WO2008122084A1 - Nucleic acid enzymes and complexes and methods for their use - Google Patents
Nucleic acid enzymes and complexes and methods for their use Download PDFInfo
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- WO2008122084A1 WO2008122084A1 PCT/AU2008/000492 AU2008000492W WO2008122084A1 WO 2008122084 A1 WO2008122084 A1 WO 2008122084A1 AU 2008000492 W AU2008000492 W AU 2008000492W WO 2008122084 A1 WO2008122084 A1 WO 2008122084A1
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- C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
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- C12Q2565/10—Detection mode being characterised by the assay principle
- C12Q2565/101—Interaction between at least two labels
- C12Q2565/1015—Interaction between at least two labels labels being on the same oligonucleotide
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/9005—Enzymes with nucleic acid structure; e.g. ribozymes
Definitions
- the present invention relates to methods that utilise multi-component nucleic acid (MNA) complexes.
- MNA complexes may be active enzymes (e.g. MNAzymes including apta-MNAzymes) with modifying activity such as ligase or cleavage activity.
- MNAzymes including apta-MNAzymes
- modifying activity such as ligase or cleavage activity.
- cascades which use MNAzymes and may also include one or more DNAzymes.
- the invention also relates to methods which use these cascades for the identification, detection and quantification of targets.
- nucleic acids have been contemplated for therapeutic entities, biosensors, nano-scale devices and tools for molecular computation.
- the methods exploit the characteristics of DNA self-assembly, electro-conductivity, information elements, amplification, switching, molecular detection and catalytic activity. Further, since DNA is robust, stable and thermostable it provides an ideal material for molecular engineering of mechanical or computational devices.
- Single stranded nucleic acids such as DNA and RNA, have the ability to fold into complex three-dimensional structures that can function as highly specific receptors (e.g. aptamers) and catalysts (e.g. ribozymes, DNAzymes). Further, the requirement for complementarity between nucleic acid strands for hybridization forms the basis for a wide range of techniques, which allow target detection (e.g. microarray analysis, Northern blotting or Southern blotting), and/or target amplification (e.g. the polymerase chain reaction). Further, hybridization provides the basis for nucleic acid nano-scale construction and for DNA based computational strategies.
- target detection e.g. microarray analysis, Northern blotting or Southern blotting
- target amplification e.g. the polymerase chain reaction
- RNA enzymes occur in nature but can be engineered to specifically recognize and modify a target RNA substrate (Haseloff and Gerlach, 1988).
- In vitro evolution techniques have facilitated the discovery and development of many more catalytic nucleic acids, including deoxyribonucleic acids often referred to as "DNA enzymes” or “DNAzymes” (reviewed Emillson and Breaker, 2002).
- DNAzymes and/or ribozymes which have the capacity to catalyse a broad range of reactions including, but not limited to, cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template- directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photo
- DNAzymes and ribozymes have been characterized which specifically cleave distinct nucleic acid sequences after hybridizing via Watson Crick base pairing.
- DNAzymes are capable of cleaving either RNA (Breaker and Joyce, 1994; Santoro and Joyce, 1997; Santoro and Joyce, 1998) or DNA (Carmi et ai, 1996) molecules.
- Ribozymes are also able to cleave both RNA (Haseloff and Gerlach, 1988) and DNA (Raillard and Joyce, 1996) target sequences.
- the "10:23" and "8: 17" DNAzymes are capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds.
- DNAzymes cleave native 3 '-5' phosphodiester linkages to create reaction products which have 2', 3 '-cyclic phosphate and 5'-hydroxyl groups (Santoro and Joyce, 1997; reviewed Emilsson and Breaker, 2002).
- DNAzymes which specifically ligate distinct nucleic acid sequences after hybridizing to two independent substrate nucleic acids have also been characterized.
- Specific deoxyribozymes can ligate 2', 3 '-cyclic phosphate and 5'- hydroxyl products and create non-native 2 '-5' linkages.
- Examples of such DNAzymes include the "7Z81" and “7Z48” ligases (Prior et al, 2004) as well as the "7Q10" DNAzyme ligase (Flynn-Charlebois et al, 2003).
- Other DNAzymes have been described (Coppins and Silverman, 2004) which can ligate 2', 3'diol and 5 '-triphosphate products and create native 3 '-5' linkages.
