Patent Application
20200299754
The present invention relates to compositions and methods for the detection of target molecules, and the amplification of detectable signals generated by detection assays. More specifically, the present invention relates to methods utilizing catalytic nucleic acid enzymes to generate and/or amplify a signal indicative of the presence of target molecules (e.g. nucleic acids and proteins), and compositions for use in the methods.
A number of assays are currently available for the detection of target nucleic acids or other molecules in a sample. Most assays rely on the use of protein enzymes, whilst a few exploit nucleic acid enzymes which catalyse modifications to nucleic acids as discussed below.
Catalytic Nucleic Acid Enzymes
Catalytic nucleic acid enzymes are non-protein enzymes capable of modifying specific substrates. Catalytic nucleic acid enzymes include DNA molecules (also known in the art as a DNAzyme, deoxyribozyme, or DNA enzyme), RNA molecules (also known in the art as a ribozyme), and multi-component nucleic acid enzymes composed of multiple DNA and/or RNA molecules (also known in the art as an MNAzyme or PlexZyme). Catalytic nucleic acid enzymes can modify specific nucleic acid substrate sequences by, for example, cleavage or ligation. A unique class of catalytic nucleic acid enzymes (known in the art as a horseradish peroxidase-mimicking DNAzyme) can catalyse peroxidase reactions that convert specific chemical substrates into their oxidated products which can for example, produce a change in colour or emit a fluorescent or chemiluminescent signal.
DNAzymes and ribozymes, capable of cleaving or ligating RNA substrates, DNA substrates and/or chimeric DNA/RNA substrates, can generally only modify a target nucleic acid substrate that meets minimum sequence requirements. For example, the substrate should exhibit sufficient base pair complementarity to the substrate binding arms of the enzyme, and also needs a specific sequence at the site of catalytic modification. Examples of such sequence requirements at the catalytic cleavage site include the requirement for a purine:pyrmidine sequence for DNAzyme cleavage (10-23 type), the requirement for a dinucleotide junction (8-17 type) and the requirement for the sequence uridine:X where X can equal A, C or U but not G, for the hammerhead ribozymes. The 10-23 DNAzyme is a DNAzyme that is capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds. This DNAzyme has a catalytic domain of 15 deoxyribonucleotides flanked by two substrate-recognition domains (arms). The 8:17 DNAzyme differs from the 10:23 DNAzyme in both the catalytic core region and cleavage site preference. The 8-17 DNAzyme does not have any stringent substrate sequence requirements and is capable of cleaving nucleic acid substrates at all four types of 5′ NG dinucleotide junctions (i.e 5′ GG, AG, CG and UG). There is an overall hierarchy of reactivity between groups of related junctions that roughly follow the order 5′ NG>NA>NC>NT where NG junctions are the most susceptible to cleavage and pyrimidine-pyrimidine junctions are the least susceptible. The 8-17 DNAyme has a catalytic domain of 14-15 deoxynucleotides and is characterized by a short intramolecular 3 base-pair stem-triloop and a 4-5 single stranded deoxunucleotide region.
Multi-component nucleic acid enzymes (MNAzymes, also known as PlexZymes) are another category of catalytic nucleic acid enzyme. These enzymes require an assembly facilitator (e.g. a target nucleic acid) for their assembly and catalytic activity. MNAzymes are composed of multiple part-enzymes, or partzymes, which self-assemble in the presence of one or more assembly facilitators and form catalytically active MNAzymes capable of modifying substrates. The partzymes have multiple domains including sensor arms which bind to the assembly facilitator (such as a target nucleic acid); substrate arms which bind the substrate, and partial catalytic core sequences which, upon assembly of multiple partzyme components, combine to provide a complete catalytic core. MNAzymes can be designed to recognize a broad range of assembly facilitators including, for example, different target nucleic acid sequences. In the presence of the assembly facilitator, a catalytically active MNAzyme can assemble from partzyme components, and then bind and catalytically modify a substrate to generate an output signal. The assembly facilitator may be a target nucleic acid present in a biological or environmental sample. In such cases, catalytic activity of the MNAzymes is indicative of the presence of the target. Several MNAzymes capable of cleaving nucleic acid substrates have been reported and additional MNAzymes which can ligate nucleic acid substrates are also known in the art (see, for example, WO/2007/041774, WO/2008/040095, WO2008/122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011-0143338)
Aptazymes are specific types of catalytic nucleic acids (DNAzymes, ribozymes or MNAzymes) which have been linked with an aptamer domain to allosterically regulate the nucleic acid enzymes such that their activity is dependent on the presence of the target analyte/ligand capable of binding to the aptamer domain. Complementary regulator oligonucleotides have been used to inhibit the activities of aptazymes in the absence of target analytes by binding to both the aptamer and part of the catalytic nucleic acid domains within the aptazymes. The inhibition of catalytic activity of aptazymes was reversible by the binding of target ligands to the aptamer portions thus promoting removal of the regulator oligonucleotide.
Signal Amplification Technologies
In order to increase the sensitivity of target detection, strategies for target amplification or signal amplification have been employed. Examples of existing methods which employ target amplification include the polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependant amplification (HDA), strand invasion based amplification (SIBA), transcript-mediated amplification (TMA), self-sustained sequence replication (3 SR), or nucleic acid sequence based amplification (NASBA). Those methods which are dependent on strand displacement for amplification, for example SDA, RCA and LAMP, require the use of polymerases which possess strand displacing activity. Signal amplification cascades that utilize nucleases including nicking endonucleases have also been described (e.g. NESA).
Many limitations are evident in existing signal amplification methods. For example, in a number of techniques the speed of the reaction is limited by the number of target DNA molecules initially present in the sample. These and other methods thus often lack the speed and sensitivity required for clinical application. Others suffer from false positive signal generation in the absence of target. Thus, there is an ongoing need for methods for detecting and quantifying nucleic acid sequences and other targets which incorporate signal amplification.
Signal amplification cascades that utilize catalytic nucleic acids have also been described. One example involves the use of an oligonucleotide consisting of two adjacent peroxidase-mimicking DNAzymes joined by a ribonucleotide junction. The ends of this oligonucleotide are linked together via a short linker DNA which hybridizes to each end, so as to form a quasi-circular structure. The formation of this structure temporarily inhibits the catalytic activity of the DNAzymes. An MNAzyme that assembles in the presence of its target assembly facilitator molecule hybridizes directly to the DNAzymes and cleaves the ribonucleotide junction between them. Cleavage of the oligonucleotide which contains the DNAzymes results in separation of the two DNAzymes from each other, and from the short linker DNA, resulting in activation of the DNAzymes. The limitations of this strategy are that amplification of signal is limited to only two DNAzymes activated for each MNAzyme cleavage event, and since these peroxidase-mimicking DNAzymes have no capacity to modify nucleic acid substrates, there is no mechanism for these DNAzymes to activate additional DNAzyme molecules. As such the strategy is unsuitable as a first step in a circular feedback cascade capable of exponential signal amplification. Further, the complementarity between the MNAzyme arms and the DNAzymes, which is necessary for the initial cleavage event to occur, may also result in the sequestering of DNAzyme molecules once they have been released from the quasi-circular structure, potentially limiting the sensitivity of the reaction.
Another approach, known as DoC, utilizes quasi-circular structures consisting of temporarily inactivated DNAzymes. The DNAzymes are inactivated by hybridization to Blocker Fragments. Cleavage of the Blocker by MNAzymes activates the DNAzymes thus initiating a signal amplification cascade. There are limitations to this approach, however, because the concentration of blocker needs to be higher than that of the DNAzymes making a substantive proportion of the MNAzyme cleavage futile. Further these quasi circular structures may be unstable and have the potential to initiate the cascade in the absence of target.
Signal amplification strategies using immobilised catalytic nucleic acid enzymes tethered to solid/fixed supports have also been described. The aim of tethering catalytic nucleic acid enzymes, their individual components, and/or their substrates to fixed/immobile supports in this manner is to promote spatial separation of the enzymes from their substrates and thereby attempt to promote reaction efficiency and/or specificity. However, signal amplification approaches relying on catalytic nucleic acid enzymes, substrates, and other components tethered to fixed/immobile solid supports are often difficult to develop and have in many cases proven sub-optimal due to inherent drawbacks. For example, there are significant difficulties in balancing (i) the need to tether substantial concentrations of catalytic nucleic enzymes, substrates, and other components to drive efficient reactions with (ii) the need to ensure that the density of these components is not too high such that molecular crowding inhibits accessibility of other reaction components due to steric hindrance. Other difficulties include (iii) undesirable conformational changes and/or potential surface interactions that may interfere with the activity of the nucleic acid catalytic domains and (iv) reduced reaction rates caused by diffusion restrictions and/or (v) the need to ensure that all unbound catalytic nucleic acids are removed to prevent cascade initiation in the absence of target.
A need exists for improved assays for the detection of target molecules. Preferably, the improved assays can facilitate increased sensitivity through improved signal amplification.
The invention described herein alleviates at least some of the problems in the prior art associated with signal amplification assays utilising reaction components tethered to fixed/immobile supports. The present inventors have devised a system capable of maintaining spatial separation of nucleic acid enzymes and their components (e.g. partzyme oligonucleotide components of MNAzymes) from their substrates without the inherent drawbacks of assays that rely on fixed/immobilised supports. The assays described herein thus achieve the enhanced reaction efficiency and/or specificity that has been sought by signal amplification assays utilising reaction components tethered to fixed/immobile supports. In general, the assays of the present invention utilise a sectively permaeable membrane to separate nucleic acid enzymes and/or their components from their substrates. The components remain mobile in the assay mix which provides various advantages over assays relying on fixed/immobile supports. Upon undergoing a modification (e.g. cleavage of a mobile support component, alteration in charge and/or size, or other), which is triggered only when target molecule is present, the enzymes/enzyme components become capable of permeating the membrane and thereby coming into contact with their substrates to modify them and provide a signal.
More specifically, the present inventors have devised a rapid detection assay utilising subZyme oligonucleotides each comprising a catalytic nucleic acid substrate component, a catalytic nucleic acid component (either a full catalytic nucleic acid or a portion thereof such as a partzyme oligonucleotide), and a component and/or property preventing the subZyme oligonucleotide from moving through an otherwise permeable membrane utilised in the assay. The subZyme oligonucleotides are mobile in solution rather than, for example, being tethered to a planar surface. This provides a number of advantages including, but not limited to, improved accessibility of catalytic nucleic acids to their substrates and increased rates of diffusion to opposite sides of the membrane leading to faster detection. The catalytic nucleic acid component of the subZyme oligonucleotide exists in a catalytically active form without, for example, any conformational constraint/s (e.g. a hybridised inhibitory molecule/oligonucleotide) that would reduce or prevent its catalytic activity in the presence of its target substrate. Providing the catalytic nucleic acid component in an uninhibited form avoids the reduction in catalytic activity associated with utilising enzymes subjected to conformational constraints. In the assays described herein, the presence of a target molecule triggers cleavage of the subZyme oligonucleotide releasing the catalytic nucleic acid component from the subZyme oligonucleotide and allowing the released catalytic nucleic acid component to permeate the membrane. Once through the membrane the released catalytic nucleic acid component can cleave other subZyme oligonucleotides comprising its substrate and thereby release other catalytic nucleic acid components. These can in turn permeate to the other side of the membrane to cleave further subZyme oligonucleotides, and so on. The assays of the present invention thus rely on the transfer of catalytically active nucleic acid enzymes, and/or components thereof such as partzyme oligonucleotides, across the membrane following an initial target recognition event. The remainder of the cleaved subZyme which includes the substrate component remains on one side of the membrane and is still unable to permeate through it. Accordingly, the assays described herein do not rely on or require the transfer of nucleic acid enzyme substrates across the membrane. Rather, they rely on the provision of catalytically active nucleic acids, and/or components thereof, as components of subZyme oligonucleotides, and their subsequent release and transfer across the membrane upon a target recognition event.
The target recognition event of the assays described herein utilises an ‘initiating’ catalytic nucleic enzyme which specifically detects the target and upon doing so effects cleavage of a subZyme oligonuleotide. The present inventors have observed that the use of initiating enzymes in the manner disclosed herein can increase the sensitivity and/or specificity of the detection assay in comparison to other methods such as, for example, those relying on target molecules to remove inhibitory component/s from catalytic nucleic acids to facilitate their activity.
The assays of the present invention may incorporate feedback loops making them capable of signal amplification, independent of the number of target molecules present. A singletarget molecule can initiate a cascade in which an initial subZyme is cleaved in a target-dependent fashion by an initiating enzyme. This releases the catalytic nucleic acid component of the subZyme which is free to permeate across the membrane. Once across the membrane, the released catalytically active nucleic acid, and/or components thereof, can access and cleave its substrate, which exists as a component of another subZyme. This event in turn releases another catalytically active nucleic acid, or components thereof (e.g. partzyme oligonucleotides), from the cleaved subZyme which can move across the permeable membrane and cleave its substrate, which again is a component of another subZyme. SubZyme cleavage events can thus be perpetuated in this manner and a feedback amplifcation loop is established that is independent of additional target molecules being present. This provides a significant advantage over, for example, linear cascades in which the amount of signal generated is more restricted by the number of target molecules present.
The present invention thus addresses at least one of the disadvantages evident in existing target molecule detection and/or signal amplification assays by encompassing, at least in part, the following listed embodiments 1-59:
A method for determining a presence of a target in a sample, the method comprising:
providing a reaction mix comprising:
a population of polynucleotide A (PA) and a population of polynucleotide B (PB), wherein
a selectively permeable barrier capable of physically separating the PA from the PB, wherein the PA and PB are unable to permeate the selectively permeable barrier, and the PA and/or PB is/are mobile in the reaction mix; and
contacting:
(i) the PA, the PB, and the oligonucleotide(s) with the sample, and using the selectively permeable barrier to separate the PA from the PB, wherein:
if the target is present in the sample, each of the two oligonucleotides hybridises to the catalytic nucleic acid substrate A of a PA polynucleotide and at least one further hybridises to the target to thereby form the catalytic nucleic acid C which cleaves the PA polynucleotide to thereby release the catalytic nucleic acid A of the PA polynucleotide which is consequently capable of movement through the permeable barrier; or
(ii) the PA, the PB, and the catalytic enzyme D with the sample, and using the selectively permeable barrier to separate the PA from the PB, wherein:
if the target is present in the sample, the target hybridises with a PA polynucleotide to form the nucleic acid duplex which comprises a recognition and cleavage site for the catalytic enzyme D,
the cleavage site for the catalytic enzyme D is not within the catalytic nucleic acid A, and
the catalytic enzyme D binds to and cleaves the nucleic acid duplex at the cleavage site releasing the catalytic nucleic acid A of the PA polynucleotide which is consequently capable of movement through the permeable barrier;
wherein:
the released catalytic nucleic acid A moves through the permeable barrier, hybridises to the catalytic nucleic acid substrate B of a PB polynucleotide, and cleaves the PB polynucleotide to thereby release the catalytic nucleic acid B of the PB polynucleotide which is consequently capable of movement through the permeable barrier;
the released catalytic nucleic acid B moves through the permeable barrier, hybridises to the catalytic nucleic acid substrate A of a second PA polynucleotide, and cleaves the second PA polynucleotide to thereby release the catalytic nucleic acid A of the second PA polynucleotide which is consequently capable of movement through the permeable barrier;
and
detecting the cleavage of any said PA and/or PB polynucleotide indicates the presence of the target in the sample.
The method according to embodiment 1, wherein:
the catalytic nucleic acid A is unable to catalytically modify catalytic nucleic acid substrate A, and/or the catalytic nucleic acid B of the second polynucleotide is unable to catalytically modify the catalytic nucleic acid substrate B.
The method according to embodiment 1 or embodiment 2, wherein the catalytic nucleic acid C is not capable of cleaving the catalytic nucleic acid substrate B of the PB polynucleotide.
The method according to any one of embodiments 1 to 3, wherein any of the PA and/or PB that are mobile in the reaction mix polynucleotides comprises:
an attached component and/or,
a region of self-complementarity providing a secondary structure, and/or
a charged moiety;
that prevents permeation of the polynucleotide across the selectively permeable barrier.
The method according to embodiment 4, wherein the attached component is:
an aptamer hybridised to a ligand, or
a bead (e.g. a magnetic bead), or
a microparticle, or
a nanoparticle, or
a charged moeity.
The method according to embodiment 4 or embodiment 5, wherein:
the cleavage of the catalytic nucleic acid substrate A releases the attached component or the region of self-complementarity from the catalytic nucleic acid A of the PA polynucleotide which is consequently capable of movement through the permeable barrier, and/or
the cleavage of the catalytic nucleic acid substrate B releases the attached component or the region of self-complementarity from the catalytic nucleic acid B of the PB polynucleotide which is consequently capable of movement through the permeable barrier.
