are listed in Tables 4 and 5, respectively. The oligonucleotides used to produce reference signals and the corresponding complementary oligonucleotides they replaced are listed in Table 6.
ACTCTTCTTCTTCACAATCA
AACAATTTAGCTCCTTTCTT
TTTCCACAAAAATCAACTCT
AAATAACAGAATAAACACCA
ATACAACCCTCAACTTTACC
GGATATCACTCATTAGTGGT
GAGCATGTTACCTACAGGTA
GAGATGGAATCGGATGCAGA
Signalling oligonucleotides were also added to the DNA carrier assembly mixture. Signalling oligonucleotides correspond to the target probe but lack the first six nucleotides at the 5′ end and the 3′ end comprises a biotin label. The signalling oligonucleotide binds to the complementary region of the capture oligonucleotide to generate capture-signalling hybrids with a 3′ toehold (i.e. overhang) that serves as a seed for the strand displacement reaction and target probe detection.
The biotinylated signalling oligonucleotides which are partially complementary to the overhang region of the capture oligonucleotides listed in Tables 4 and 5 are listed in Tables 7 and 8, respectively.
After preparation of the linear DNA scaffold and the oligonucleotide sets, the DNA carrier was assembled by mixing, heating up and cooling down. First, the solutions were prepared as following: 8 μL cut M13mp18 DNA scaffold (100 nM); 12 μL oligonucleotide mix (each oligo 200 nM, oligos after phosphorylation); 6 μL 100 mM MgCl2; 2.9 μL 100 mM Tris-HCl (pH=8.0), 10 mM EDTA; 11.1 μL Mili-Q water. The mixture was heated to 70° C. for 30 s followed by a linear cooling ramp to 25° C. over 50 minutes.
The annealed DNA carrier sample is then purified using a 100 kDa Amicon filter by mixing the annealed mixture with 460 μL washing buffer (10 mM Tris-HCl (pH=8.0), 0.5 mM MgCl2) and centrifugated at 9,000×G for 10 minutes. This step was repeated twice. The purified DNA carrier was retrieved by spinning for 2 minutes at 1,000×G.
The MS2 probes were cut from ˜3.6 kb MS2 RNA. Firstly, cutting oligonucleotides adjusted to the target probes were annealed to target RNA by mixing the cutting oligonucleotides listed in Table 4 at a concentration of 1 μM in 1×RNase H reaction buffer and with 330 nM of target RNA. The mixture was heated to 65° C. for 5 minutes, then incubated at 21° C. for 5 minutes and immediately transferred to ice.
In the next step, 1.5 units of RNase H was added to mixture. The mix was incubated for 20 minutes at 37° C. to allow RNase H to cut RNA strands in the RNA:DNA hybrid regions where cutting oligonucleotides were bound to the target RNA. The enzyme was temperature-inactivated at 65° C. for 20 minutes.
The strand displacement reaction (SDR) was performed in a buffer that contains 10 mM MgCl2 and 100 mM NaCl with DNA carrier concentration of 6.72 nM and different excess of target probes with known concentration or ten times excess of expected probe concentration after RNase H cutting of MS2 RNA for 10 minutes at room temperature.
All samples were run on a 1% (w/v) agarose gel prepared in fresh 1×TBE buffer with autoclaved Milli-Q water for 180 minutes, at 70V on ice. The RNA sample was loaded on the gel and fresh 1×purple loading dye without SDS (NEB) or 1× orange dye was used. The gel was poststained in 1×GelRed buffer (Biotium) for 10 minutes and imaged with a GelDoc-It™ (UVP).
The presence or absence of target probes was determined using nanopore detection. 14±3 nm glass nanopores were fabricated by pulling quartz capillaries with filaments (0.5 mm outer diameter and 0.2 inner diameters, Sutter Instrument, USA) using a laser-assisted puller (P-2000, Sutter Instruments). DNA carriers were diluted to 0.3-1 nM concentration in 4M LiCl (pH 9.4) with 9 times excess of monovalent streptavidin to each biotin site on DNA carrier and measured using Axopatch 200B current amplifier at the constant voltage of 600 mV. The same filters and the nanopore setup were used as described in Bell, N. A. W. & Keyser, U. F. Nat. Nanotechnol. 11, 645-651 (2016).
Data analysis of nanopore current traces for each sample was performed using LabView algorithms. Briefly, single DNA events were extracted by setting a current drop threshold to 0.8 nA and minimal duration of 0.5 ms and the event charge deficit (ECD) i.e. event area in the range from 50 to 1000 fC. The characteristics of events were determined using the procedure described in Bell, N. A. W. & Keyser, U. F. Nat. Nanotechnol. 11, 645-651 (2016).