- catalytic nucleic acids have similar basic structures with multiple domains including a conserved catalytic domain ("catalytic core") flanked by two non- conserved substrate-binding domains ("arms”), which specifically recognize and hybridise to the substrate.
- Examples of nucleic acids with this basic structure include, but are not limited to, the hammerhead ribozyme, the 10:23 and 8:17 DNAzymes, the "7Z81", “7Z48” and “7Q10” DNAzyme ligases, the "UVlC” thymine dimer photoreversion DNAzyme and the "DAB22" carbon-carbon bond forming DNAzyme.
- catalytic nucleic acids are typically uni-molecular although examples of catalytic nucleic acids with more than one component are known but these required extensive engineering (Tabor et al, 2006, Kurata et al, 2000).
- Catalytic nucleic acids have been shown to tolerate only certain modifications in the area that forms the catalytic core (Perreault et al., 1990; Perreault et al, 1991; Zaborowska et al, 2002; Cruz et al, 2004; Silverman, 2004). Depending on the stringency of the reaction conditions, some degree of mismatch may be tolerated within the substrate arms. However, the requirement for Watson Crick base pairing is sufficiently strict so as to have enabled the development of protocols that use catalytic nucleic acids to facilitate the discrimination of closely related sequences (Cairns et al., 2000) (WO 99/50452).
- Target amplification and detection technologies such as PCR have been widely used in research and/or in clinical diagnostics.
- protein enzymes e.g. DNA polymerase, RNA polymerase, reverse transcriptase, and or ligase.
- DNA polymerase e.g. DNA polymerase, RNA polymerase, reverse transcriptase, and or ligase.
- the inclusion of protein enzymes increases the complexity and cost of reagent manufacture and decreases the shelf life of kits containing reagents.
- Other associated technical challenges include contamination by replicons (target amplicons) from previous reactions leading to false positive signal, and/or background signal caused by replication of primer sequences (primer-dimers) or background caused by target-independent ligation.
- nucleic acid enzyme cascades which comprise a plurality of nucleic acid enzymes and in particular at least one multicomponent nucleic acid enzyme (MNAzyme).
- MNAzyme multicomponent nucleic acid enzyme
- the present invention provides detection methods involving nucleic acid enzyme cascades which incorporate at least one nucleic acid enzyme with ligase activity.
- nucleic acid enzymes with ligase activity allows formation of components for nucleic acid complexes, such as assembly facilitators, partzymes, substrates, DNAzymes or components thereof.
- a method for the detection of a target comprising a) providing a first nucleic acid complex comprising components which require association with said target to facilitate formation of a first nucleic acid enzyme, wherein said first nucleic acid complex further comprises at least one substrate, and b) providing at least one component of a second nucleic acid complex capable of associating with a product of a reaction catalysed by the first nucleic acid enzyme and forming a second nucleic acid complex capable of functioning as a second nucleic acid enzyme wherein at least one of said nucleic acid enzymes has ligase activity and at least one of said nucleic acid enzymes is an MNAzyme, and wherein said method comprises: i) contacting a sample putatively containing a target with the first nucleic acid complex, wherein said target associates with said first nucleic acid complex to facilitate formation of said first nucleic acid enzyme, and ii) contacting a product of the reaction cataly
- the method may further comprise providing at least one component of a third nucleic acid complex capable of associating with a product of a reaction catalysed by the first or second nucleic acid enzyme or both and forming a third nucleic acid complex capable of functioning as a third nucleic acid enzyme wherein said method comprises contacting a product of the reaction catalysed by the first or second nucleic acid enzyme or both with said at least one component of the third nucleic acid complex thereby permitting formation of a third nucleic acid complex which functions as said third nucleic acid enzyme, and wherein activity of the first, second, or third nucleic acid enzymes or any combination thereof is indicative of the presence of said target.
- the method may further comprise providing at least one component of at least one additional nucleic acid complex capable of associating with a product of at least one of said first, second or third nucleic acid enzymes and forming at least one additional nucleic acid complex capable of functioning as at least one additional nucleic acid enzyme wherein said method comprises contacting a product of the reaction catalysed by at least one of said first, second or third nucleic acid enzymes with said at least one component of the at least one additional nucleic acid complex thereby permitting formation of at least one additional nucleic acid complex which functions as said at least one additional nucleic acid enzyme, and wherein activity of the first, second, third or at least one additional nucleic acid enzymes or any combination thereof is indicative of the presence of said target.