The method according to any one of embodiments 1 to 6, wherein the catalytic nucleic acid A and/or the catalytic nucleic acid B is a DNAzyme or a ribozyme.
The method according to any one of embodiments 1 to 7, wherein:
the catalytic nucleic acid A and/or the catalytic nucleic acid B is selected from the group consiting of an 8-17 DNAzyme, a 10-23 DNAzyme, a 9-86 DNAzyme, a 12-91 DNAzyme, a GR-5 DNAzyme, a 17E DNAzyme, an RFD-EC1 DNAzyme, an F-8 DNAzyme, a 39-E DNAzyme, an E2 DNAzyme, an Mg5 DNAzyme, an A43 DNAzyme, a DAB22 DNAzyme, a PS2.M DNAzyme, a hammerhead ribozyme, an L-histidine-dependent DNAzyme, and an HRP DNAzyme.
The method according to any one of embodiments 1 to 8, wherein:
the catalytic nucleic acid A and the catalytic nucleic acid B are each a different variety of DNAzyme.
The method according to any one of embodiments 1 to 9, wherein:
the catalytic nucleic acid A is a 10-23 DNAzyme and the catalytic nucleic acid B is an 8-17 DNAzyme; and
the catalytic nucleic acid substrate A is an 8-17 DNAzyme substrate and the catalytic nucleic acid substrate B is an 10-23 DNAzyme substrate; or
(ii) the catalytic nucleic acid A is a 8-17 DNAzyme and the catalytic nucleic acid B is an 10-23 DNAzyme; and
the catalytic nucleic acid substrate A is an 10-23 DNAzyme substrate and the catalytic nucleic acid substrate B is an 8-17 DNAzyme substrate.
The method according to any one of embodiments 1 to 10, wherein:
the two oligonucleotides are provided, each capable of hybridising specifically to the catalytic nucleic acid substrate A of a PA polynucleotide and at least one being capable of hybridising specifically to the target, to thereby form the catalytic nucleic acid C capable of cleaving the catalytic nucleic acid substrate A of the PA polynucleotide;
the target comprises or consist of a protein, analyte, glycoprotein, lipid, lipoprotein, cell, virus, bacterium, archeon, fungus, antibody, metabolite, pathogen, toxin, contaminant, poison, small molecule, polymer, metal ion, metal salt, prion, or any derivative, portion or combination thereof;
the catalytic nucleic acid C comprises an aptamer portion capable of binding to the target;
the method further comprises contacting the sample with an inhibitor that hybridises to the aptamer to thereby render catalytic nucleic acid C inactive;
the inhibitor has lower binding affinity for the aptamer portion than the target; and
when present in the sample the target binds to the aptamer displacing the inhibitor and rendering the catalytic nucleic acid C active, thereby facilitating cleavage of the catalytic nucleic acid substrate A of the PA polynucleotide.
The method according to any one of embodiments 1 to 11, wherein:
the catalytic nucleic acid C is a multi component nucleic acid enzyme (MNAzyme), and
the two oligonucleotides are two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target.
The method according to any one of embodiments 1 to 10, wherein:
The method according to embodiment 12, wherein:
the target comprises or consists of a protein, analyte, glycoprotein, lipid, lipoprotein, cell, virus, bacterium, archeon, fungus, antibody, metabolite, pathogen, toxin, contaminant, poison, small molecule, polymer, metal ion, metal salt, prion, or any derivative, portion or combination thereof;
at least one of the two partzyme oligonucleotides comprises a DNA, RNA or peptide aptamer portion capable hybridising specifically to the target;
the method further comprises contacting the sample with an MNAzyme assembly facilitator oligonucleotide facilitating self-assembly of the two partzyme oligonucleotides into an MNAzyme, and an inhibitor molecule that hybridises to the aptamer rendering the MNAzyme catalytically inactive;
the inhibitor has lower binding affinity for the aptamer portion than the target; and
the target hybridises to the aptamer portion removing the inhibitor and rendering the MNAzyme catalytically active.
The method according to any one of embodiments 1 to 10, wherein:
the target is a polynucleotide, the catalytic enzyme D is an endonuclease or an exonuclease.
The method according to any one of embodiments 1 to 15, wherein
the release of the catalytic nucleic acid A from the catalytic nucleic acid substrate A of the PA polynucleotide and/or the release of the catalytic nucleic acid B from the catalytic nucleic acid substrate B of the PB polynucleotide separates a fluorophore from a quencher thereby providing a signal to facilitate the detection of the target.
The method according to any one of embodiments 1 to 16, wherein the detection comprises
isolating the released catalytic nucleic acid A and/or the released catalytic nucleic acid B from the reaction mix;
applying the isolated catalytic nucleic acid(s) to a second reaction mix comprising catalytic nucleic acid substrate A and/or catalytic nucleic acid substrate B under conditions suitable for cleavage of the catalytic nucleic acid substrates by the catalytic nucleic acids; and detecting cleavage of the catalytic nucleic acid substrates by the catalytic nucleic acids.
The method according to any one of embodiments 1 to 17, wherein the detection comprises quantifying the released catalytic nucleic acid A and/or released catalytic nucleic acid B in real time.
The method according to any one of embodiments 1 to 18, comprising:
the cleavage of fewer than five, fewer than six, fewer than seven, fewer than eight, fewer than nine, fewer than ten, fewer than fifteen, or fewer than twenty, of the PA polynucleotides, and/or
the cleavage of fewer than five, fewer than six, fewer than seven, fewer than eight, fewer than nine, fewer than ten, fewer than fifteen, or fewer than twenty, of the PB polynucleotides,
to facilitate the detection of the target.
A method for determining a presence of a target in a sample, the method comprising:
(a) providing a reaction mix comprising:
The method according to embodiment 20, wherein:
each PC polynucleotide comprises a partzyme oligonucleotide B2 and a catalytic nucleic acid substrate B that is cleavable by catalytic nucleic acid A,
the released catalytic nucleic acid A cleaves the PC polynucleotide to thereby release the partzyme oligonucleotide B2 of the PC polynucleotide, and
detecting the cleavage of any PC polynucleotide indicates the presence of the target in the sample.
The method according to embodiment 20, wherein
each PC polynucleotide comprises a partzyme oligonucleotide B2 and a catalytic nucleic acid substrate A, and
if the target is present in the sample, the substrate arm of each of the partzyme oligonucleotides C1 and C2 hybridises to the catalytic nucleic acid substrate A of the PC polynucleotide and at least one of the partzyme oligonucleotides C1 and C2 hybridises to the target forming the catalytic nucleic acid C which cleaves the PC polynucleotide to thereby release the partzyme oligonucleotide B2 of the PC polynucleotide.
The method according to embodiment 20, wherein if the target is present in the sample, the target hybridises with a PC polynucleotide to form the nucleic acid duplex which comprises a recognition and cleavage site for the catalytic enzyme D,
the cleavage site for the catalytic enzyme D is not within the catalytic nucleic acid A of the PC polynucleotide, and
the catalytic enzyme D binds to and cleaves the nucleic acid duplex at the cleavage site to thereby release the partzyme oligonucleotide B2 of the PC polynucleotide.
The method according to any one of embodiments 20 to 22, wherein any of the PA, PB and/or PC polynucleotides that are mobile in the reaction mix comprises:
an attached component and/or,
a region of self-complementarity providing a secondary structure, and/or
a charged moiety;
that prevents permeation of the polynucleotide across the selectively permeable barrier
The method according to embodiment 24, wherein the attached component is:
an aptamer hybridised to a ligand, or
a bead (e.g. a magnetic bead), or a microparticle,
or a nanoparticle, or a charged moeity.
The method according to embodiment 24 or embodiment 25, wherein:
the cleavage of the catalytic nucleic acid substrate A releases the attached component or the region of self-complementarity from the catalytic nucleic acid A of the PA polynucleotide which is consequently capable of movement through the permeable barrier,
and/or the cleavage of the catalytic nucleic acid substrate B releases the attached component or the region of self-complementarity from the PB polynucleotide which is consequently capable of movement through the permeable barrier.
The method according to any one of embodiments 20 to 26, wherein:
the catalytic nucleic acid C is a multi component nucleic acid enzyme (MNAzyme), and
the two oligonucleotides are two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target.
The method according to embodiment 27, wherein:
the target comprises or consists of a protein, analyte, glycoprotein, lipid, lipoprotein, cell, virus, bacterium, archeon, fungus, antibody, metabolite, pathogen, toxin, contaminant, poison, small molecule, polymer, metal ion, metal salt, prion, or any derivative, portion or combination thereof;
at least one of the two partzyme oligonucleotides comprises a DNA, RNA or peptide aptamer portion capable hybridising specifically to the target;
the method further comprises contacting the sample with an MNAzyme assembly facilitator oligonucleotide facilitating self-assembly of the two partzyme oligonucleotides into an MNAzyme, and an inhibitor molecule that hybridises to the aptamer rendering the MNAzyme catalytically inactive;
the inhibitor has lower binding affinity for the aptamer portion than the target; and
the target hybridises to the aptamer portion removing the inhibitor and rendering the MNAzyme catalytically active.
The method according to embodiment 27, wherein:
the target comprises or consists of a polynucleotide; and
the two partzyme oligonucleotides each hybridise to the polynucleotide during the self-assembly to form the MNAzyme.
The method according to any one of embodiments 1 to 29, wherein:
the reaction mix further comprises a population of the catalytic nucleic acid substrate A which is mobile in the reaction mix, each catalytic nucleic acid substrate A of the population comprising a fluorophore molecule and a quencher molecule;
and the catalytic nucleic acid B and/or the catalytic nucleic acid C cleaves members of the population of catalytic nucleic acid substrate A, separating the fluorophore from the quencher to thereby providing a signal facilitating the detection of the target.
A composition for the detection of a target in a sample, the composition comprising:
a population of polynucleotide A (PA) and a population of polynucleotide B (PB), wherein
a selectively permeable barrier capable of separating the PA from the PB.
A composition comprising:
(i) a population of polynucleotide A (PA), a population of polynucleotide B (PB) and a population of polynucleotide C (PC), wherein
(ii) an assembly facilitator oligonucleotide capable of hybridising to a sensor arm of partzyme oligonucleotide B1 and to a sensor arm of partzyme oligonucleotide B2;
(iii) a selectively permeable barrier capable of physically separating the PA from the PB, wherein the PA and PB are unable to permeate the selectively permeable barrier,
wherein hybridisation of the partzyme oligonucleotide B1 substrate arm and the partzyme oligonucleotide B2 substrate arm to the catalytic nucleic acid substrate A, and hybridisation of the partzyme oligonucleotide B1 sensor arm and the partzyme oligonucleotide B2 sensor arm to the assembly facilitator, forms a catalytic nucleic acid B capable of cleaving the catalytic nucleic acid substrate A.
The composition according to embodiment 32, wherein:
each PC polynucleotide comprises a partzyme oligonucleotide B2 and said catalytic nucleic acid substrate B capable of cleavage by catalytic nucleic acid A; or
each PC polynucleotide comprises a partzyme oligonucleotide B2 and said catalytic nucleic acid substrate A.
The composition according to embodiment 31, wherein any of the PA and/or PB comprises:
an attached component and/or,
a region of self-complementarity providing a secondary structure, and/or
a charged moiety;
that prevents permeation of the polynucleotide across the selectively permeable barrier.
The composition according to embodiment 32 or embodiment 33, wherein any of the PC comprises:
an attached component and/or,
a region of self-complementarity providing a secondary structure, and/or
a charged moiety;
that prevents permeation of the polynucleotide across the selectively permeable barrier.
The composition according to embodiment 34 or embodiment 35, wherein the attached component is:
an aptamer hybridised to a ligand, or
a bead (e.g. a magnetic bead), or
a microparticle, or
a nanoparticle, or
a charged moeity.
The composition according to any one of embodiments 31 to 36, wherein:
the catalytic nucleic acid A is unable to catalytically modify catalytic nucleic acid substrate A, and/or
the catalytic nucleic acid B of the second polynucleotide is unable to catalytically modify the catalytic nucleic acid substrate B.
The composition of any one of embodiments 31 to 37, further comprising two oligonucleotides each capable of hybridising specifically to the catalytic nucleic acid substrate A of a PA polynucleotide and at least one being capable of hybridising specifically to the target, to thereby form a catalytic nucleic acid C capable of cleaving the catalytic nucleic acid substrate A of the PA polynucleotide.
The composition according to embodiment 38, wherein:
the catalytic nucleic acid C is a multi component nucleic acid enzyme (MNAzyme), and
the two oligonucleotides are two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target.
The composition of any one of embodiments 31 to 36, further comprising:
a catalytic enzyme D capable of cleaving one or more stands of a nucleic acid duplex formed by hybridisation of the target to the PA;
wherein:
at least a portion of the PA polynucleotide shares sequence complementarity with the target such that it is capable of hybridising with the target to form a nucleic acid duplex comprising a recognition and cleavage site for the catalytic enzyme D, and
the cleavage site for the catalytic enzyme D is not within the catalytic nucleic acid A of the PA polynucleotide.
The composition according to embodiment 40, wherein:
the target is a polynucleotide,
the catalytic enzyme D is an endonuclease or an exonuclease.
The composition according to embodiment 38, wherein the target comprises or consists of a protein, analyte, glycoprotein, lipid, lipoprotein, cell, virus, bacterium, archeon, fungus, antibody, metabolite, pathogen, toxin, contaminant, poison, small molecule, polymer, metal ion, metal salt, prion, or any derivative, portion or combination thereof;
the catalytic nucleic acid C comprises an aptamer portion capable of binding to the target;
the composition further comprises an inhibitor that hybridises to the aptamer; and
the inhibitor has lower binding affinity for the aptamer portion than the target.
a selectively permeable barrier capable of separating the PA from the PB.
The composition according to embodiment 38 or embodiment 39, wherein:
the target comprises or consists of a polynucleotide; and
the two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target can each hybridise to the polynucleotide of the target and the catalytic nucleic acid A substrate of the PA polynucleotide, and thereby self-assemble to form the MNAzyme.
The composition according to embodiment 39, wherein:
the target comprises or consist of a protein, analyte, glycoprotein, lipid, lipoprotein, cell, virus, bacterium, archeon, fungus, antibody, metabolite, pathogen, toxin, contaminant, poison, small molecule, polymer, metal ion, metal salt, prion, or any derivative, portion or combination thereof;
at least one of the two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target comprises a DNA, RNA or peptide aptamer portion capable hybridising specifically to the target;
the composition further comprises an MNAzyme assembly facilitator oligonucleotide to which the two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target can each hybridise facilitating self-assembly of the MNAzyme;
the composition further comprises an inhibitor molecule that can hybridise to the aptamer thereby rendering the MNAzyme catalytically inactive;
the inhibitor has lower binding affinity for the aptamer portion than the target; and
the target can hybridise to the aptamer portion to remove the inhibitor and render the MNAzyme catalytically active.
The composition according to any one of embodiments 31 to 44, further comprising catalytic nucleic acid A released by cleavage of the PA polynucleotide by the catalytic nucleic acid C and/or by the catalytic nucleic acid B.
The composition according to any one of embodiments 31 to 45, further comprising catalytic nucleic acid B released by cleavage of the PB polynucleotide by the catalytic nucleic acid A.
The composition according to any one of embodiments 31 to 46, wherein:
the catalytic nucleic acid A and/or the catalytic nucleic acid B is a DNAzyme or a ribozyme.
The composition according to any one of embodiments 31 to 47, wherein:
the catalytic nucleic acid A and/or the catalytic nucleic acid B is selected from the group consiting of an 8-17 DNAzyme, a 10-23 DNAzyme, a 9-86 DNAzyme, a 12-91 DNAzyme, a GR-5 DNAzyme, a 17E DNAzyme, an RFD-EC1 DNAzyme, an F-8 DNAzyme, a 39-E DNAzyme, an E2 DNAzyme, an Mg5 DNAzyme, an A43 DNAzyme, a DAB22 DNAzyme, a PS2.M DNAzyme, a hammerhead ribozyme, an L-histidine-dependent DNAzyme, and an HRP DNAzyme.
The composition according to any one of embodiments 31 to 48, wherein:
the catalytic nucleic acid A and the catalytic nucleic acid B are each a different variety of DNAzyme.