Firstly, the structure of the DNA carrier with and without streptavidin-biotin complex was validated. The DNA carrier comprises two labels that are used to identify the location of the capture oligonucleotides. In the positive control (
The displacement of signalling oligonucleotide by target probes (i.e. strand displacement reaction (SDR)) was validated with synthetic SARS-COV-2 RNA and DNA oligonucleotides using nanopore readout. The effect of the probe to DNA carrier ratio on the level of the SDR was assessed for ratios from 0 to 10 times. The SDR mix was incubated for 10 minutes. The occupied fraction refers to the absolute number of detected downward peaks between two references. An occupied fraction of 1 means that the probe is not present, and that the signalling oligonucleotide is not displaced, and an occupied fraction of 0 means that the probe is present, and, in all events, the signalling oligonucleotide is displaced.
The data for RNA and DNA 20 nt target probes are plotted in
The following experiments were performed with constant 10:1 probe to DNA carrier ratio with different incubation times for the SDR. Here, RNA probes (
The specificity of the DNA carrier for target probes was then verified in the complex mixture of human total RNA. For the negative control, the level of the SDR did not significantly change with incubation with 300 ng of human total RNA with ˜2.4 ng of DNA carrier under the same SDR conditions (
RNase H cutting of the viral RNA was optimized with MS2 RNA phage by designing three probes (M1-M3) to be cut from RNA and with corresponding three sites on the DNA carrier (
The efficiency of RNase H cutting was estimated with native 2% (w/v) agarose gel electrophoresis as illustrated in
Methods known in the art for randomly fragmenting RNA include, for example, incubating RNA in the presence of MgCl2 at high temperature. However, incubation of MS2 RNA in 10 mM MgCl2 at 94° C. for 15 minutes was less efficient at fragmenting MS2 RNA to produce short, e.g. 20 nt, probes (
The SDR with cut MS2 probes M1-M5 (see Table 2) were tested with the DNA carrier and nanopores. In ten times excess of MS2 RNA after cutting, displacement of sites varied according to the position of each probe in 3D RNA structure. The blank control (no probes added) has all five sites occupied as indicated in
Based on the results described herein, the inventors identified the following criteria for the optimisation of target probes: (a) a GC content of 40-60%; (b) a terminal region that has a GC content of 40-60%; (c) a high specificity, i.e. low similarity to other nucleic acids that may be present in the sample to limit cross-hybridization; and/or (d) located in unstructured i.e. unhybridized regions of the target nucleic acid.
The method of the invention was used to detect the presence or absence of SARS-COV-2 in the presence of total nucleic acid isolated from a patient swab sample. The method step are as follows:
Results on the detection of SARS-COV-2 sequences in the presence of human total RNA background are shown in
Human patient swab samples that had previously been tested using RT-PCR were incubated with capture oligonucleotides for 10-30 minutes (for positive and negative samples). The results are shown in
Short RNA Probe Cleavage from Long RNA
To test the hypothesis that the position of a probe in a folded RNA affects the free concentration of that probe in the solution, the inventors used MS2 RNA (10165948001 Roche). MS2 RNA is single-stranded and 3569 nucleotide long. The inventors designed multiple short probes based on their position in folded RNA (Dai et al. Nature. 2017; 541 (7635): 112-116, and The Vienna RNA Web suite). Two example probes, M1 and M2, were designed to be in the unpaired RNA region (M1) or the paired RNA region (M2) of MS2.
The probe cutting process is described in Example 1 and includes: annealing cutting DNA oligonucleotides MXA and MXB (where X is 1 or 2) to MS2 RNA; RNaseH cutting of the DNA: RNA hybrid sites; and validating data using polyacrylamide gel electrophoresis (PAGE) and nanopore detection.
The results of cutting for these two probes are shown in
To confirm that the additional band corresponds to the target probe, the inventors added oligonucleotide cM1 (which has a 30 nt tail) which is complementary to the target probe. In the presence of cM1, the gel shift confirms that the additional band indeed corresponds to free target probe M1 (
The same experiments were repeated for the M2 probe (
These results indicate that target probes positioned in an unpaired region (e.g. M1) are more efficiently cleaved and released in solution than target probes positioned in a paired region (e.g. M2). Cleavage and release of target probes into solution is essential for successful detection of target probes by toehold-mediated SDR.
Table 9 demonstrates the occupied fractions for DNA carrier comprising five detection sites for five MS2 20 nt probes (M1-M5) in the presence of different samples. Sample 1 (blank control) indicates that in the absence of probes the occupied fraction is high. Sample 2 (probes M4 and M5 present) and sample 3 (probes M1, M4 and M5 present) indicate a reduced occupied fraction for sites corresponding to the probes present in the sample, indicating detection of these probes.
RNA Probe Cleavage from SARS-COV-2 RNA Isolated from a Patient Sample
Using the same protocol as for MS2 RNA targets the inventors designed three probes (S1, S2, and S3) present in the conserved domain of SARS-COV-2 RNA and predicted to be partially unpaired.
Table 10 demonstrates the occupied fractions for: sample 1 is a negative control with a negative patient swab sample; sample 2 is a positive control with S1, S2 and S3 20 nt probes added instead of patient sample in the estimated similar concentration with the negative patient swab sample 1; and sample 3 is a positive sample with probes S1, S2 and S3 cut from a positive patient samples.