- the target may be a nucleic acid.
- At least one of said components may further comprise at least one aptamer and wherein said method may further comprise providing at least one assembly facilitator and at least one assembly inhibitor of said first nucleic acid complex wherein said target associates with said aptamer allowing said first nucleic acid complex to function as a first nucleic acid enzyme.
- the target is a non-nucleic acid target.
- the assembly facilitator may be an additional target to be detected.
- a method for the detection of a first assembly facilitator using a cascade comprising a) providing components for a first MNAzyme, at least a first MNAzyme substrate, a first DNAzyme ligase, a first DNAzyme ligase substrate, a second assembly facilitator, at least a second MNAzyme substrate, and a first partzyme for a second MNAzyme wherein said method comprises: b) associating said first assembly facilitator with said components for said first MNAzyme under conditions permitting catalytic activity of said first MNAzyme thus facilitating the catalytic activity of said first MNAzyme thereby allowing modification of said first MNAzyme substrate wherein; the modification of the first MNAzyme substrate results in the production of a second DNAzyme ligase substrate; and wherein the first DNAzyme ligase assembles with the first and second DNAzyme ligase substrates; and wherein catalytic activity of the DNAzyme
- a method for the detection of a first assembly facilitator using a cascade comprising a) providing components for a first MNAzyme, at least a first MNAzyme substrate, a DNAzyme ligase, a first DNAzyme ligase substrate, at least a second MNAzyme substrate, and first and second partzymes for a second MNAzyme and wherein said method comprises: b) associating said first assembly facilitator with said components for the first MNAzyme under conditions permitting catalytic activity of said first MNAzyme thus facilitating the catalytic activity of said first MNAzyme thereby allowing modification of said first MNAzyme substrate wherein; the modification of the first MNAzyme substrate results in the production of a second DNAzyme ligase substrate; and wherein the first DNAzyme ligase assembles with the first and second DNAzyme ligase substrates; and wherein catalytic activity of the DNAzyme ligase produces a
- a method for the detection of a first assembly facilitator using a cascade comprising a) providing components for a first MNAzyme, at least a first MNAzyme substrate, components for a second MNAzyme wherein said second MNAzyme has ligase activity, a second assembly facilitator for the second MNAzyme, a second MNAzyme substrate, a third assembly facilitator for a third MNAzyme, at least a third MNAzyme substrate, and a first partzyme for the third MNAzyme and wherein said method comprises: b) associating said first assembly facilitator with said components for said first MNAzyme under conditions permitting catalytic activity of said first MNAzyme thus facilitating the catalytic activity of said first MNAzyme thereby allowing modification of said first MNAzyme substrate wherein; the modification of the first MNAzyme substrate results in the production of a fourth MNAzyme substrate; and wherein the second MNAzyme assembly facilitator facilitates assembly of
- a product of the reaction catalysed by the third MNAzyme may be a component required to permit function of a fourth nucleic acid enzyme.
- a product of the reaction catalysed by the fourth nucleic acid enzyme may be a component which participates in a reaction catalysed by the second or third MNAzyme or both.
- the modification of any one of said substrates, or a combination thereof provides a detectable effect.
- the modification of at least one substrate may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation,
- the first assembly facilitator may be a target to be identified, detected or quantitated.
- the DNAzyme ligase may be selected from the group comprising DNAzyme ligases 7Z81, 7Z48, 7Q10 and 9DBl.
- a product of the second MNAzyme may be the second DNAzyme ligase substrate, and wherein the DNAzyme ligase facilitates the ligation of the first and the second DNAzyme ligase substrates thereby producing further second partzyme for the second MNAzyme.
- a product of the second MNAzyme may be the second DNAzyme ligase substrate, and wherein the DNAzyme ligase facilitates the ligation of the first and the second DNAzyme ligase substrates thereby producing further second assembly facilitator for the second MNAzyme.
- a product of the third MNAzyme may be the fourth MNAzyme substrate, and wherein the second MNAzyme facilitates the ligation of the second and the fourth MNAzyme substrates thereby producing a further second partzyme for the third MNAzyme.
- a product of the third MNAzyme may be the fourth MNAzyme substrate, and wherein the second MNAzyme facilitates the ligation of the second and the fourth MNAzyme substrates thereby producing a further third assembly facilitator for the third MNAzyme.