The composition according to any one of embodiments 31 to 49, wherein:
(i) the catalytic nucleic acid A is a 10-23 DNAzyme and the catalytic nucleic acid B is an 8-17 DNAzyme; and
the catalytic nucleic acid substrate A is an 8-17 DNAzyme substrate and the catalytic nucleic acid substrate B is a 10-23 DNAzyme substrate; or
(ii) the catalytic nucleic acid A is an 8-17 DNAzyme and the catalytic nucleic acid B is a 10-23 DNAzyme; and
the catalytic nucleic acid substrate A is a 10-23 DNAzyme substrate and the catalytic nucleic acid substrate B is an 8-17 DNAzyme substrate.
The composition according to any one of embodiments 31 to 50, wherein the PA and/or PB and/or PC are mobile in the composition.
The composition according to any one of embodiments 31 to 51, wherein the PA polynucleotides and/or PB polynucleotides and/or the PC polynucleotides each comprise a fluorophore and a quencher.
The composition according to any one of embodiments 31 to 52, further comprising a population of the catalytic nucleic acid substrate A which is mobile in the composition, each catalytic nucleic acid substrate A of the population comprising a fluorophore molecule and a quencher molecule, wherein the catalytic nucleic acid B is capable of cleaving members of the population of catalytic nucleic acid substrate A, separating the fluorophore from the quencher to thereby providing a signal facilitating the detection of the target.
The composition according to embodiment 38 or embodiment 39, further comprising a population of the catalytic nucleic acid substrate A which is mobile in the composition, each catalytic nucleic acid substrate A of the population comprising a fluorophore molecule and a quencher molecule, wherein the catalytic nucleic acid C is capable of cleaving members of the population of catalytic nucleic acid substrate A, separating the fluorophore from the quencher to thereby providing a signal facilitating the detection of the target.
A polynucleotide comprising
The polynucleotide according to embodiment 55, wherein the attached component is:
an aptamer hybridised to a ligand, or
The polynucleotide according to embodiment 55 or embodiment 56, wherein the catalytic nucleic acid is a DNAzyme or a ribozyme.
The polynucleotide according to embodiment 57, wherein the DNAzyme is a 10-23 DNAzyme or an 8-17 DNAzyme.
The polynucleotide according to embodiment 55 or embodiment 56, wherein the component thereof is a partzyme oligonucleotide component of an MNAzyme.
Preferred embodiments of the present invention will now be described by way of example only, with reference to the accompanying figures wherein:
Certain terms and phrases are used herein which shall have the meanings set forth as follows.
As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an MNAzyme” also includes a plurality of MNAzymes. Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
Unless indicated differently, the terms “comprising” and “having” mean “including principally, but not necessarily solely”. Furthermore, variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings. Thus, for example, a sample “comprising” target molecule A may consist exclusively of target molecule A or may include one or more different type/s of target molecules (e.g. target molecule B and/or target molecule C).
The terms “target” and “target molecule” as used herein refer to any molecule capable of detection by the molecular complexes described herein including, but not limited to, nucleic acids, proteins, glycoproteins, lipids, lipoproteins, viruses, bacteria, archaea, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, small molecules, polymers, metal ions, metal salts, small organic compounds, whole cells and entire organisms. For example, a target may be a nucleic acid which serves as an assembly facilitator to direct the assembly of an MNAzyme, or any molecule capable of binding to an aptamer causing activation of a catalytic nucleic acid comprising the aptamer (e.g. apta-MNAzyme or other aptazyme). Target nucleic acids include, but are not limited to, DNApolynucelotides and RNA polynucleotides.
The terms “catalytic nucleic acid”, “catalytic nucleic acid molecule”, “catalytic nucleic acid enzyme” and “nucleic acid enzyme” are used interchangeably herein and have the same meaning. These terms encompass any nucleic acid, or portion thereof, capable of the specific recognition and catalytic modification of one or more substrates (e.g. nucleic acid substrates). For example, the substrate or substrates may be nucleic acids, and the catalytic modification may be ligation or cleavage. Catalytic nucleic acid enzymes as used herein include DNA molecules or DNA-containing molecules, RNA or RNA-containing molecules, and DNA-RNA or DNA-RNA-containing molecules. Non-limiting examples of catalytic nucleic acid enzymes include DNAzymes (also known as DNA enzymes and deoxyribozymes), ribozymes (also known as RNA enzymes and RNAzymes) and multi-component nucleic acid enzymes (MNAzymes) also known in the art in the art as PlexZymes.
As used herein, the term “subZyme oligonucleotide” or “subZyme” refers to an oligonucleotide comprising a component that is a catalytic nucleic acid or a portion thereof (e.g. a partzyme oligonucleotide), and a component that is a catalytic nucleic acid substrate. The catalytic nucleic acid of a subZyme oligonucleotide is unable to catalytically modify the catalytic nucleic acid substrate component of the same subZyme oligonucleotide. The catalytic nucleic acid or portion thereof, and catalytic nucleic acid substrate may be in an uninterrupted sequence along the subZyme oligonucleotide, or may be separated, for example, by intervening nucleotides and/or by any suitable linker available in the art. A subZyme oligonucleotide may comprise deoxyribonucleotide and/or ribonucleotide bases, and/or analogues, derivatives, variants, fragments or combinations thereof.
The subZyme oligonucleotide may optionally comprise additional molecule/s which may, by way of non limiting example, aid in its separation or purification (e.g. from a reaction mix) including, but not limited to, beads, microparticles and enzymes. The SubZyme oligonucleotide may comprise an additional functional group enabling attachment of the oligonucleotide to surfaces. In some embodiments of the invention and by way of non-limiting example, a subZyme oligonucleotide may comprise a 10-23 DNAzyme and a substrate for an 8-17 DNAzyme, or an 8-17 DNAzyme and a substrate for a 10-23 DNAzyme. By way of non-limiting example, a SubZyme may comprise catalytic nucleic acids selected from the group consiting of an 8-17 DNAzyme, a 10-23 DNAzyme, a 9-86 DNAzyme, a 12-91 DNAzyme, a GR-5 DNAzyme, a 17E DNAzyme, an RFD-EC1 DNAzyme, an F-8 DNAzyme, a 39-E DNAzyme, an E2 DNAzyme, an Mg5 DNAzyme, an A43 DNAzyme, a DAB22 DNAzyme, a PS2.M DNAzyme, a hammerhead ribozyme, an L-histidine-dependent DNAzyme, and an HRP DNAzyme. SubZyme oligonucleotides/subZymes according to the present invention may comprise a portion of a catalytic nucleic acid, such as for example a partzyme oligonucleotide. The portion of the catalytic catalytic nucleic acid may be capable of partial or complete enzymatic activity as compared to the complete nucleic acid from which it is derived. Alternatively, the portion of the catalytic nucleic acid may not be capable of catalytic activity until combined with other portion/s of the parent catalytic nucleic acid from which it is derived (e.g. a partzyme oligonucleotide).
The subZyme oligonucleotide may optionally comprise additional molecule/s which may, by way of non limiting example, aid in detection. Examples include, but are not limited to, fluorophore and quencher moietys for fluorescence detection; gold or silver particles for SPR and/or colorimetric detection; enzymes for electrochemical and/or pH detection and enzymes and functional groups for luminescence detection.
As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably and have the same meaning, referring to a single-stranded or double-stranded polymer of deoxyribonucleotide and/or ribonucleotide bases, or analogues, derivatives, variants, fragments or combinations thereof including, but not limited to, DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, locked nucleic acids, bridged nucleic acids, peptide nucleic acids, derivatives thereof, amplicons thereof or any combination thereof. By way of non-limiting example, the source of a given nucleic acid as described herein may be any one or more of a synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, or archael source.
As used herein, the terms “oligonucleotide” and “oligo” are used interchangeably and have the same meaning, referring to, a single-stranded polymer of deoxyribonucleotide and/or ribonucleotide bases, or analogues, derivatives, variants, fragments or combinations thereof. Non-limiting examples of oligonucleotides according to the present invention include nucleic acid targets for detection by the methods and compositions described herein; catalytic nucleic acid enzymes (e.g. DNAzymes, ribozymes, MNAzymes); MNAzyme components such as partzyme oligonucleotides; aptamers; SubZyme oligonucleotides; and nucleic acid enzyme substrates (for example, those which can be modified by an MNAzyme, DNAzyme and/or ribozyme). Oligonucleotides may comprise at least one addition or substitution, including but not limited to any one or more of those set out in Table 1 below. An oligonucleotide as referred to herein may be synthesised by any method including, for example, by chemical synthesis (e.g. from component nucleotides, or the addition of nucleotide(s) to a pre-existing fragment of the oligonucleotide). An oligonucleotide can also be constructed by ligating or otherwise joining multiple fragments of the oligonucleotide. A “ligation product” as referred to herein is a nucleic acid comprising an oligonucleotide composed of two or more oligonucleotides that have been joined (ligated) together, for example, by a ligase enzyme.
The terms “nucleotide” and “nucleotide residue” and “base” as used herein have the same meaning and encompass nucleotides comprising the bases A, C, G, T, or U, as well as derivatives or analogues thereof (non-limiting examples of which are listed in Table 1).
The term “derivative” as used herein in relation to a nucleic acid or nucleotide includes any functionally equivalent nucleic acid or nucleotide, including any fusion molecule produced integrally (e.g. by recombinant means) or added post-synthesis (e.g. by chemical means). Such fusions may comprise oligonucleotides of the invention with RNA or DNA added thereto or conjugated to a polypeptide (e.g. puromycin or other polypeptide), a small molecule (e.g., psoralen), or an antibody.
The term “analogue” as used herein in relation to a nucleic acid or nucleotide includes a compound having a physical structure that is related to a DNA or RNA molecule or residue, and may be capable of forming a hydrogen bond with a DNA or RNA residue or an analogue thereof (i.e. it is able to anneal with a DNA or RNA residue or an analogue thereof to form a base-pair), but such bonding is not so required for said compound to be encompassed within the term “analogue”. Such analogues may possess different chemical and biological properties to the ribonucleotide or deoxyribonucleotide residue to which they are structurally related. Methylated, iodinated, brominated or biotinylated residues are examples of analogues. Active DNAzymes have been described which contain nucleotide analogues, including deoxyinosine, C-5-immidazole deoxyuridine, 3-(aminopropynyl)-7-deaza-dATP, 2′-O-methyl RNA, 2′O-methyl cap. Other analogues could also be compatible with catalytic activity of catalytic nucleic acid enzymes such as DNAzymes, ribozymes and MNAzymes. Alteration of a nucleic acid with catalytic activity, for example by substitution of one base for another, by substitution of an analogue for a base, or alteration of the sugar component or phosphodiester backbone, can be straight forward for the skilled artisan. For example, alterations can be made during synthesis or by modification of specific bases after synthesis. Empirical testing of catalytic nucleic acids incorporating alterations such as base changes or base analogues allows for assessment of the impact of the altered sequences, or specific analogues, on catalytic activity. Analogues of the bases A, C, G, T and U are known in the art, and a subset is listed in Table 1. Non-limiting examples of analogues which can inhibit nuclease digestion are also well known in the art. Such analogues can be strategically placed within oligonucleotides to prevent cleavage by an exonuclease and/or an endonuclease.
The terms “multi-component nucleic acid enzyme” and “MNAzyme” are used interchangeably herein and will be taken to have the same meaning. They are formed from two or more partzyme oligonucleotides which, only in the presence of an MNAzyme assembly facilitator oligonucleotide or an MNAzyme assembly facilitator polynucleotide (for example, a target nucleic acid), assemble to form a catalytically active nucleic acid enzyme that is capable of catalytically modifying one or more substrates. For example, partzyme oligonucleotides A and B may each bind to a target nucleic acid by complementary base pairing with the nucleic acid target. The MNAzyme only forms when the sensor arms of partzymes A and B hybridise adjacent to each other on the target nucleic acid. The substrate arms of the MNAzyme engage the substrate, the modification of which (e.g. cleavage or ligation) is catalysed by the catalytic core of the MNAzyme, formed by the interaction of the partial catalytic domains on partzyme oligonucleotides A and B. It will be understood that terms “multi-component nucleic acid enzyme” and “MNAzyme” as used herein encompass all known MNAzymes and modified MNAzymes including those disclosed in any one or more of PCT patent publication numbers WO/2007/041774, WO/2008/040095, WO2008/122084, WO2012/065231, WO/2013/033792, WO/2013/123552, and WO2013/188912, related U.S. Pat. Nos. 8,394,946, 8,945,836, 9,127,311, 8,962,238, and 9,506,108, and related US patent publication numbers 20140017669, 20160348161, and 20160083785 (the contents of each of these documents are incorporated herein by reference in their entirety). Non-limiting examples of MNAzymes and modified MNAzymes encompassed include those with cleavage catalytic activity (as exemplified herein), disassembled or partially assembled MNAzymes comprising one or more assembly inhibitors, MNAzymes comprising one or more aptamers (“apta-MNAzymes”), MNAzymes comprising one or more truncated sensor arms and optionally one or more stabilising oligonucleotides, MNAzymes comprising one or more activity inhibitors, multi-component nucleic acid inactive proenzymes (MNAi), and MNAzymes with ligase catalytic activity (“MNAzymes ligases”).
The term “aptazyme” as used herein, refers to a catalytic nucleic acid (e.g. a DNAzyme, a ribozyme or an apta-MNAzyme) which has been modified to incorporate an aptamer domain to allosterically regulate its activity such that it is dependent on the presence of a target analyte. Methods for incorporating an aptamer into a catalytic nucleic acid enzyme or catalytic nucleic acid enzyme component, include, but are not limited to, direct conjugation of the aptamer to one or more domains of the catalytic nucleic acid or catalytic nucleic acid component, incorporation of the aptamer into a non-functional region of the catalytic nucleic acid, or conjugation of the aptamer adjacent to a functional region of the catalytic nucleic acid. An aptamer of an aptazyme may be wholly or partially hybridised to an inhibitory molecule to inhibit catalytic activity of the aptazyme in the absence of the analyte. The inhibitory molecule may have reduced binding affinity for the aptamer in comparison to the target molecule.
The terms “assembly facilitator molecule”, “assembly facilitator”, “MNAzyme assembly facilitator oligonucleotide”, “feedback assembly facilitator” and “MNAzyme assembly facilitator” are used interchangeably herein and refer to nucleic acids that can hybridise with a sensor arm of one or more partzyme oligonucleotides, and thereby facilitate the assembly of a catalytically active MNAzyme. Assembly facilitators may facilitate the assembly of MNAzymes which have cleavage, ligase or other enzymatic activities. An assembly facilitator may be a single molecule or comprise multiple separate molecules, which hybridise to a sensor arm of one or more partzymes oligonucleotide/s. The assembly facilitator may be a target to be detected or quantified (e.g. a nucleic acid selected from the group consisting of DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, tRNA, mRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons, or any combination thereof).
As used herein, the terms “partzyme”, “partzyme component”, and “partzyme oligonucleotide” are used interchangeably and have the same meaning, each referring to DNA-containing and/or RNA-containing oligonucleotide, two or more of which, only in the presence of an MNAzyme assembly facilitator, can self-assemble into an “MNAzyme.” A partzyme oligonucleotide, comprises three domains: a “catalytic” domain, which forms part of the catalytic core of the MNAzyme which catalyses the modification of a substrate; a “sensor arm” domain, which associates with and/or binds to an assembly facilitator (e.g. a target nucleic acid); and a “substrate arm” domain, which associates with and/or binds to a substrate.
The terms “substrate”, and “substrate molecule”, are used interchangeably herein and refer to any molecule capable of recognition and catalytic modification by a catalytic molecule (e.g. a catalytic nucleic acid enzyme or a protein enzyme). A substrate may comprise, for example, a single-stranded or double-stranded nucleic acid capable of specific recognition and catalytic modification by a catalytic nucleic acid enzyme. A substrate that is catalytically modified may be detected by indirect and/or direct means. For example, the catalytic modification of a substrate may be detected indirectly by virtue of one or more subsequent steps in a cascade that rely on the substrate being catalytically modified. Additionally or alternatively, the catalytic modification of a substrate may be detected, for example, by directly detecting one or more modified substrate products and/or any other signal directly generated by modification of the substrate (e.g. a fluorescent signal generated by cleaving the substrate and thereby spatially separating previously paired fluorophore and quencher molecules present on the unmodified substrate). A substrate that can be detected directly upon catalytic modification by a catalytic molecule is also referred to herein as a “reporter substrate”, a “reporter probe substrate”, a “reporter probe” or a “probe”.