ATCGAAGCGCAGTAAGGATG
AGGCTCCCTCAGTTGCAATG
AGGAGAATTAGTCTGAGTCT
C. elegans miRNA Detection
miRNAs play an essential role in the development of C. elegans and it is described in the literature that more than one miRNA is necessary to influence development. The method of the invention was used to detect miRNAs in C. elegans. Five miRNAs (miR-58, miR-1, miR-71, miR-70, miR-72) that have previously been described to play a role in the development of C. elegans (Kato, M. et al. Genome Biol 10, R54 (2009)) were analysed using the methods of the invention. Results on the detection of the miRNA sequences in the presence of C. elegans total RNA background are shown in
Simultaneous identification of multiple respiratory viruses and SARS-COV-2 single-nucleotide variants Simultaneous detection of multiple viruses is of great clinical relevance for respiratory virus diagnostics. Firstly, respiratory viruses can have a similar clinical manifestation and co-infection with multiple viruses are common and increase the severity and mortality of respiratory diseases. The inventors employed a DNA carrier for multiplexed target identification of five different respiratory viruses including SARS-COV-2, influenza A type, Respiratory Syncytial Virus (RSV), parainfluenza A type, and rhinoviruses (sequences are listed in Tables 15-17, and design principles are provided below). The design of DNA carriers for multiple respiratory viruses/viral groups is shown in
The ability to detect and track viral variants is important for both clinical decision making and epidemiology. A particular difficulty when detecting viral variants is that they may deviate from the wild-type virus by only a single nucleotide variation. To confirm that the methods of the invention could detect and differentiate between single nucleotide variants (SNVs), the inventors designed DNA carriers to detect emerging SARS-COV-2 variants (sequences are listed in Tables 18-21, and design principles are provided below). The DNA carrier has five capture oligonucleotide sites specific to a reference strain first isolated in Wuhan (B by PANGOLIN nomenclature); European strain B.1; and three variants of the European strain defined as variants of concern B.1.1.7 (alpha), and B.1.351 (beta), and B.1.617 (delta) first detected in United Kingdom, South Africa, and India, respectively (see
DNA carrier for multiple respiratory viruses is prepared as described above. Capture oligo sequences (Table 17), biotin signalling strand sequences (Table 16), and target probe sequences (Table 15) for each virus are provided below. Target probes for Influenza A, Parainfluenza A, and Rhinoviruses are common to a group of these viruses rather than a single variant.
DNA carrier for multiple SARS-COV-2 variants is prepared as described above. Capture oligo sequences (Table 21), biotin signalling strand sequences (Table 19), and target probes sequences for wildtype (Table 20) and variants (Table 18) are provided below.
GTATGAAAATGCCTTTTTAC
CCGGTATAGATTGTTTAGGA
GGTGTTAACTGCACAGAAGT
CTTATGGTGTTGGTTACCAA
TAAAGGTTTTAATTGTTACT
CCTGTATAGATTGTTTAGGA
GATGTTAACTGCACAGAAGT
CTAATGGTGTTGGTTACCAA
TGAAGGTTTTAATTGTTACT
DNA carriers were designed to detect the presence of wild type SARS-COV-2 and the presence of two single nucleotide and single amino acid variants, N501T and N501S. DNA carriers for SARS-COV-2 N501 RNA virus variants was prepared as described herein. The sequences of SARS-COV-2 N501 RNA, capture oligos, biotinylated strand, and cutting oligos for RNase H cutting are provided in Table 23.
For nanopore sensing, SARS-COV-2 RNA (S:N501 in Table 23) controls were used for the detection with DNA carriers. Firstly, cutting oligos were mixed with a SARS-COV-2 N501 RNA in a ratio 1:1:1 and the mixture was heated to 70° C. for 5 minutes. RNase H (5,000 units/ml, NEB) was added, mixed, and heated for 20 minutes at 37° C. to allow the enzyme to cut RNA in the DNA: RNA hybrid to effectively releases target RNA probes. RNase H was thermally inactivated by incubation at 65° C. for 10 min.
DNA carrier was mixed with cut SARS-COV-2 RNA (S:N501 in Table 23, protocol see above) at ten times excess in 10 mM MgCl2 and 100 mM NaCl. The mixture (5 μL) was incubated at room temperature (˜10 min) until prepared for nanopore measurement.
Nanopore readouts confirmed that the SARS-COV-2 capture oligos were specific to the variants they were designed to identify. The N501T target probe displaced the signalling oligo annealed to the N501T capture oligo, and not the N501S or the wild-type capture oligo as evidenced by the presence of the N501S and wild-type signals in the presence of N501T target probe (
These results further confirm that the method of the invention can be used to discriminate between RNA probes containing single-nucleotide variants (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2112088.6 | Aug 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/052171 | 8/23/2022 | WO |