- a pair of isolated nucleic acids as catalytic core sequences incorporated into a partzyme pair for an MNAzyme ligase wherein said pairs of nucleic acids are selected from the group consisting of TGGAGGTGGGCTC (SEQ ID NO : 47) and ACG (SEQ ID NO: 48), GGAGGTGGGCTC (SEQ ID NO : 49) and ACGT (SEQ ID NO: 50), GGAGGTTAGCTC (SEQ ID NO : 71) and ACGGCGGAGTGATTG (SEQ ID NO : 72), TGGGAGGTTAGCTC (SEQ ID NO : 73) and ACGGCGGAGTGAT (SEQ ID NO : 74), ATTGGGAGGTTAGCTC (SEQ ID NO : 75) and ACGGCGGAGTG (SEQ ID NO : 76), TGATTGGGAGGTTAGCTC (SEQ ID NO : 77) and ACGGCGGAG
- an MNAzyme ligase to ligate two substrates wherein said substrates are ligated by said MNAzyme ligase to form a product following the engagement of said substrates by the substrate arms of said MNAzyme ligase.
- the product may associate with the components of at least one nucleic acid complex.
- the product may be selected from the group comprising assembly facilitator, partzyme, substrate, DNAzyme, stabiliser arm, activity inhibitor, assembly inhibitor, or component thereof.
- the product may participate in a cascade.
- a method for the detection of a plurality of targets comprising: a) providing a plurality of first nucleic acid complexes each comprising components which require association with one of said plurality of targets to facilitate formation of a plurality of first nucleic acid enzymes, wherein each of said plurality of first nucleic acid complexes further comprises at least one substrate, and b) providing at least one component of each of a plurality of second nucleic acid complexes capable of associating with a plurality of products of reactions catalysed by the plurality of first nucleic acid enzymes and forming a plurality of second nucleic acid complexes capable of functioning as a plurality of second nucleic acid enzymes, wherein at least one of said nucleic acid enzymes has ligase activity and at least one of said nucleic acid enzymes is an MNAzyme, and wherein said method comprises: i) contacting a sample putatively containing said plurality of targets with
- At least one of the products of the reactions catalysed by any one of the nucleic acid enzymes associates with the components of any one of the preceding nucleic acid complexes thereby permitting at least one of the nucleic acid complexes to function as further nucleic acid enzyme.
- oligonucleotides as multi-component nucleic acid enzyme ligase (MNAzyme ligase) substrates wherein said substrates are modified by an MNAzyme ligase following the engagement of said substrates by the substrate arms of said MNAzyme ligase.
- MNAzyme ligase multi-component nucleic acid enzyme ligase
- a method for the detection of at least two targets comprising: a) providing at least two nucleic acid complexes each comprising components which require association with at least one of said targets to facilitate formation of at least two nucleic acid enzyme complexes, and b) contacting a sample putatively containing said at least two targets with said at least two nucleic acid complexes, wherein said targets associate with said nucleic acid complexes to facilitate formation of said nucleic acid enzyme complexes, and wherein at least one product created by the activity of each nucleic acid enzyme complex is required for formation of an additional nucleic acid enzyme complex, and wherein activity of the additional nucleic acid enzyme complex is indicative of the presence of said at least two targets.
- At least one of said nucleic acid enzyme complexes may have ligase activity. Further, at least one of said nucleic acid enzyme complexes may be an MNAzyme complex.
- MNAzyme ligase an MNAzyme with ligase activity to switch an active MNAzyme to an MNAi complex; wherein said active MNAzyme is formed in the presence of at least a first assembly facilitator component and a second assembly facilitator component and wherein said MNAzyme ligase forms in the presence of at least a third assembly facilitator and wherein said MNAzyme ligase facilitates the ligation of said second assembly facilitator component with an activity inhibitor component to form an activity inhibitor; and wherein said activity inhibitor associates with one or more components of said MNAzyme to form an MNAi complex.
- MNAzyme ligase MNAzyme with ligase activity
- At least one of the assembly facilitators, activity inhibitor component or activity inhibitor may be nucleic acid molecules.
- at least one of the assembly facilitators, activity inhibitor component or activity inhibitor may be comprised of DNA or an analogue thereof.