As used herein the term “aptamer” encompasses a nucleic acid or peptide sequence that has the ability to recognize and bind to one or more ligands with high affinity and specificity due to their higher level structure (e.g. a 3-D binding domain or pocket). Aptamers can bind various ligands, non-limiting examples of which include nucleic acid, proteins, prions, small organic compounds, or entire organisms. Preferred aptamers herein are short single-strand DNA or RNA oligomers which can be isolated, for example, from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. Aptamers can be generated to recognise almost any target molecule, ranging from small molecules such as amino acids, or antibiotics to proteins, nucleic acid structures or whole cells.
The term “ligand” as used herein refers to any molecule capable of binding to an aptamer with high affinity and specificity, including but not limited to, proteins, prions, polypeptides, peptides or nucleic acids, glycoproteins, lipids, lipoproteins, viruses, bacteria, archaea, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, small molecules, polymers, metal ions, metal salts, small organic compounds, whole cells and entire organisms. A ligand may also be referred to as a “target analyte” or “analyte”.
Reference herein to “hybridisation” between two or more nucleic acids, or, to two or more nucleic acids that are “hybridised”, will be understood to require complementary base pairing between all or a portion of the nucleic acids.
The term “selectively permeable barrier” refers to a material (e.g. a membrane) that allows the passage of particular substances through it (in one direction or in both directions) while preventing other different substances from doing so. The selectively permeable barrier can thus be designed to maintain physical separation between two non-identical molecules. The selectively permeable barrier may achieve the physical separation based on its properties including, but not limited to, those which facilitate charge-based separation, weight-based separation, size-based separation, shape-based separation, separation based on lipid solubility, and/or separation based on affinity to carrier molecules present in the membrane. By way of non-limiting example the selectively permeable barrier may comprise any one or more of polyethersulfone, polycarbonate, nitrocellulose, regenerated cellulose, cellulose acetate, polyamide, propylene, nylon, aluminium oxide or polytetrafluoroethylene.
The following abbreviations are used herein and throughout the specification:
RE: restriction enzyme/endonuclease
NE: nicking enzyme/endonuclease
DSN: duplex-specific nuclease
LAMP: loop-mediated isothermal amplification
RCA: rolling circle amplification
TMA: transcript-mediated amplification
3SR: self-sustained sequence replication
NASBA: nucleic acid sequence based amplification
MNAzyme: multi-component nucleic acid enzyme
DNAzyme or Dz: deoxyribonucleic acid enzyme;
PCR: polymerase chain reaction;
F: fluorophore dye molecule;
Q: quencher molecule;
JOE or 6-JOE: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein;
FAM or 6-FAM: 6-Carboxyfluorescein.
TxR: texas red
Oligo: oligonucleotide
IB: Iowa Black
IDT: Integrated DNA Technologies
SPR: Surface Plasmon Resonance
Sub: Substrate
Pz: Partzyme
Cat: Catalytic Nucleic Acid
PES: polyethersulfone
MB: Magnetic Bead
The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those of ordinary skill in the art to practice the present invention. Features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention or the present invention as a whole. Hence, the following detailed description does not limit the scope of the present invention, which is defined only by the claims.
As discussed above, numerous limitations are evident in currently available assays for target molecule detection and methods designed to amplify the signals generated by such assays. One or more of these limitations are addressed by the compositions, kits and methods of the present invention.
Compositions, methods and kits are provided for the detection, identification and/or quantification of a target.
Provided herein are compositions and kits for carrying out the methods of the invention. By way of non-limiting example only, the compositions and kits may comprise any one or more of catalytic nucleic acid enzymes (e.g. MNAzymes and/or partzyme oligonucleotide component/s thereof, DNAzymes, and/or ribozymes), subZyme oligonucleotide/s, substrates for catalytic nucleic acid enzymes, and/or oligonucleotides which comprise one strand of a recognition site for a protein enzyme).
Various components of the compositions and kits may be provided in a functionally inactivated form. Additionally or alternatively, various components of the compositions and kits may be provided in a functionally active form.
Non-limiting examples of components suitable for inclusion in the compositions and kits are provided below.
Catalytic Nucleic Acid Enzymes
Compositions and kits of the present invention may comprise one or more different types of catalytic nucleic acid enzymes and/or one or more components thereof (e.g. one or more partzyme oligonucleotides and/or assembly facilitator oligonucleotides) and/or the complement of catalytic nucleic acid enzymes or components thereof.
The substrates for catalytic nucleic acids may be capable of providing a detectable signal upon catalytic modification. For example, the substrate may comprise one or more detectable labels (e.g. a fluorophore and quencher).
The compositions and kits may comprise any suitable catalytic nucleic acid enzyme(s) (non-limiting examples of which include DNAzymes, MNAzymes, ribozymes and/or aptazymes), and/or components thereof (e.g. partzyme(s) and/or assembly facilitator(s)), and/or those which are a component of larger entities such as subZyme oligonucleotides.
For example, compositions and kits of the present invention may comprise DNAzymes. Any suitable DNAzyme may be utilised. The DNAzymes may be known/existing DNAzymes or newly generated by in vitro selection. The DNAzymes may be capable of cleaving or ligating either RNA or DNA molecules. Divalent metal ions such as, for example, Ba2+, Sr2+, Mg2+, Ca2+, Ni2+, Co2+, Mn2+, zn2+, and/or Pb2+ may be provided as co-factors for the DNAzymes. The DNAzymes may comprise a catalytic domain (catalytic core) flanked by two non-conserved substrate binding domains (“hybridizing arms”), which are regions of sequence that specifically bind to a substrate. Non-limiting examples of suitable DNAzymes include 10-23 DNAzymes which comprise a catalytic domain of 15 deoxyribonucleotides flanked by two substrate-recognition arms, and 8-17 DNAzymes.
Additionally or alternatively, the compositions and kits may comprise ribozymes. Any suitable ribozyme may be utilised. The ribozymes may be natural ribozymes or artificially generated ribozymes. The ribozymes may be capable of cleaving or ligating either RNA or DNA molecules. Divalent metal ions such as, for example, Ba2+, Sr2+, Mg2+, Ca2+, Ni2+, Co2+, Mn2+, Zn2+, and/or Pb2+ and/or monovalent cations may be provided as co-factors for the ribozymes. The ribozymes may comprise a catalytic domain (catalytic core) flanked by two non-conserved substrate binding domains (“hybridizing arms”), which are regions of sequence that specifically bind to a substrate. Alternatively, other ribozyme structures are contemplated wherein the structures may comprise separate target and substrate binding arms and a catalytic core. Non-limiting examples of suitable ribozymes include hammerhead ribozymes, hairpin ribozymes, branching ribozymes, maxizymes, Group I ribozymes, Group II intron ribozymes, HDV ribozyme, RNase P, CPEB3 ribozyme, glniS ribozyme, peptidyl transferase 23S rRNA, VS ribozyme, CoTC ribozyme and GIR1 leadzyme.
Additionally or alternatively, compositions and kits of the present invention may comprise any one or more of MNAzymes, partzyme oligonucleotide components capable of forming catalytically active MNAzymes, MNAzyme assembly facilitator oligonucleotides/polynucleotides, and/or MNAzyme substrates. As well known to those in the field, MNAzymes are catalytically active nucleic acid enzymes which self-assemble from two or more partzymes upon hybridisation to an appropriate assembly facilitator (e.g. a target). Each partzyme oligonucleotide component comprises a partial catalytic core, which upon assembly of the MNAzyme combine to form a single catalytic core capable of modifying a substrate.
Non-limiting examples of suitable MNAzymes and methods for their generation are disclosed, for example in any one or more of PCT patent publication numbers WO/2007/041774, WO/2008/040095, WO2008/122084, WO2012/065231, WO/2013/033792, WO/2013/123552, and WO2013/188912, related U.S. Pat. Nos. 8,394,946, 8,945,836, 9,127,311, 8,962,238, and 9,506,108, and related US patent publication numbers 20140017669, 20160348161 and 20160083785 (the contents of each of these documents are incorporated herein by reference in their entirety). Suitable MNAzymes include those with cleavage catalytic activity, those with ligation activity, disassembled or partially assembled MNAzymes comprising one or more assembly inhibitors, MNAzymes comprising one or more aptamers (“apta-MNAzymes”), MNAzymes comprising one or more truncated sensor arms and optionally one or more stabilizing oligonucleotides, MNAzymes comprising one or more activity inhibitors, multi-component nucleic acid inactive proenzymes (MNAi), and MNAzymes with ligase catalytic activity (“MNAzyme ligases”), each of which is described in detail in one or more of WO/2007/041774, WO/2008/040095, WO2008/122084, US 2007-0231810, US 2010-0136536, and/or US 2011-0143338. The partzyme oligonucleotides self-assemble in the presence of an MNAzyme assembly facilitator to form an MNAzyme. In some embodiments, the presence of an MNAzyme can be detected, and is indicative of the presence of a target, because the MNAzyme forms only in the presence of the target, wherein the target comprises the assembly facilitator.
As known to those skilled in the field, MNAzyme structures are based on one or more DNAzymes (e.g. 10-23 and 8-17 DNAzymes) and/or ribozymes. The MNAzymes may comprise ribonucleotide bases and/or deoxyribonucleotide bases and/or analogues thereof. For example, one or more of a sensor arm, a substrate arm, or the catalytic core of the MNAzyme may comprise one or more ribonucleotide bases and/or one or more deoxyribonucleotide bases and/or one or more analogues thereof. In some embodiments the MNAzyme comprises at least one deoxyribonucleotide base, or its analogue, within the catalytic core of the MNAzyme. The deoxyribonucleotide base, or its analogue, may be required for catalytic activity.
MNAzymes of the compositions and kits may contain one or more substitutions such as analogues, derivatives, modified or altered bases, ribonucleotides, alterations of the sugar or phosphate backbone, various deletions, insertions, substitutions, duplications or other modifications, or any combination of these, well known to those skilled in the art. Such modifications, substitutions, deletions, insertions, etc may be made in the sensor and/or substrate arms and/or in the catalytic core portions such that the molecule retains catalytic activity. Substitutions and modifications to arms that bind the substrate or assembly facilitator may be well tolerated and allow tailoring of the molecules to different substrates/assembly facilitators. For example, modification of the sensor arms allows tailoring to different assembly facilitators, while modification of the substrate arms allows tailoring to different substrates.
Additionally or alternatively, compositions and kits of the present invention may comprise one or more components of a catalytic nucleic acid enzyme. For example, the compositions and kits may comprise individual component(s) of an MNAzyme (e.g. one or more partzyme oligonucleotides, and/or one or more assembly facilitator oligonucleotides).
By way of non-limiting example, the compositions and kits may comprise individual partzyme(s) which, upon recognition of a target molecule, are capable of self-assembly to form a catalytically active MNAzyme capable of modifying one or more substrates. The MNAzyme so formed may be designed to assemble only upon hybridisation of partzyme sensor arms to certain assembly facilitators (which may be specific target molecule(s)) and/or to only catalytically modify certain specific substrate(s) capable of hybridisation to substrate arm(s) of the MNAzyme. Accordingly, MNAzymes included in the compositions and kits may be designed to initiate a detection and/or signal amplification cascade according to the present invention by virtue of requiring the presence of a target molecule in order to self-assemble and catalytically modify one or more substrate/s into a product required for the cascade to occur.
For example, by altering only the sensor arms of the partzymes, but by leaving the substrate arms unchanged, a large variety of MNAzymes specific for various targets can be designed all of which may utilize a universal MNAzyme substrate for detection. The skilled artisan will appreciate the advantages that this offers in terms of eliminating the need for customized or unique substrates for each target. Each new target requires only one or more changes in one or more of the sensor arm portions; the substrate arm portion and the catalytic core portion can remain constant. Thus, a single MNAzyme substrate can be used for a single target using an MNAzyme, and multiple targets in a series of assays using altered MNAzymes. A plurality of MNAzyme substrates allows multiplexing to detect multiple targets in a single assay using multiple MNAzymes, one for each target. Such multiplexed methods of using MNAzymes are readily accomplished in solution or with attachment to a support system. It is contemplated herein that multiplexed assays can thus be accomplished in systems involving attaching one or more of the substrate, or the MNAzyme partzymes or assembly facilitator, or additional enzyme activities, to a support as described herein.
Similarly, the MNAzymes may be engineered to specifically hybridise to and catalytically modify certain substrates. For example, by altering only the substrate arms of the partzymes, but by leaving the sensor arms unchanged, a large variety of MNAzymes specific for a given target can be designed which recognise and catalytically modify a series of different MNAzyme substrates. The substrate may be a reporter substrate capable of providing a detectable signal upon catalytic modification by the MNAzyme.
In certain embodiments, MNAzymes of the compositions and kits may be engineered to specifically hybridise to and catalytically modify a universal or generic substrate. Universal MNAzyme substrates may be used to allow rapid assay development by allowing facile design changes to create new MNAzymes which recognize different targets. The substrate arm portion and the catalytic core portion of the partzymes may remain unchanged, with changes only to the sensor arm portion of one or more partzymes required for new targets. Universal substrate sequences are provided and thus the same substrate can be incorporated in assays for many different targets. Further, the same substrate can be incorporated into the methods in various embodiments herein, including assays where the substrate is free in solution or is tethered or attached to a support. A series of universal substrates can be used in a multiplex reaction allowing simultaneous detection of multiple targets. MNAzyme strategies using universal substrates offer a major advantage over detection technologies such as TaqMan® or Beacons or Hybridization probes which require the design and use of probes specific for each new target. Since the MNAzyme substrate is universal and useful for any target, cleavage of this universal MNAzyme substrate allows for the generation and amplification of a signal in the presence of any target.
DNAzymes, SubZymes, ribozymes, partzymes, assembly facilitator oligonucleotides/polynucleotides and/or MNAzyme substrates included in compositions and kits of the present invention may comprise an aptamer which is capable of binding to a target analyte. Preferred aptamers may comprise short single-stranded DNA or RNA oligomers or peptides that can be isolated from complex libraries of synthetic nucleic acids or peptides by an iterative process of adsorption, recovery, and re-amplification. Aptamers may therefore be generated against almost any target analyte, ranging from small molecules such as amino acids or antibiotics, to protein and nucleic acid structures. In preferred embodiments, aptamers include, for example, nucleic acid binding molecules which are preferably generated by evolution and selection techniques. The aptamers may comprise DNA molecules, RNA molecules or a combination of both including, but not limited to, the nucleotide analogues as per, for example, Table 1 above.
Strategies for combining the use of aptamers with ribozymes or DNAzymes are known in the art. Such molecules are generally chimeric and contain both the aptamer domain and DNAzyme or ribozyme domain, and are activated by the presence of the target ligand. The aptazyme functional activity may be switched on in response to the aptamer domain binding to its analyte. Strategies for generating aptazymes include, but are not limited to, the fusion of the ribozyme or DNAzyme and the aptamer domains together via a communication domain. The communication domain can be evolved via in vitro selection methods to improve its ability to allow for ribozyme or DNAzyme activity only in the presence of the target analyte. Another exemplary strategy involves the incorporation of the aptamer into a non-functional stem loop or hairpin that merely plays a structural role in the ribozyme or DNAzyme. Aptamers may also be linked to a DNAzyme or ribozyme and both the aptamer domain and enzyme domain may be partially hybridized to a regulator oligonucleotide, which is used to inhibit the catalytic activity of the enzyme domain in the absence of the analyte. In the presence of the analyte, the aptamer can bind to the analyte, releasing the regulator oligonucleotide from the enzyme domain and restoring its catalytic activity. In this case, the presence of the analyte may remove the regulator oligonucleotide from the DNAzyme or ribozyme and restore its catalytic activity. Aptamers can also be used to bridge two or more components of a DNAzyme or ribozyme together such that the enzyme is then capable of modifying its substrate. A unique class of DNAzymes also exists that contains an aptamer for hemin and in its presence can mimic the activities of peroxidase, catalysing various chemical substrates to generate fluorescent, chemiluminescent, and colorimetric signals.
Strategies for combining the use of aptamers with MNAzymes are also known in the art. Aptazymes which contain MNAzyme components linked to aptamer may also be referred to as Apta-MNAzymes. For example at least one partzyme of a MNAzyme may incorporate an aptamer (an apta-partzyme) as well as a complementary sequence capable of forming a hairpin and therefore inhibiting MNAzyme assembly. An analyte or target to be detected may bind to the apta-partzyme, thus enabling assembly of an active MNAzyme. In the absence of a target analyte the apta-partzyme adopts a hairpin structure which inhibits assembly of an active MNAzyme. In the presence of target analyte, the target analyte binds to the aptamer domain of the apta-partzyme, thus disrupting the hairpin structure and allowing the apta-partzyme to participate in assembly of an active MNAzyme.
In other embodiments the aptamer may be present as part of an assembly facilitator oligonucleotide that incorporates an aptamer as well as complementary inhibitor sequence capable of forming a hairpin structure. In the absence of a target analyte, the assembly facilitator oligonucleotide adopts a hairpin structure which inhibits the ability of this component to direct the assembly of active MNAzymes. In the presence of target analyte, the target analyte binds to the aptamer domain of the assembly facilitator, thus disrupting the hairpin structure and allowing the component to direct the assembly of a catalytically active MNAzyme. One skilled in the art will appreciate that the aptamer may be incorporated into either end of the assembly facilitator molecule or molecules. Further it will be appreciated that multiple aptamers could be incorporated into one or more of the partzyme oligonucleotide components.
In further embodiments an aptamer sequence may be incorporated at the end of a partzyme (forming an ‘apta-partzyme’) in a configuration whereby an active initiating Apta-MNAzyme is only formed in the presence of the target analyte. In this case the partzymes required for the detection strategy include; a standard partzyme; an apta-partzyme which is a partzyme with an aptamer incorporated into one of its ends; an assembly facilitator which binds to both the apta-partzyme and the partzyme enabling assembly of an active initiating Apta-MNAzyme (in the presence of target analyte); a substrate; and an assembly inhibitor which hybridises to the apta-partzyme in a region which spans at least part of the aptamer sequence and part of the substrate binding arm of the partzyme sequence. In the absence of a target the assembly inhibitor oligonucleotide binds to the apta-partzyme preventing cleavage of the reporter probe substrate. In the presence of a target, the target binds to the aptamer sequence of the apta-partzyme, preventing the binding of the assembly inhibitor and allowing the binding and cleavage of the MNAzyme substrate by the initiating Apta-MNAzyme. As such, an active initiating Apta-MNAzyme can only form and modify a MNAzyme substrate in the presence of target analyte.
It will also be appreciated by persons skilled in the art that one or more aptamers may be incorporated into any of the oligonucleotide components, including the partzyme oligonucleotide/s, the assembly facilitator oligonucleotide or the MNAzyme substrate.
Catalytic nucleic acid enzymes and/or components thereof (e.g. DNAzymes, ribozymes, MNAzymes, partzyme oligonucleotides, assembly facilitator oligonucleotides, substrates, and/or aptazymes) in compositions and kits of the present invention may be provided as a component of a molecular complex hybridised with other molecule(s) by complementary base pairing.
In some embodiments, the compositions and kits may comprise all of the components necessary to activate a molecular switch as described herein, with the exception of a co-factor necessary for the catalytic function of an initiator catalytic nucleic acid enzyme and/or a catalytic nucleic acid enzyme of the switch (e.g. divalent metal ions such as, for example, Ba2+, Sr2+, Mg2+, Ca2+, Ni2+, Co2+, Mn2+, Zn2+, and/or Pb2+, and/or monovalent cations).
SubZyme Oligonucleotides
Compositions and kits of the present invention may comprise one or more SubZyme oligonucleotides. In general, a subZyme oligonucleotide according to the present invention comprises at least one catalytic nucleic acid or a portion thereof (e.g. a partzyme oligonucleotide), and at least one catalytic nucleic acid substrate. In a number of embodiments, the catalytic nucleic acidor portion thereof in a subZyme oligonucleotide is unable to catalytically modify the catalytic nucleic acid substrate component of the same SubZyme oligonucleotide.
The catalytic nucleic acid or portion thereof, and the catalytic nucleic acid substrate may be arranged consecutively on the subZyme oligonucleotide without intervening sequence or linker/s. Accordingly, they may constitute an uninterrupted sequence along the subZyme oligonucleotide.
Alternatively, the catalytic nucleic acid or portion thereof, and catalytic nucleic acid substrate of the subZyme oligonucleotide may be separated, for example, by intervening nucleotides and/or by any suitable linker or spacer molecule available in the art.
No particular limitation exists in relation to the number of intervening nucleotides, which may be more than one nucleotide, more than two nucleotides, more than three nucleotides, more than four nucleotides, more than five nucleotides, more than ten nucleotides, more than fifteen nucleotides, more than twenty nucleotides, less than two nucleotides, less than three nucleotides, less than four nucleotides, less than five nucleotides, less than ten nucleotides, less than fifteen nucleotides, less than twenty nucleotides, one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, ten nucleotides, fifteen nucleotides, or twenty nucleotides.
No particular limitation exists in relation to the particular form or characteristics of a linker or spacer that may be present in a SubZyme oligonucleotide separating a catalytic nucleic acid or portion thereof, and a catalytic nucleic acid substrate of the SubZyme oligonucleotide. Spacers and linkers can be used to provide varying amounts of separation between the nucleic acid substrate and catalytic nucleic acid components. Examples include, but are not limited to, carbon chains, polyethylene glycols (PEG), poly-A, poly-T, abasic furans, abasic oligonucleotides or photocleavable PC modifications.
Linkers and spacers can also be incorporated between an oligonucleotide and a surface to which it is attached. No particular limitation exists in relation to the particular form or characteristics of a linker or spacer separating a given SubZyme oligonucleotide from a given surface. By creating greater distance, the linkers and spacers can mitigate steric or charge affects. Examples of linkers and spacers that can be used between SubZyme oligonucleotides and solid surfaces include, but are not limited to, carbon, dextran, TEG, PEG, poly-A tail, poly-T tail, PNA, DNA or LNA.
A SubZyme oligonucleotide may comprise deoxyribonucleotide and/or ribonucleotide bases, and/or analogues, derivatives, variants, fragments, linkers, spacers or combinations thereof.
SubZymes can be attached to surfaces using a variety of different attachment chemistries. There are a range of oligonucleotide modifications that enable immobilization of SubZymes onto surfaces including, but not limited to, the following oligonucleotide functional groups amine, carboxyl, disulfide, hydrazide, thiol, acrydite, biotin, NHS ester (azide), cholesterol TEG, digoxigenin, aminooxy, adenylation, desthiobiotin, hexynyl, maleimide, alkyne, dithiol, octadiynyl, aldehyde and epoxy. Alternatively SubZymes may be ligated to, or hybridized with, other oligonucleotides already immobilized on a surface.
The SubZyme oligonucleotides may optionally comprise or be attached to additional molecule/s which may, by way of non limiting example, aid in its separation or purification (e.g. from a reaction mix). These may include by way of non-limiting example magnetic, latex, glass, agarose or silica beads. In some embodiments of the invention and by way of non-limiting example, a SubZyme oligonucleotide may comprise a 10-23 DNAzyme and a substrate for an 8-17 DNAzyme, or an 8-17 DNAzyme and a substrate for a 10-23 DNAzyme. In other embodiments of the invention a SubZyme oligonucleotide may comprise a partzyme oligonucleotide and a substrate for an 8-17 DNAzyme, or a partzyme oligonucleotide and a substrate for a 10-23 DNAzyme.
The subZyme oligonucleotide may optionally comprise or be attached to additional molecule/s which may, by way of non limiting example, aid in the detection using detection systems including, but not limited to, electrochemical, fluorescent, colorimetric, pH, SPR, magnetic resonance and luminescence. Non-limiting examples of additional molecules include magnetic beads, gold particles, silver particles, enzymes, fluorophores, chemical groups, quenchers and quantum dots.
The subZyme oligonucleotide may optionally comprise additional sequence which is complementary to the target to be detected. In preferred embodiments the additional target-specific sequence may include nucleotides required to form a recognition site for a protein endonuclease or exonuclease. By way of non-limiting example, the additional target specific sequence may include one strand of a double stranded recognition site for a restriction enzyme.
SubZyme oligonucleotides/subZymes according to the present invention may comprise a portion of a catalytic nucleic acid (e.g. a partzyme oligonucleotide). The portion of the catalytic nucleic acid may be capable of partial or complete enzymatic activity as compared to the complete nucleic acid from which it is derived. Alternatively, the portion of the catalytic nucleic acid may not be capable of catalytic activity until combined with other portion/s of the parent catalytic nucleic acid from which it is derived (e.g. another partzyme oligonucleotide and/or assembly facilitator). In some embodiments of the invention and by way of non-limiting example, a SubZyme oligonucleotide may comprise a 10-23 partzyme oligonucleotide and a substrate for another different 10-23 DNAzyme,a 10-23 Partzyme and a substrate for an 8-17 DNAzyme, an 8-17 Partzyme and a substrate for a 10-23 DNAzyme, or an 8-17 Partzyme and a substrate for another different 8-17 DNAzyme.
Exoncleases and endonucleases
Compositions and kits of the present invention may comprise one or more exonucleases and/or endonucleases. Non-limiting examples of suitable endonucleases include restriction endonucleases, Mung Bean nuclease, Endonuclease IV (E. coli), RNase A, RNase I (E. coli), RNase III (E. coli) or RNase H (E. coli). Non-limiting examples of suitable exonucleases include Exonuclease I (E. coli), Exonuclease III (E. coli), Exonuclease VII and T7 Exonuclease. The endonucleases may be restriction enzymes that recognize and cleave both strands of a double stranded nucleic acid duplex, or may be Nicking enzymes that recognize a double stranded nucleic acid duplex but only cleaves only strand.
Oligonucleotides
Oligonucleotides included in compositions and kits of the present invention, such as DNAzymes, SubZyme oligonucleotides, ribozymes, aptazymes, MNAzymes, their respective substrates, and MNAzyme components (e.g. partzyme oligonucleotides, assembly facilitator oligonucleotides) may contain one or more substitutions such as analogues (e.g. those listed in Table 1), derivatives, modified or altered bases, ribonucleotides, alterations of the sugar or phosphate backbone, various deletions, insertions, substitutions, duplications or other modifications, or any combination of these, well known to those skilled in the art. Such modifications, substitutions, deletions, insertions, etc may be made at any position provided the oligonucleotide retains its function. Substitutions and modifications to the oligonucleotides may be well tolerated and allow tailoring of the molecules to function under certain conditions or for improvement of the efficiency of reaction. The skilled addressee will appreciate that the DNAzymes, ribozymes, SubZyme oligonucleotides and their substrates, MNAzymes and their substrates, and MNAzyme components (e.g. partzymes, assembly facilitator oligonucleotides/polynucleotides) may comprise either deoxyribonucleotides or ribonucleotides, or both. The oligonucleotides may comprise at least one deoxyribonucleotide, and may in some cases consist of deoxyribonucleotides and/or analogues thereof
Catalytic Nucleic Acid Substrates
Compositions and kits of the present invention may comprise substrates capable of modification by catalytic nucleic acid enzymes (e.g. DNAzymes, ribozymes, aptazymes and/or MNAzymes or apta-MNAzymes).
The substrate may be any single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogues, derivatives, variants, fragments or combinations thereof, which is capable of being recognized, acted upon or modified by an enzyme including a catalytic nucleic acid enzyme.
The substrate may be modified by various enzymatic activities including but not limited to cleavage or ligation, wherein modification of the substrate by the enzyme may provide a detectable effect indicative of the catalytic activity of the enzyme.
Substrates for nucleic acid enzymes may also comprise or be attached to non-nucleic acid constituents such as, for example, an amino acid, peptide or protein or any chemical constituent outlined in Table 6.1 of (“New strategies in Chemical synthesis and Catalysis”, B. Pignataro in Wiley-VCH, 2012).
The substrate may be a reporter substrate comprising one or more features to facilitate the quantification and/or detection of a modified form of the substrate arising due to the catalytic activity of an enzyme. Reporter substrates can be free in solution or bound (or “tethered”), for example, to a surface, or to another molecule. A reporter substrate can be labelled by any of a large variety of means including, for example, fluorophores (with or without one or more additional components, such as quenchers), radioactive labels, gold and/or silver particles, biotin (e.g. biotinylation) or chemiluminescent labels. These various labels may provide a means of generating a detectable signal upon modification (e.g. cleavage) by a catalytic nucleic acid enzyme.
The substrate may be a universal substrate that is recognized by and acted on catalytically by a plurality of catalytic nucleic acid enzymes. The universal substrate may be tethered to a solid support. Substrates included in compositions and kits of the present invention may be provided as discrete components capable of recognition and modification by a catalytic nucleic acid. Other aspects of the present invention relate to substrates that can be recognized and cleaved by more than one catalytic nucleic acid. By way of example, a single nucleic acid substrate may be cleavable by both a DNAzyme and an MNAzyme provided the substrate binding arms of the DNAzyme, and the substrate binding arms of the partzyme components of the MNAzyme, are both complementary to the same said single nucleic acid substrate. In such cases the specific catalytic core sequence of the DNAzyme, and the catalytic core portions of the partzyme pair of the MNAzyme, may be compatible with cleavage at a “cleavage” site within the substrate.
Selectively Permeable Barriers
Compositions and kits of the present invention may comprise selectively permeable barrier/s for the separation of various components for use in the methods described herein. By virtue of its properties, the selectively permeable barrier will generally allow the passage of certain assay components through it while preventing others from doing so. The selectively permeable barrier may allow two-way passage of components for use in the methods described herein.
By way of non limiting example only, the selectively permeable barrier may serve to separate any one or more of the following assay component/s from one or more other assay component/s: MNAzymes, MNAzyme component/s (e.g. assembly facilitator oligonucleotides, partzyme oligonucleotides), MNAzyme substrates, DNAzymes, DNAzyme substrates, ribozymes, ribozyme substrates, SubZyme oligonucleotides.
In certain embodiments, the selectively permeable barrier may serve to physically separate different types of SubZyme oligonucleotides from one another. In some embodiments the selectively permeable barrier is used to physically separate a first SubZyme oligonucleotide from a non-identical second SubZyme oligonucleotide.
The first SubZyme oligonucleotide may comprise a catalytic nucleic acid enzyme sequence (cat A′) and a component that is a substrate for a catalytic nucleic acid enzyme (sub A′). The second SubZyme oligonucleotide may comprise a catalytic nucleic acid enzyme sequence (cat B′) and a component that is a substrate for a catalytic nucleic acid enzyme (sub B′). Cat A may be incapable of catalytically modifying (e.g. cleaving) sub A, and may be capable of catalytically modifying (e.g. cleaving) sub B. Cat B may be incapable of catalytically modifying (e.g. cleaving) sub B, but may be capable of catalytically modifying (e.g. cleaving) sub A.
In other embodiments, a SubZyme oligonucleotide may comprise a portion of a catalytic nucleic acid enzyme (e.g. a partzyme oligonucleotide), and a component that is a substrate for a catalytic nucleic acid enzyme. The portion of the catalytic nucleic acid enzyme may not be capable of hybridising to the substrate of the SubZyme oligonucleotide.
The selectively permeable barrier may achieve the physical separation of assay components based on properties of the barrier including, but not limited to, those which facilitate charge-based separation, size-based separation, shape-based separation, weight-based separation, separation based on lipid solubility, separation based on molecule mobility and/or separation based on affinity to carrier molecules present in the membrane.
In some embodiments, the selectively permeable barrier can achieve physical separation of assay components by virtue of size restriction. For example, the barrier may comprise pores of a certain maximum size that prevents specific assay components from moving through the barrier. For example, an assay component such as a SubZyme oligonucleotide may comprise a physical feature (e.g. a secondary structure arising from a region of self-complementarity) and/or may be attached to an entity (e.g. a bead or microparticle) that is too large to move through the pores of the barrier.
In other embodiments, the selectively permeable barrier can achieve physical separation of assay components by virtue of charge characteristics. For example, the barrier may comprise pores with a certain electrical charge (e.g. ion channels) that prevents specific assay components from moving through the barrier. For example, an assay component such as a SubZyme oligonucleotide may comprise a certain charge and/or may be attached to an entity (e.g. a bead) with a certain charge that prevents it moving through pores of the barrier with the same or similar charge.
In still other embodiments, the selectively permeable barrier can achieve physical separation of assay components by virtue of lipid solubility characteristics. For example, the barrier may comprise lipids that prevent certain assay components from moving through the barrier if they are lipophobic. For example, an assay component such as a SubZyme oligonucleotide may comprise lipophobic elements and/or may be attached to an entity (e.g. a bead) with lipophobic characteristics that prevents it moving through the lipophilic barrier.
In still other embodiments, the selectively permeable barrier can achieve physical separation of assay components by virtue of shape restriction. For example, the barrier may comprise pores of a certain shape that prevents specific assay components from moving through the barrier. For example, an assay component such as a SubZyme oligonucleotide may comprise a physical feature (e.g. a secondary structure arising from a region of self-complementarity) and/or may be attached to an entity (e.g. a bead or microparticle) of a shape that is incompatible with movement through the pores of the barrier.