- a multicomponent nucleic acid enzyme with ligase activity comprising at least two or more oligonucleotide components wherein at least a first oligonucleotide component and a second oligonucleotide component self-assemble in the presence of an MNAzyme ligase assembly facilitator to form a catalytically active multi-component nucleic acid enzyme with ligase activity (MNAzyme ligase),
- each of said at least first and said second oligonucleotide components comprise a substrate arm portion, a catalytic core portion, and a sensor arm portion; wherein upon self-assembly, the sensor arm portion of said first and second oligonucleotide components act as sensor arms of the MNAzyme ligase, the substrate arm portion of the first and second oligonucleotide components act as substrate arms of the MNAzyme ligase, and the catalytic core portion of the first and second oligonucleotide components act as a catalytic core of the MNAzyme ligase; and wherein the sensor arms of the MNAzyme ligase interact with said MNAzyme ligase assembly facilitator so as to maintain the first and second oligonucleotide components in proximity for association of their respective catalytic core portions to form the catalytic core of the MNAzyme ligase, said catalytic core capable of ligating at least two substrates, and wherein said
- ligation of the substrates may provide a detectable effect.
- composition comprising at least two oligonucleotide partzymes each comprising at least a catalytic core portion, a sensor arm portion, and a reporter arm portion, and at least three further oligonucleotide components comprising an assembly facilitator oligonucleotide and at least two substrate nucleic acids, wherein said composition is capable of forming a multicomponent nucleic acid complex capable of ligating the substrate nucleic acids.
- At least one of said oligonucleotide components, assembly facilitator or substrates of the composition may comprise DNA or an analogue thereof.
- composition comprising at least two or more oligonucleotide components wherein at least a first oligonucleotide component and a second oligonucleotide component self-assemble in the presence of an MNAzyme assembly facilitator to form a catalytically active multi-component nucleic acid enzyme with ligase activity (MNAzyme ligase)
- MNAzyme ligase catalytically active multi-component nucleic acid enzyme with ligase activity
- each of said at least first and said second oligonucleotide components comprise a substrate arm portion, a catalytic core portion, and a sensor arm portion; wherein upon self-assembly, the sensor arm portion of said first and second oligonucleotide components act as sensor arms of the MNAzyme ligase, the substrate arm portion of the first and second oligonucleotide components act as substrate arms of the MNAzyme ligase, and the catalytic core portion of the first and second oligonucleotide components act as a catalytic core of the MNAzyme ligase; and wherein the sensor arms of the MNAzyme ligase interact with said MNAzyme assembly facilitator so as to maintain the first and second oligonucleotide components in proximity for association of their respective catalytic core portions to form the catalytic core of the MNAzyme ligase, said catalytic core capable of ligating at least one substrate to another, and wherein said substrate arms of
- At least one of said oligonucleotide components, assembly facilitator or substrates of the composition may comprise DNA or an analogue thereof.
- a multicomponent nucleic acid enzyme with ligase activity wherein at least one oligonucleotide component of said MNAzyme further comprises an aptamer.
- at least one of the assembly facilitator or substrates further comprises an aptamer.
- assembly of the MNAzyme ligase may be directed by the presence or absence of one or more analytes capable of binding the aptamer.
- At least one of said oligonucleotide components or assembly facilitator may be comprised of DNA or an analogue thereof.
- said assembly facilitator may be a target to be identified, detected or quantitated.
- said target may be a nucleic acid.
- said nucleic acid may be selected from the group comprising 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, derivatives thereof, amplicons thereof or any combination thereof.
- the source of the nucleic acid may be selected from the group comprising synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, archael sources or any combination thereof.
- the method may further comprise a step of amplifying the assembly facilitator.
- the step of amplifying may comprise one or more of: polymerase chain reaction (PCR), strand displacement amplification, loop-mediated isothermal amplification, rolling circle amplification, transcription-mediated amplification, self-sustained sequence replication, ligase chain reaction, nucleic acid sequence based amplification, or reverse transcription polymerase chain reaction (RT- PCR).
- PCR polymerase chain reaction
- strand displacement amplification loop-mediated isothermal amplification
- rolling circle amplification transcription-mediated amplification
- transcription-mediated amplification self-sustained sequence replication
- ligase chain reaction nucleic acid sequence based amplification
- RT- PCR reverse transcription polymerase chain reaction
- At least one of said assembly facilitator, said oligonucleotide components or substrates for the reaction or a combination thereof may be comprised of more than one molecule.