In further embodiments, the selectively permeable barrier can achieve physical separation of assay components by virtue of affinity to carrier molecules present within the barrier. For example, the barrier may comprise carrier molecules that facilitate the transport of specific assay components through the barrier, but not others. For example, an assay component such as a SubZyme oligonucleotide may comprise a physical feature and/or may be attached to an entity (e.g. a bead) having characteristics that facilitate attachment to a carrier molecule present in the barrier, and thereby facilitate transport of the subZyme oligonucleotide through the barrier.
The skilled person will recognise that the physical separation of assay components by the selective permeable barrier can be achieved by any standard means known in the art with no particular limitation to those specifically described herein.
The selectively permeable barrier may be made of any suitable material/s known in the art. In general, the material/s will be compatible with the working components of the assay. Non-limiting examples of suitable materials include polyethersulfone, polycarbonate, nitrocellulose, regenerated cellulose, cellulose acetate, polyamide, propylene, nylon, aluminium oxide or polytetrafluoroethylene.
Detectable Labels
Components within the compositions and kits of the present invention may comprise detectable labels capable of generating output signal determined by fluorescence spectroscopy, surface plasmon resonance, mass spectroscopy, electrochemistry, NMR, electron spin resonance, polarization fluorescence spectroscopy, circular dichroism, immunoassay, chromatography, radiometry, photometry, scintigraphy, electronic methods, UV, visible light or infra red spectroscopy, enzymatic methods or any combination thereof.
Nanoscopic and microscopic gold particles attached to oligonucleotides can be used to generate colourimetric signals whereby gold particles in close proximetery appear blue and when separated appear red. For, example separation may be achieved by cleavage of a substrate or SubZyme by an MNAzyme or DNAzyme. Oligonucleotides can be labelled with fluorophore and quencher dyes which when separated can result in an increase in florescence. For, example separation may be achieved by cleavage of a substrate or SubZyme by an MNAzyme or DNAzyme.
Oligonucletides may be labelled with gold or silver particles at one end and attached to a sensor surface at the other end to facilitate measurement by surface plasmon resonance. For example, an attached particle may be released from the surface of the sensor by cleavage of a substrate or SubZyme by an MNAzyme or DNAzyme resulting in a detectable shift in surface plasmon resonance. Oligonucleotides can be attached to a surface at one end and attached to enzymes involved in electrochemical reactions at the other end. Release of the enzyme from the surface so that it can partake in an electrochemical reaction may be achieved by cleavage of a substrate or SubZyme by an MNAzyme or DNAzyme.
Detection and Signal Amplification Methods
The present invention provides various methods for the detection, identification, and/or quantification of at least one target. Further, the present invention provides various cascades for the amplification of signals generated by these methods.
The methods utilise compositions of the present invention and components thereof. In many embodiments the methods include signal amplification cascades.
Exemplary Assay According to the Present Invention
An exemplary scheme according to embodiments of the present invention is illustrated in
After a period of incubation uncleaved SubZyme-MB and cleaved MB fragments can be separated from released DNAzymes (Cat A and Cat B). As illustrated in
As depicated in
Cascade Inititation
The skilled person will recognize that the signal amplification cascades of the present invention may be used to detect any target (s) including nucleic acid and non-nucleic acid targets (e.g. proteins, peprtides, analytes atc.). The mechanism of initiation of the cascade in the presence of target can also vary. By way of non-limiting example and with reference to
In the scheme shown in
In the exemplary scheme in
The skilled person will recognise that there are many other enzymes in addition to restriction enzymes and their subtype nicking enzymes which could cleave PA following hybridization of a target to an optional target specific region within PA. By way of non-limiting example, Ribonuclease H (RNAse H) could be employed to initiate the cascade if PA contained, for example, four or more ribonucleotides in the target specific region. RNAse H is a non-sequence specific endoclease enzyme that can catalyze the cleavage of RNA in an RNA/DNA duplex. Formation of target SubZyme hybrids could provide the RNA/DNA duplexes that are cleavable by RNAse H enzymes, whilst leaving the other single stranded regions of the SubZyme and the target intact.
As a second non-limiting example, duplex-specific nuclease (DSN) could be employed to initiate the cascade in the presence of RNA or ssDNA targets. DSN is a nuclease enzyme that shows a strong preference for cleaving DNA in DNA-RNA duplexes and double stranded DNA, yet, it is inherently inactive towards single stranded DNA, single stranded RNA and double stranded RNA. Formation of target and SubZyme hybrids could provide the RNA/DNA duplexes that are cleavable by DSN enzymes, whilst leaving the other single stranded regions of the SubZyme and the target intact.
The skilled person will recognise there are many other enzymes in addition to endonucleases such as restriction enzymes and their subtype nicking enzymes which may be used to cleave the PA polynucleotide following hybridization with a target. By way of non-limiting examples exonucleases such as exonuclease III or T7 could be employed to initiate the cascade. Exonuclease III enzyme removes nucleotides from 3′ termini of blunt or recessed termini or DNA duplexes, but is not active on single stranded DNA. T7 Exonuclease removes nucleotides from the 5′ end of DNA duplexes or DNA/RNA duplexes. Formation of target subZyme hybrids could provide duplexes that are cleavable by exonuclease III or T7, whilst leaving the other single stranded regions of the subZyme intact.
In the exemplary scheme depicted in
In the exemplary scheme depicted in
In the exemplary scheme depicted in
Methods Using Multiple Enzymes to Analyze Multiple Targets
The skilled person will recognize that the methods provided herein may be used to detect a single target per reaction, or to detect multiple targets in a single reaction. When detecting multiple targets, one or more MNAzymes may be used depending on the assay and what is to be detected. For example, a single MNAzyme may suffice when detecting multiple related structures such as, for example, a group of sequences sharing a critical sequence (recognized by the MNAzyme) and varying only, for example, in length, or in sequence outside of the critical sequence. Any sequence with the critical sequence could be detected. Multiple MNAzymes are contemplated to be useful when detecting related sequences differing by as little as a single nucleotide or even where vastly different targets are to be detected, and it is desirable to know the presence or absence of each.
Detection of Ionic Compounds
In another strategy, methods are provided for determining the absence of ionic compounds, detecting the presence of ionic compounds, and/or quantifying ionic compounds, in a sample (e.g. an environmental sample or a biological sample). The ionic compounds may be monovalent or divalent ions. The ionic compounds may be metal ion cofactors required for the activity of a catalytic nucleic acid enzyme.
The methods comprise providing a molecular complex as described herein (a molecular switch) and at least one additional component capable of activating the switch to thereby provide a detectable signal. The functional activity of the additional component may be reliant on the presence of the ionic compound in a sample to be tested. For example, the methods may comprise contacting a sample suspected of containing ionic compounds with a molecular switch comprising a first catalytic nucleic acid enzyme hybridised to and functionally inactivated by a blocker oligonucleotide, and an initiator catalytic nucleic acid enzyme (e.g. a DNAzyme, ribozyme, assembled MNAzyme, or components capable of assembly into an MNAzyme).
The ionic compound may additionally be a co-factor required for catalytic activity of the first catalytic nucleic acid enzyme of the complex.
Consequently the methods may utilise catalytic nucleic acids (e.g. DNAzymes, ribozymes, aptazymes and/or MNAzymes) which require a specific metal ion cofactor for catalytic activity, to detect the presence or determine the absence of the metal ion in a sample (e.g. environmental or biological samples). For example, the methods may facilitate DNAzyme-mediated detection of Pb2+ in an environmental sample such as water.
Aptamers
Persons skilled in the art will readily appreciate that the methods described herein may be performed with aptamers, wherein said aptamers may facilitate the detection, identification and/or quantification of targets including targets other than nucleic acids.
Methods of using MNAzymes to detect targets, including non-nucleic acid entities are contemplated. Such methods may use aptamers which may comprise a nucleic acid or protein, polypeptide, or peptide or combination thereof that has the ability to recognize one or more ligands. Aptamers may bind target ligands, for example, proteins, polypeptides, peptides or nucleic acids, glycoproteins, lipids, lipoproteins, cells, viruses, bacteria, archaea, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, entire organisms, small molecules, polymers, metal ions, metal salts, prions or any derivatives, portions or combinations thereof, or any other entity.
Preferred aptamers herein may comprise short single-stranded DNA or RNA oligomers or peptides that can be isolated from complex libraries of synthetic nucleic acids or peptides by an iterative process of adsorption, recovery, and reamplification. Aptamers may therefore be generated against almost any target/ligand, ranging from small molecules such as amino acids or antibiotics, to protein and nucleic acid structures. In some embodiments, aptamers include, for example, nucleic acid binding molecules which are preferably generated by evolution and selection techniques. Aptamers may comprise DNA or RNA molecules, or a combination of both, including but not limited to the nucleotide analogues as per, for example, Table 1 above.
Persons skilled in the art will appreciate that the aptamer may be incorporated into a DNAzyme, ribozyme or any of the MNAzyme components. DNAzymes and ribozymes which are coupled to aptamers are known in the art as aptazymes. Such aptazymes may have their catalytic activity switched on or off by the presence of ligands with affinity to their aptamer components. Further it will be appreciated that multiple aptamers can be incorporated into one or more of the partzyme oligonucleotide components.
In further exemplary embodiments an aptamer sequence may be incorporated at the end of a partzyme (apta-partzyme) in a configuration whereby an active MNAzyme is only formed in the presence of the target analyte. In this case the partzymes for the MNAzyme detection strategy include; a standard partzyme; an apta-partzyme which is a partzyme with an aptamer incorporated into one of its ends; an assembly facilitator which binds to both the apta-partzyme and the partzyme enabling assembly of an active MNAzyme (in the presence of target); a substrate; and an assembly inhibitor which hybridises to the apta-partzyme in a region which spans at least part of the aptamer sequence and part of the partzyme sequence. In the absence of a target the assembly inhibitor binds to the apta-partzyme thus blocking binding (and cleavage) of the reporter probe substrate. In the presence of a target, the target binds to the aptamer sequence of the apta-partzyme, preventing the binding of the assembly inhibitor and allowing the binding and cleavage of the MNAzyme substrate. As such, an active MNAzyme can only form and modify an MNAzyme substrate in the presence of target.
Methods using Insoluble Supports
It is also to be understood that generally the methods of the present invention may utilise insoluble supports on which one or more reaction component/s such as subZymes, DNAzymes, MNAzyme components (substrate, partzyme or assembly facilitator/target) are attached. For example, methods for detecting targets using an MNAzyme, whereby a reaction component (e.g. a SubZyme, DNAzyme, MNAzyme, or component/s thereof) may be anchored to a support, are contemplated herein. The support may be an insoluble material or a matrix which retains the reaction component and excludes it from freely moving in the bulk of the reaction mixture. Alternatively, the support may be an insoluble material or a matrix that retains the reaction component but otherwise allows mobility of the reaction component in the reaction mixture as it is not fixed to another surface. There are advantages in using signal amplification approaches where catalytic nucleic acid enzymes, substrates, and other components are immobilised onto mobile entities, for example beads, rather than fixed supports/surfaces. For example, beads have an increased surface area to volume ratio which permitts improved surface density of tethered components. Other advantages include miniaturisation which can facilitate (i) enhanced reaction rates by faster diffusion, and (ii) reduced reaction volumes which ultimately lowers reagent costs. Further, beads exhibit a high degree of practicality with respect to their production and incorporation into devices. A skilled person will appreciate that the support can be selected from a wide variety of matrices, polymers, and the like, in a variety of forms including beads convenient for use in microarrays, as well as other materials compatible with the reaction conditions. In certain embodiments, the support may be a plastic material, such as plastic beads or wafers, metallic material, magnetised material, or that of the well or tube in which a particular assay is conducted. In certain embodiments the support may be microcarriers or nanocarriers. In certain embodiments the support may be encoded.
Optimisation of Methods
The skilled person will readily understand that the methods described herein may be optimized using a variety of experimental parameters in order to enhance the detection, identification and/or quantification of a target. The particular experimental parameters that are optimized, and the level of such optimization, will depend upon the particular method being employed and the particular target being sought to be detected, identified and/or quantified. Such parameters include, but are not limited to time, temperature, pH, concentration and identity of salts and buffers, concentrations of oligonucleotides, co-factors, detergents, cations and other reagents including, but not limited to, dimethylsulfoxide (DMSO), EDTA, ATP, glycerol, length of complementarity, GC content and melting point (Tm) of nucleic acids components of MNAzymes, SubZymes, DNAzymes, substrates.
In some embodiments, for example, those methods involving detection of specific nucleic acid sequences, experimental parameters including the temperature at which the method is performed, may be optimized so as to discriminate between binding of an MNAzyme component to a target nucleic acid that does or does not comprise a sequence variation. The temperature at which such methods may be performed may be in the range of about 20° C. to about 96° C., about 20° C. to about 75° C., 20° C. to about 60° C. or about 20 to about 55° C.
In certain embodiments, optimized reactions for practicing the methods described herein are provided. In such optimized reactions, the signal detected is increased by up to 10%, 20%, or 30% above un-optimized reactions. More preferred reaction conditions may improve signal detected by at least 35%, or 40%, and preferably up to 50% or more. In other embodiments, optimized reactions may provide an increase of catalytic activity of more than 50%, and up to 66%, 75% or even 100%. In still other embodiments, a fully optimized reaction method may offer 100%, 200% or even 300% or more increase in signal detection. Other preferred reaction conditions can improve the catalytic activity by up to 1000% or more over methods practiced with unoptimized reaction conditions. A highly preferred reaction condition for optimizing the methods provided herein is the inclusion of certain divalent cations. The catalytic activity of most nucleic acid enzymes and protein nucleic acid-modifying enzymes may be influenced in a concentration-dependent fashion by the concentration of divalent cations. Preferred optimized reactions are optimized for one or more of Ba2+, Sr2+, Mg2+, Ca2+, Ni2+, Co2+, Mn2+, Zn2+, and Pb2+.
Kits
The present invention also provides kits for practising the methods disclosed herein. Typically, kits for carrying out the methods of the present invention contain all the necessary reagents to carry out the method.
The kits may comprise any composition according to the present invention or component(s) thereof. By way of non-limiting example only, the kits may comprise catalytic nucleic acid enzymes (e.g. MNAzymes and/or partzyme components thereof, DNAzymes, SubZymes, aptazymes and/or ribozymes). The kits may optionally contain endonucleases or exonucleases.
The kits may be fragmented kits or combined kits as defined herein. Fragmented kits comprise reagents housed in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. Such kits may also include a container which will accept the test sample, a container which contains the reagents used in the assay, containers which contain wash reagents, and containers which contain a detection reagent. Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods. Kits and methods of the invention may be used in conjunction with automated analysis equipment and systems, for example, including but not limited to, real time PCR machines, spectrophotometer, electrochemical platforms or Surface Plasmon Resonance platforms.
For example, the kit may comprise a first container and second container. The first container may comprise catalytic nucleic acids and/or portions thereof (e.g. MNAzymes, DNAzymes, partzyme oligonucleotides) and/or a SubZymes). The second container may comprise catalytic nucleic acid substrates, and/or different type/s of catalytic nucleic acids and/or portions thereof, and/or different types of SubZymes, as compared to the contents of the first container). The kits may be fragmented kits or combined kits as defined herein.
The kits may also comprise one or more containers, containing for example, wash reagents, and/or other reagents as required in the performance of the methods of the invention.
For application of detection, identification or quantitation of different targets, a single kit of the invention may be applicable, or alternatively different kits, for example containing reagents specific for each target, may be required. Methods and kits of the present invention find application in any circumstance in which it is desirable to detect, identify or quantitate any entity.
The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.
Sequences and Annotations
The Examples below refer to various nucleotide sequences, in a number of cases by sequence identification number (SEQ ID NO). The specific seqeunces utilised in the following Examples are set out in Table 2 below.
Reagents
Streptavidin-functionalised Dynabeads (M-270) were purchased from Invitrogen. The characteristics and properties of streptavidin-functionalised Dynabeads (M-270) provided by the manufacturer are as following: superparamagnetic, hydrophilic bead surface, 2.8 μm in diameter, size distribution (CV<3%), no blocking proteins used, isoelectric point of pH 4.5, highly charged (−50 mV at pH7), iron content of 14%, low aggregation of beads in high salt solutions.
Streptavidin functionalized BioMAG Plus beads (1.5 μm), ProMag beads (1 μm), ProMag HP beads (3 μm) and COMPEL Magnetic beads (6 μm and 8 μm) were purchased from Bangs Laboratories, Inc. The characteristics and properties of these beads provided by the manufacturer are as following: spherical shape, iron oxide crystals dispersed in a polymer matrix, respective magnetite contents of 26.5 wt %, 16 wt %, >90 wt %, 5.7 wt % and wt % and respective densities of 1.8 g/cm3, 1.4 g/cm3, 2.5 g/cm3, 1.1 g/cm3 and 1.1 g/cm3. All beads are supplied at 1% solids (w/v) except for the BioMAG Plus beads which are supplied at 5.0 mg/mL solids.