- the method may further comprise determination of the presence of said catalytic activity during or after said amplification.
- the self assembly of the MNAzyme ligase may require contact of the assembly facilitator with one or both of said first and second oligonucleotide components.
- the method may further comprise providing at least a third oligonucleotide component that contacts at least a portion of either or both of the first and second oligonucleotide components to self-assemble the MNAzyme ligase.
- said third oligonucleotide component may be comprised of more than molecule.
- an isolated nucleic acid sequence capable of forming part of a catalytic core of an MNAzyme ligase comprising the sequence selected from the group comprising TGGAGGTGGGCTC (SEQ ID NO : 47), ACG (SEQ ID NO: 48), GGAGGTGGGCTC (SEQ ID NO : 49), ACGT (SEQ ID NO: 50), GGAGGTTAGCTC (SEQ ID NO : 71), ACGGCGGAGTGATTG (SEQ ID NO : 72), TGGGAGGTTAGCTC (SEQ ID NO : 73), ACGGCGGAGTGAT (SEQ ID NO : 74), ATTGGGAGGTTAGCTC (SEQ ID NO : 75), ACGGCGGAGTG (SEQ ID NO : 76), TGATTGGGAGGTTAGCTC (SEQ ID NO : 77), ACGGCGGAG (SEQ ID NO 78), AGTGATTGGGAGGTTAGCTC (SEQ ID NO : 47), ACG (SEQ ID NO:
- a method for the detection of a first assembly facilitator using a cascade comprising components for a first MNAzyme, a first MNAzyme substrate, components for a second MNAzyme, a second assembly facilitator for the second MNAzyme, a second MNAzyme substrate, a third assembly facilitator for the third MNAzyme, at least a third MNAzyme substrate, and a first partzyme for the third MNAzyme wherein association of said first assembly facilitator with said components for a first MNAzyme under conditions permitting catalytic activity of said first MNAzyme facilitates the catalytic activity of said first MNAzyme thereby cleaving the said first MNAzyme substrate wherein; the cleavage of the first MNAzyme substrate results in the production of a fourth MNAzyme substrate; and wherein the second MNAzyme assembly facilitator facilitates assembly of the second MNAzyme with the second and fourth MNAzyme substrates; and wherein cata
- the modification of the first, second or at least a third MNAzyme substrates or any combination thereof may provide a detectable effect.
- the modification of the at least a third MNAzyme substrate may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of
- the method further comprises an amplification cascade wherein a product of the third MNAzyme is the fourth MNAzyme substrate, and wherein the second MNAzyme facilitates the ligation of the second and the fourth MNAzyme substrates thereby producing a further second partzyme for the third MNAzyme.
- a method for the detection of a first assembly facilitator using a cascade comprising a components for a first MNAzyme, a first MNAzyme substrate, components for a second MNAzyme, a second MNAzyme assembly facilitator, a second MNAzyme substrate, components for a third MNAzyme and at least a third MNAzyme substrate, wherein association of said first assembly facilitator with said components for a first MNAzyme under conditions permitting catalytic activity of said first MNAzyme facilitates the catalytic activity of said first MNAzyme thereby cleaving the said first MNAzyme substrate wherein; the cleavage of the first MNAzyme substrate results in the production of the fourth MNAzyme substrate; and wherein the second MNAzyme assembly facilitator facilitates assembly of the second MNAzyme with the second and fourth MNAzyme substrates; and wherein catalytic activity of the second MNAzyme produces a third MNAzyme assembly facilitator
- the modification of the first, second or at least a third MNAzyme substrate or any combination thereof may provide a detectable effect.
- the modification of the at least a third MNAzyme substrate may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion and phosphoramidate cleavage.
- the method further comprises an amplification cascade wherein a product of the third MNAzyme is the fourth MNAzyme substrate, and wherein the second MNAzyme facilitates the ligation of the second and the fourth MNAzyme substrates thereby producing further third assembly facilitator for the third MNAzyme.