NaOH (5 M) was purchased from Australian Chemical Reagents (ACR). Tris buffer (pH 8.0), NaCl (5M), MgCl2 (1M) and nuclease free water were purchased from Ambion. 10×PCR Buffer II was purchased from Applied Biosystems. Nitrocellulose membrane was purchased from Bio-Rad, polyethersulfone membrane and polycarbonate membrane were purchased from Sterlitech and double sided sticky tape was purchased from Sellotape.
Human genomic DNA was purchased from Promega (193 ng/μL). All synthetic oligonucleotides were purchased from Integrated DNA Technologies (IDT) and prepared as 20 μM stock solutions using nuclease free water. Chlamydia trachoniatis (CT) (serovar D) TNA samples were obtained in vitro, using standard tissue culture techniques as described below.
The human epithelial cell line (HEp-2) (ATCC® CCL-23™) were grown in Dulbecco's Modified Eagle medium (DMEM) (Sigma Aldrich) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Sigma Aldrich), 100 mg/mL Streptomycin (Gibco®, Invitrogen Corporation), 50 mg/mL Gentamicin (Gibco® by Life Technologies) and 20 mM Glutamine (Sigma Aldrich), incubated at 37° C. with 5% CO2.
Chlamydia trachoniatis was inoculated on a HEp-2 monolayer, present on a T75 flask (Nunc™, Thermo Fisher) with a Multiplicity of Infection (MOI) of 1. The infection was completed by centrifugation-assisted inoculation at 500 g for 30 minutes at a temperature of 28° C. and subsequently incubated. At 4 hours post-infection (PI), DMEM was replaced with addition of 1 mg/mL of Cychlohexamide (Sigma Aldrich) and again incubated at 37° C. with 5% CO2 until harvested. At an exponential growth phase, 24 hours PI, cells were manually harvested using a cell scraper and sucrose-phosphate-glutamate (SPG) buffer (250 mM sucrose, 10 nM sodium phosphate and 5 mM L-glutamate). Harvested material was stored for further processing at −80° C. Total nucleic acid samples (TNA) were extracted using the QIAamp MinElute Virus Spin Kit (QIAgen) following the standard protocol.
Preparation of SubZymeBead Complexes (SubZyme Bead)
Assays described in the Examples below employed SubZymes A to K (SEQ ID No: 1-12) which were attached to Beads using one of the following two protocols. Examples 2-7 used Attachment Method One (3A), and Examples 10-11 and 13-20 used Attachment Method Two (3B). When a SubZyme sequence is attached to a Bead its name includes ‘-Bead’. When a SubZyme sequence is attached to a magnetic bead its name includes ‘-MB’ (e.g. SubZyme A attached to a magnetic bead is named “SubZyme A-MB”).
Attachment Method 1:
The M270 Dynabead storage vial was vortexed and thoroughly mixed to ensure that the magnetic beads (MBs) were homogenously dispersed. Dynabeads were washed three times to remove the preservative and to denature any ribonucleases by washing two times with 100 μL of ‘Wash Buffer A’ (100 mM NaOH, 50 mM NaCl) for three mins each and one time with 100 μL of ‘Wash Buffer B’ (100 mM NaOH) for three mins. SubZyme A and SubZyme B were immobilised onto the MBs using the highly specific biotin-streptavidin interaction. The washed MBs were incubated in a 50 μL final volume containing ‘Incubation Buffer’ (2 M NaCl, 10 mM Tris-HCl pH 7.5) and 5 μM of either SubZyme A or SubZyme B for one hour at room temperature. The incubation mixtures were measured using a spectrophotometer (Trinean Xpose) at A260 before and after incubation to determine the immobilisation efficiencies. The incubation solutions were removed and discarded and the SubZyme-MB complexes were thoroughly washed to remove excess SubZyme molecules. A series of three 5 minute washes were performed in 200 μL of ‘Incubation Buffer’ at 55° C., followed by two 5 minute washes in 200 μL of ‘Reaction Buffer’ (15 mM MgCl2, 1×PCR Buffer II) at 55° C.
The final wash solutions were collected and incubated with 200 nM of the complementary substrate to ensure that the unbound SubZyme strands were thoroughly removed. Quality control tests were performed using the functionalised beads to estimate the concentration of attached SubZyme-MB complexes and to ensure that the DNAzyme component remained catalytically active. Finally, the SubZyme-MBs were stored dry at 5° C. in the fridge.
Attachment Method 2:
The Bead storage vials were vortexed and thoroughly mixed to ensure that the Beads were homogenously dispersed. Beads were washed three times to remove the preservative using 150 μL of Buffer C (1.5 M NaCl, 10 mM Tris-HCl pH 7.5). SubZymes were immobilised onto the Beads using the highly specific biotin-streptavidin interaction. The washed Beads were incubated in a 50 μL final volume containing ‘Buffer C’ (1.5 M NaCl, 10 mM Tris-HCl pH 7.5) and 150 nM of SubZyme for five minutes at room temperature. The incubation solutions were removed and discarded and the SubZyme-Bead complexes were thoroughly washed to remove excess SubZyme molecules. SubZyme-Beads were washed once with 400 μL of Buffer C (1.5 M NaCl, 10 mM Tris-HCl pH 7.5) followed by five washes with 400 μL of nuclease free water and two washes performed in 150 μL of ‘Reaction Buffer’ (45 mM MgCl2, 1×PCR Buffer II) at 55° C. Finally, the SubZyme-Beads were re-suspended in 20 of nuclease free water and stored at 5° C. in the fridge. Magnetic beads were washed using a magnetic rack to retain magnetic beads and discarding the supernatant. Non-magnetic beads were washed by centrifuging the silica bead solution for 3 minutes at 10,000 rpm to pellet the beads followed by removing and discarding the supernatant.
Preparation of Permeable barriers (“T-Bags”)
Some assays described in the Examples below employed permeable barriers (T bags,) that were prepared using one of the following two protocols. Examples 2-7 used T bag Method One (3A) and Examples 10-20 used T bag Method Two (3B).
T Bag Method 1:
Some of the permeable barriers (T bags) were manually prepared according to the diagram depicted in
T Bag Method 2:
Some of the permeable barriers (T bags) were manually prepared according to the diagram depicted in
In this experiment, the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NO: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), TFRC Partzyme A1 (SEQ ID NO: 15), TFRC Partzyme B1 (SEQ ID NO: 16) and Target TFRC Oligo (SEQ ID NO: 17). The sequences are listed in the Sequence Listing. With reference to
Each reaction contains 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 1×PCR Buffer II, 5.6 μL of SubZyme A-MB, 1 T-Bag containing SubZyme B-MB and 15 mM MgCl2 in a 125 μL final volume. Reactions either contained target TFRC oligo at final concentrations of 1 pM, 100 fM, 1 fM and 500 aM; or lacked target (no DNA control). Reactions were incubated at 50° C. on a heat block in 2 mL reaction tubes (Eppendorf) for either 15 minutes (
After the initial incubation, samples were transferred from the heat-block and the T bags were removed and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme-MBs and cleaved partial substrate-MB fragments from cleaved “free” DNAzymes 1 and 2 present in the supernatant. Two aliquots of 48 of the supernatant were transferred to separate wells in a 96 well plate (Bio-Rad). To each of these wells, 2 μL of substrate mix containing equimolar concentrations of dual labelled Substrate A and Substrate B were added (0.15 μM and 0.2 μM final concentrations for
The results shown in
These results are consistent with the following scenario. In the presence of target oligonucleotide the partzymes align and form a MNAzyme. The MNAzyme cleaves SubZyme A-MB in solution separating the 8-17 DNAzyme from the surface of the MB. The “free” 8-17 DNAzyme can subsequently migrate through the T bag selectively permeable membrane where it can cleave SubZyme B-MB causing separation of the 10-23 DNAzyme from the surface of the MB. The 10-23 DNAzyme is then free to migrate out of the T bag permeable membrane into solution where it can cleave SubZyme A-MB and continue the cascade. After a period of incubation a magnet is used to separate the un-cleaved SubZyme-MBs and cleaved partial substrate-MB fragments from the cleaved ‘free’ DNAzymes.
Fluorescence from the Texas Red channel was measured using a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 5 seconds for a total of 201 or 150 cycles (
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NO: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), TFRC Partzyme A1 (SEQ ID NO: 15), TFRC Partzyme B1 (SEQ ID NO: 16), Target TFRC Oligo (SEQ ID NO: 17), Off-target Oligo Bla_KPC (SEQ ID NO: 18) and Off-target Oligo AF-CTcds2_2 (SEQ ID NO: 19). The sequences are listed in the Sequence Listing. With reference to
Each reaction contained 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 1×PCR Buffer II, 2.5 μL of SubZyme A-MB, 1×T-Bag containing SubZyme B-MB and 15 mM MgCl2 in 125 μL final volume. Reactions further contained either 1 pM of Target TFRC Oligo or 1 nM off target Oligo Bla_KPC or 1 nM of off target Oligo CTcds2_2 or no DNA target. The reactions were incubated at 50° C. for 20 minutes in 2 mL reaction tubes on a heat block with intermittent mixing via manual inversion. After the initial incubation, the samples were transferred from the heat-block and the T bags were removed and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme-MBs and cleaved partial substrate-MB fragments from cleaved “free” DNAzymes. Two aliquots of 48 μL of the supernatant were transferred to a 96 well plate (Bio-Rad) and 2 μL of substrate mix containing equimolar concentrations of Substrate A and Substrate B (0.15 μM final concentration) were added to each of the wells. The plate was sealed and briefly centrifuged. The plate was placed on a CFX96 real time PCR detection system (Bio-Rad) with acquisition every 5 seconds for 150 cycles at a constant temperature of 50′C. Fluorescence from the Texas Red channel was monitored in real time. The results in
The results in
The results presented in
In this experiment it was demonstrated that the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NO: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), OXA Partzyme A (SEQ ID NO: 20), OXA Partzyme B (SEQ ID NO: 21) and Target OXA Oligo (SEQ ID NO: 22). The sequences are listed in the Sequence Listing.
Each reaction contained 0.2 μM OXA Partzyme A, 0.2 μM OXA Partzyme B, 1×PCR Buffer II, 2.45 μL of SubZyme A-MB, 1×T-Bag containing SubZyme-MB and 15 mM MgCl2 in 125 μL final volume. The reactions were incubated at 50° C. in 2 mL reaction tubes for 20 minutes on a heat block with intermittent mixing via manual inversion. Reactions either contained 10 pM of the Target OXA Oligo or no DNA.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme A-MBs and cleaved partial substrate-MB fragments from cleaved DNAzymes freed into solution. Two aliquots of 48 μL of the supernatant were transferred to separate wells in a 96 well plate and 2 μL of substrate mix containing equimolar concentrations of Substrate A and Substrate B (0.2 μM final concentration) were added to each of the wells. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 5 seconds for 150 cycles at a constant temperature of 50° C. The results shown in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NOo: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), TFRC Partzyme A1 (SEQ ID NO: 15) and TFRC Partzyme B1 (SEQ ID NO: 16). The sequences are listed in the Sequence Listing.
Each of the reactions contained 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 1×PCR Buffer II, 1 μL of SubZyme A-MB, 1×T-Bag containing SubZyme B-MB, and 15 mM MgCl2 in 70 μL final volume. Reactions also contained 772 ng of genomic DNA which had been heat denatured at 95° C. for 2 minutes. The reactions were performed in the absence (Reaction in
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate the un-cleaved SubZyme A-MBs and partial cleaved substrate A-MB fragments from cleaved DNAzymes freed into solution. One 48 μL aliquot of supernatant from each reaction was transferred to a 96 well plate (Bio-Rad) and then mixed with 2 μL of substrate mix containing equimolar concentrations of Substrate A and Substrate B (0.2 μM final concentration) and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 111 cycles at a constant temperature of 50° C.
The results presented in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NO: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), TFRC Partzyme A1 (SEQ ID NO: 15), TFRC Partzyme B1 (SEQ ID NO: 16), TFRC Partzyme A2 (SEQ ID NO: 23), TFRC Partzyme B2 (SEQ ID NO: 24), TFRC Partzyme A3 (SEQ ID NO: 25) and TFRC Partzyme B3 (SEQ ID NO: 26). The sequences are listed in the Sequence Listing.
The reactions contained 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 0.2 μM TFRC Partzyme A2, 0.2 μM TFRC Partzyme B2, 0.2 μM TFRC Partzyme A3, 0.2 μM TFRC Partzyme B3, 1×PCR Buffer II, 1 μL of SubZyme A-MB, 1×T bag containing SubZyme B-MB, and 15 mM MgCl2 in 70 μL final volume. Reactions also contained, or lacked, human genomic DNA which had been subjected to heat denaturation at 95° C. for 2 minutes before its addition to the reaction. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block with intermittent mixing via manual inversion.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme A-MBs and cleaved partial substrate A-MB fragments from DNAzymes freed into solution. A 48 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate mix containing equimolar concentrations of Substrate A and Substrate B (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 120 cycles at a constant temperature of 50° C.
The results presented in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NO: 1), PB (SubZyme B) (SEQ ID NO: 2), Substrate A (SEQ ID NO: 13), Substrate B (SEQ ID NO: 14), TFRC Partzyme A1 (SEQ ID NO: 15) and TFRC Partzyme B1 (SEQ ID NO: 16). The sequences are listed in the Sequence Listing.
Each of the reactions contain 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 1×PCR Buffer II, 2.5 μL of SubZyme A-MB, 1×T-Bag comprising SubZyme B-MB, and 15 mM MgCl2 in 125 μL final volume. Reactions ether lacked genomic DNA or contained 965 ng or 579 ng of human genomic DNA which had been heat denatured at 95° C. for 2 minutes. The reactions were incubated at 50° C. for 20 minutes in 2 mL reaction tubes on a heat block with intermittent mixing via manual inversion. The experiment was performed in the presence of 965 ng (solid line), 579 ng (dashed line) or absence (dotted line) of human genomic DNA.
After the initial incubation, the samples were transferred from the heat-block and the T bag was removed from the tube and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate the un-cleaved SubZyme A-MBs and cleaved partial substrate A-MB fragments from DNAzymes freed into solution. Two 48 μL aliquots of the supernatant were transferred to separate wells in a 96 well plate (Bio-Rad) and mixed with 2 of substrate containing equimolar concentrations of Substrate A and Substrate B (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 5 seconds for 150 cycles at a constant temperature of 50° C. The results are the averages from duplicates that were plotted using Microsoft Excel (Version 14).
In this experiment, the long-term stability of two exemplary SubZymes were compared. SubZyme A is comprised of an 8-17 catalytic nucleic acid component and a 10-23 catalytic nucleic acid substrate component. SubZyme C is comprised of a 10-23 catalytic nucleic acid component and a 10-23 catalytic nucleic acid substrate component. The results obtained are shown in
The oligonucleotides specific to this experiment include; PA (SubZyme A) (SEQ ID NOo: 1), PA (SubZyme C) (SEQ ID NO: 3), TFRC Partzyme A1 (SEQ ID NO: 15), TFRC Partzyme B1 (SEQ ID NO: 16), Target TFRC Oligo (SEQ ID NO: 17), Target TFRC Oligo 4 (SEQ ID NO: 27) and DNAzyme A (SEQ ID NO: 28). The sequences are listed in the sequence listing.
SubZyme C is comprised of a 10-23 catalytic nucleic acid component and a 10-23 catalytic nucleic acid substrate component, the substrate component contains an internal fluorescein and two internal ZEN Quencher moieties. Reactions A, B, C and D contain 0.2 μM TFRC Partzyme A1, 0.2 μM TFRC Partzyme B1, 0.2 μM SubZyme C, 1×PCR Buffer II, and 25 mM MgCl2 in a 20 μL final reaction volume. All reactions were performed in duplicate at 52° C. in a Bio-Rad® CFX96 thermocycler and fluorescence was measured in FAM channel with acquisition taking place every 5 seconds (scan mode: SYBR/FAM Only) for a total of 201 cycles for reactions A and B and 200 cycles for reactions C and D. Reactions A and C do not contain Target TFRC Oligo. Reaction B contains 1581 pg (5 nM) of Target TFRC Oligo 4 and Reaction D contains 512 pg (2 nM) of Target TFRC Oligo.
SubZyme A is comprised of an 8-17 catalytic nucleic acid component and a 10-23 catalytic nucleic acid substrate component, the substrate component contains an internal fluorescein and two internal ZEN Quencher moieties. Reactions E, F, G and H contain 0.2 μM SubZyme A, 1×PCR Buffer II, and 25 mM MgCl2 in a 20 μL final reaction volume. All reactions were performed in duplicate at 50° C. in a Bio-Rad® CFX96 thermocycler and fluorescence signal was measured in FAM channel with acquisition taking place every 5 seconds (scan mode: All channels) for a total of 201 cycles. Reactions E and G do not contain any initiating DNAzyme and Reactions F and H each contain 503 pg (2.5 nM) of initiating DNAzyme.
The results from reactions A and B are shown in
The results from reactions E and F are shown in
The following example highlights some of the issues encountered when SubZymes are tethered onto planar (immobile) surfaces but were not evident when SubZymes were tethered onto mobile three-dimensional surfaces including, but not limited to, magnetic beads. In this experiment, SubZyme activity is monitored after immobilization onto glass slides. The activity of the immobilized SubZyme (Reaction B) is compared with an untethered (free) DNAzyme (Reaction A) and the results obtained are shown in
The oligonucleotides specific to this experiment include; SubZyme D (SEQ ID NO: 4), Substrate C (SEQ ID NO: 29) and DNAzyme B (SEQ ID NO: 30). The sequences are listed in the Sequence Listing.
The control reaction (Reaction A) was performed using a blank aldehyde slide that had been washed according to the washing procedure described in Stralis-Pavese et al (2011), Nat Protoc. 2011; 6(5):609-24. The test reaction (Reaction B) was performed on a VSS-25 Vantage Silyated Aldehyde slide that had been functionalised with SubZyme D, also using methods described in Stralis-Pavese et al (2011) Nature Protocols, 6(5), 609-624. Each reaction contains 0.4 μM Substrate C, 0.02% Tween-20, 25 mM MgCl2 and 1×PCR Buffer II in a 25 μL final volume. Reaction A contained 50 nM of DNAzyme D which is homologous to the catalytic nucleic acid component of SubZyme D. All reactions occurred within Hybriwell adhesive chambers (Grace Bio-Labs, 611204) placed on top of the slide. The slides were incubated on top of a heat-block set at 54° C. Fluorescence was measured before and after 30 minutes incubation using a Leica fluorescent microscope with the following settings; GFP2 emission, PMT Gain set at 3.5 and an exposure time of 59.7 sec. The images were processed using Image J analysis software where the 8 bit green channels were isolated and the RGB values were measured. The images are shown in
The results shown in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme E) (SEQ ID NO: 5), Substrate D (SEQ ID NO: 31) and Target ompA Oligo 1 (SEQ ID NO: 32). The sequences are listed in the Sequence List. SubZyme E is attached to magnetic beads (SubZyme E-MB) as described in Example One—Attachment Method 2.
All reactions contained 1×NEB Buffer 3.1, 5 mM MgCl2 and 0.5 μL of SubZyme E-MBs in 55 μL final volume. Reactions A contained 4 units Nt.BstNBI and 2 nM Target ompA oligo 1. Reactions B contained 2 nM Target ompA oligo 1 and lacked Nt. BstNBI. Reactions C contained 4 units of Nt. BstNBI and lacked Target ompA oligo 1. Reactions were incubated at 55° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the samples were briefly centrifuged. A 48 μL aliquot of the reaction solution was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate mix containing 0.2 μM (final concentration) of Substrate D. The plate was sealed and briefly centrifuged. Fluorescence from the FAM channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results shown in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; SubZyme F (SEQ ID NO: 6), Substrate E (SEQ ID NO: 33) and initiating DNAzyme A (SEQ ID NO: 28). The sequences are listed in the Sequence List.
The reactions contained 1×PCR Buffer II, 45 mM MgCl2 in a 60 μL final volume. Reactions contained 1×T-Bag made of polycarbonate material (either 0.8 μm or 2 μm) containing SubZyme F-MBs. Reactions contained, or lacked, 2 nM of DNAzyme A. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the samples were briefly centrifuged. A 48 aliquot of the reaction solution was transferred to a 96 well plate (Bio-Rad) and mixed with 2 of Substrate E (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results shown in
The following example highlights the importance of empirically testing the use of different membrane materials for the construction of T bags to determine if the material itself can impact upon the catalytic activity of the reaction constituents. Further, this example indicates that the presence of some membrane materials such as polyethersulfone (PES) may enhance the catalytic activity of DNAzymes.
The oligonucleotides specific to this experiment include; PA (SubZyme G) (SEQ ID NO: 7), Substrate F (SEQ ID NO: 34) and DNAzyme C (SEQ ID NO: 35). The sequences are listed in the Sequence Listing. In this example, PA (SubZyme G) is attached to magnetic beads (SubZyme G-MB) as described in Example One—Attachment Method 2.
The reactions contained 1×PCR Buffer II, 1 μL SubZyme G-MBs, 45 mM MgCl2 in a 60 μL final volume and either lacked or contained 2 nM of DNAzyme C. Reactions either contained or lacked 2×3 mm discs of PES membrane and were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the samples were briefly centrifuged. Using a magnetic rack to retain the magnetic beads (uncleaved SubZyme G-MBs and MB-partial substrate portion of cleaved SubZyme G-MBs), a 45 μL aliquot of the reaction solution was transferred to a 96 well plate (Bio-Rad) and mixed with 5 μL of Substrate F (0.2 μM final concentration). The PES membrane was discarded. The plate was sealed and briefly centrifuged. Fluorescence from Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C. All reactions were performed in duplicate and the results are presented in
In this experiment the addition of PES membrane to reactions of DNAzyme cleavage of SubZyme-MB appears to enhance DNAzyme activity. This was observed as a faster increase in fluorescence over time for reactions containing PES membrane compared with reactions lacking membrane. This indicates faster cleavage of fluorescent substrates by DNAzymes following their release from the surface of magnetic beads.
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PB (SubZyme F) (SEQ ID NO: 6), Substrate E (SEQ ID NO: 33) and DNAzyme A (SEQ ID NO: 28). The sequences are listed in the Sequence Listing.
The reactions contained 1×PCR Buffer II, 45 mM MgCl2 in a 60 μL final volume. Reactions contained 1×T bag made of PES material (either 0.65 μm, 0.8 μm or 1.2 μm) containing 0.6 μL of SubZyme F-MBs. Reactions contained, or lacked, 2 nM of DNAzyme A. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the samples were briefly centrifuged. A 48 μL aliquot of the reaction solution was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of Substrate E (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results shown in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme H) (SEQ ID NO: 8), PB (SubZyme I) (SEQ ID NO: 9), Substrate G (SEQ ID NO: 36), Substrate H (SEQ ID NO: 37), ompA Partzyme A1 (SEQ ID NO: 38), ompA Partzyme B1 (SEQ ID NO: 39), Target ompA Oligo 2 (SEQ ID NO: 40), Off-Target Oligo p273 (SEQ ID NO: 41) and Off-Target Oligo PPIA (SEQ ID NO: 42). The sequences are listed in the Sequence Listing.
The reactions contained 5 nM ompA Partzyme A1, 5 nM ompA Partzyme B1, 1×PCR Buffer II, 0.5 μL of SubZyme H-MB, 1×T bag containing 0.5 μL of SubZyme I-MB, and 45 mM MgCl2 in 70 μL final volume. T bags were made of polycarbonate membrane of 0.8 μm pore size. Reactions either contained target ompA oligo 2 at final concentrations of 100 fM, 10 fM and 1 fM, or 1 nM off target Oligo p273, or 1 nM of off target Oligo PPIA, or lacked target (no DNA control). Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme H-MBs and cleaved partial substrate H-MB fragments from DNAzymes freed into solution. A 48 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate mix containing equimolar concentrations of Substrate G and Substrate H (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the FAM channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results shown in
These results are consistent with the following scenario. In the presence of target oligonucleotide the Partzymes align and form an active initiating MNAzyme. The MNAzyme cleaves SubZyme H-MB in solution separating the 8-17 DNAzyme from the surface of the MB. The “free” 8-17 DNAzyme can subsequently migrate through the T bag selectively permeable membrane where it can then cleave SubZyme I-MB causing separation of the 10-23 DNAzyme from the surface of the MB. The 10-23 DNAzyme is then free to migrate out of the T bag permeable membrane into solution where it can cleave SubZyme H-MB and continue the cascade. After a period of incubation a magnet can separate the un-cleaved SubZyme-MBs and cleaved partial substrate-MB fragments from the cleaved ‘free’ DNAzymes. The free DNAzymes (10:23 and 8:17) can be transferred into a second reaction chamber containing fluorescent substrates, within, the free DNAzymes can cleave substrates and generate fluorescence signal.
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme H) (SEQ ID NO: 8), PB (SubZyme I) (SEQ ID NO: 9), Substrate G (SEQ ID NO: 36), Substrate H (SEQ ID NO: 37), ompA Partzyme A1 (SEQ ID NO: 38), ompA Partzyme B1 (SEQ ID NO: 39), and Target ompA Oligo 2 (SEQ ID NO: 40). The sequences are listed in the Sequence Listing.
The reactions contained 5 nM ompA Partzyme A1, 5 nM ompA Partzyme B1, 1×PCR Buffer II, 0.5 μL of SubZyme H-MB, 1×T bag containing 0.5 μL of SubZyme I-MB, and 25 mM MgCl2 in 70 μL final volume. T bags were made of polycarbonate membrane of 0.8 μm pore size. Reactions either lacked DNA (No DNA control), or contained 2 fM of target ompA oligo, or contained Chlamydia trachomatis TNA which was the TNA had been subjected to heat denaturation at 95° C. for 2 minutes before its addition to the reaction. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme H-MBs and cleaved partial substrate H-MB fragments from DNAzymes freed into solution. A 48 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate mix containing equimolar concentrations of Substrate G and Substrate H (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the FAM channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results presented in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme H) (SEQ ID NO: 8), (PB) SubZyme I (SEQ ID NO: 9), Substrate H (SEQ ID NO: 37), ompA Partzyme A1 (SEQ ID NO: 38), ompA Partzyme B1 (SEQ ID NO: 39), and Target ompA Oligo 2 (SEQ ID NO: 40). The sequences are listed in the Sequence Listing.
The reactions contained 5 nM ompA Partzyme A1, 5 nM ompA Partzyme B1, 1×PCR Buffer II, 0.5 μL of SubZyme H-MB, 1×T bag containing 0.5 μL of SubZyme I-MB, and 25 mM MgCl2 in 60 μL final volume. T bags were made of polycarbonate membrane of 0.8 μm pore size. Reactions either lacked DNA (No DNA control), or contained 500 pM or 2 pM of target ompA oligo. Reactions were incubated at 50° C. for 12 minutes in 2 mL tubes on a heat block.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme H-MBs and cleaved partial substrate H-MB fragments from DNAzymes freed into solution. A 46 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 4 μL of substrate Substrate H (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the FAM channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results presented in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme J) (SEQ ID NO: 10), PB (SubZyme K1) (SEQ ID NO: 11), PC (SubZyme K2) (SEQ ID NO: 12), Substrate E (SEQ ID NO: 33), ompA Partzyme A1 (SEQ ID NO: 38), ompA Partzyme B1 (SEQ ID NO: 39), Target ompA Oligo 2 (SEQ ID NO: 40) and AF-B3 (Feedback Assembly Facilitator) (SEQ ID NO: 43). The sequences are listed in the Sequence Listing.
The reactions contained 50 nM ompA Partzyme A1, 50 nM ompA Partzyme B1, 1×PCR Buffer II, 45 mM MgCl2. 2 nM SubZyme K2, 2 nM Feedback Assembly Facilitator, 0.6 of PA (SubZyme J-MB), 1×T bag containing 0.6 μL of PB (SubZyme K1-MB) in 60 μL final volume. Reactions either lacked DNA (No DNA control), or contained 2 nM, 200 pM, 50 pM, 10 pM or 1 pM of target ompA oligo. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme J-MBs, cleaved partial substrate J-MB fragments and un-cleaved SubZyme K1-MBs from DNAzymes and Partzymes freed into solution. A 48 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate Substrate E (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results presented in
This experiment demonstrates that SubZymes composed entirely of 8-17 catalytic nucleic acid components can be used to execute a cross-catalytic signal amplification cascade using SubZymes and selectively permeable barriers. The strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme L) (SEQ ID NO: 44), PB (SubZyme M) (SEQ ID NO: 45), Substrate E (SEQ ID NO: 33) and DNAzyme D (SEQ ID NoO: 46). The sequences are listed in the Sequence List. SubZyme L and SubZyme M were attached to magnetic beads (SubZyme L-MB and SubZyme M-MB) as described in Example One—Attachment Method Two. The T bags were manually prepared using polycarbonate membrane (0.8 μm) according to the diagram depicted in
The reactions contained 1×PCR Buffer II, 1.5× concentration of SubZyme L-MB, 1×T bag containing 1.5× concentration of SubZyme M-MB, and 45 mM MgCl2 in 60 μL final volume. Reactions either lacked initiating DNA (No DNAzyme control), or contained 2 nM, 200 pM or 20 pM of initiating DNAzyme (DNAzyme D). Reactions were incubated at 48° C. for 60 minutes in 2 mL tubes on a heat block.
After the initial incubation, the samples were transferred from the heat-block and the T bags were removed from the tubes and discarded. Samples were briefly centrifuged and the tubes were placed on a magnetic rack to separate un-cleaved SubZyme L-MBs and cleaved partial substrate L-MB fragments from DNAzymes freed into solution. A 48 μL aliquot of the supernatant was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of substrate Substrate E (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 48° C.
The results presented in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PB (SubZyme F) (SEQ ID NO: 6), Substrate E (SEQ ID NO: 33) and DNAzyme A (SEQ ID NO: 28). The sequences are listed in the Sequence Listing.
The reactions contained 1×PCR Buffer II, 45 mM MgCl2 in a 60 μL final volume. Reactions contained 1×T bag made of either polycarbonate material (0.8 μm) or PES material (0.8 μm) containing 0.6 μL of SubZyme F-SBs. Reactions contained, or lacked, 2 nM of DNAzyme A. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block. After the initial incubation, the samples were transferred from the heat-block and the samples were briefly centrifuged. A 48 μL aliquot of the reaction solution was transferred to a 96 well plate (Bio-Rad) and mixed with 2 μL of Substrate E (0.2 μM final concentration). The plate was sealed and briefly centrifuged. Fluorescence from the Texas Red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every 10 seconds for 150 cycles at a constant temperature of 50° C.
The results shown in
In this experiment the strategy outlined in
The oligonucleotides specific to this experiment include; PA (SubZyme J) (SEQ ID NO: 10), PB (SubZyme K1) (SEQ ID NO: 11), PC (SubZyme K2) (SEQ ID NO: 12), Substrate E (SEQ ID NO: 33), ompA Partzyme A1 (SEQ ID NO: 38), ompA Partzyme B1 (SEQ ID NO: 39), Target ompA Oligo 2 (SEQ ID NO: 40) and AF-B3 (Feedback Assembly Facilitator) (SEQ ID NO: 43). The sequences are listed in the Sequence Listing.
T bag reactions contained 50 nM ompA Partzyme A1, 50 nM ompA Partzyme B1, 1×PCR Buffer II, 45 mM MgCl2, 2 nM Feedback Assembly Facilitator (AF-B3), 0.6 μL of PA (SubZyme J-MB), 0.6 μL of PC (SubZyme K2-MB) and 1×T bag containing 0.6 μL of PB (SubZyme K1-MB) in 60 μL final volume. MNAzyme control reactions contained 50 nM ompA Partzyme A1, 50 nM ompA Partzyme B1, 1×PCR Buffer II and 45 mM MgCl2 in 60 μL final volume. Reactions either lacked DNA (No DNA control), or contained 500 pM, 200 pM, 100 pM or 50 pM of target ompA oligo. Reactions were incubated at 50° C. for 20 minutes in 2 mL tubes on a heat block.
After incubation, the samples were removed from the heat-block and were briefly centrifuged. An aliquot (40 μL) of the solution was transferred to a 96 well plate (Bio-Rad), the plate was sealed and briefly centrifuged. Fluorescence from the Texas red channel was measured in a CFX96 real time PCR detection system (Bio-Rad) with acquisition taking place every ten seconds for five cycles at a constant temperature of 50° C.
The results in
The present application claims priority from Australian provisional application number 2017901321 filed on 11 Apr. 2017, the entire contents of which are incorporated herein by cross reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017901321 | Apr 2017 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2018/000052 | 4/11/2018 | WO | 00 |