- a method for the detection of a first assembly facilitator using a cascade comprising components for a first MNAzyme with cleavage activity, a first MNAzyme substrate, a first DNAzyme with ligase activity (DNAzyme ligase), a first DNAzyme ligase substrate, a second assembly facilitator, at least a second MNAzyme substrate, and a first partzyme for a second MNAzyme wherein association of said first assembly facilitator with said components for a first MNAzyme under conditions permitting catalytic activity of said first MNAzyme facilitates the catalytic activity of said first MNAzyme thereby providing modification of said first MNAzyme substrate wherein; the modification of the first MNAzyme substrate results in the production of the second DNAzyme ligase substrate; and wherein the first DNAzyme ligase assembles with the first and second DNAzyme ligase substrates; and wherein catalytic activity of the DNA
- At least one of said oligonucleotide components, assembly facilitator or substrates may comprise DNA or an analogue thereof.
- the first assembly facilitator may be a target to be identified, detected or quantitated.
- the modification of the first or second MNAzyme substrate or ligase substrates or any combination thereof may provide a detectable effect.
- the catalytic activity of the second MNAzyme may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion and phosphoramidate cleavage.
- the modification of the second MNAzyme substrate may be cleavage.
- the ligation of the substrates may provide a detectable effect.
- the method further comprises an amplification cascade wherein a product of the second MNAzyme is the second DNAzyme ligase substrate, and wherein the DNAzyme ligase facilitates the ligation of the first and second DNAzyme ligase substrates thereby producng a further second partzyme for the second MNAzyme.
- the DNAzyme ligase may be selected from the group comprising DNAzyme ligases 7Z81, 7Z48 and 7Q10.
- a method for the detection of a first assembly facilitator using a cascade comprising components for a first MNAzyme with cleavage activity, a first MNAzyme substrate, a first DNAzyme with ligase activity (DNAzyme ligase), a first DNAzyme ligase substrate, components for a second MNAzyme and at least a second MNAzyme substrate, and wherein association of said first assembly facilitator with said components for a first MNAzyme under conditions permitting catalytic activity of said first MNAzyme facilitates the catalytic activity of said first MNAzyme thereby providing modification of said first MNAzyme substrate wherein; the modification of the first MNAzyme substrate results in the production of a second DNAzyme ligase substrate; and wherein the first DNAzyme ligase assembles with the first and second DNAzyme ligase substrates; and wherein catalytic activity of the DNAzyme ligase produces a
- At least one of said oligonucleotide components, assembly facilitator or substrates may comprise DNA or an analogue thereof.
- the modification of the first or second MNAzyme substrate or ligase substrates or any combination thereof may provide a detectable effect
- the first assembly facilitator may be a target to be identified, detected or quantitated.
- the catalytic activity of the second MNAzyme may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion and phosphoramidate cleavage.
- the DNAzyme ligase may be selected from the group comprising DNAzyme Hgases 7Z81, 7Z48 and 7Q10.
- the modification of the second MNAzyme substrate may be cleavage.
- the ligation of the substrates may provide a detectable effect.
- the method further comprises an amplification cascade wherein a product of the second MNAzyme is the second DNAzyme ligase substrate, and wherein the DNAzyme ligase facilitates the ligation of the first and second DNAzyme ligase substrates thereby producing further second assembly facilitator for the second MNAzyme.
- a method for the detection of a first assembly facilitator using a cascade comprising components for a first MNAzyme with cleavage activity (a first MNAzyme cleaver), a first cleavable MNAzyme substrate, components for a first MNAzyme with ligase activity (a first MNAzyme ligase), a first MNAzyme ligase assembly facilitator, a first MNAzyme ligase substrate, a second assembly facilitator for a second MNAzyme cleaver, a second cleavable MNAzyme substrate, and a first partzyme for a second MNAzyme cleaver wherein association of said first assembly facilitator with said components for a first MNAzyme cleaver under conditions permitting catalytic activity of said first MNAzyme cleaver facilitates the catalytic activity of said first MNAzyme cleaver thereby providing modification of said first cleavable M
- At least one of said oligonucleotide components, assembly facilitators or substrates may comprise DNA or an analogue thereof.
- the first assembly facilitator may be a target to be identified, detected or quantitated.
- the modification of the first or second cleavable MNAzyme substrate or both may provide a detectable effect.
- the ligation of the substrates may provide a detectable effect.
- the method further comprises an amplification cascade wherein the product of the second MNAzyme is the second MNAzyme ligase substrate, and wherein the MNAzyme ligase facilitates the ligation of the first and second MNAzyme ligase substrates thereby producing a further second partzyme for the second MNAzyme cleaver.
- a method for the detection of a first assembly facilitator using a cascade comprising components for a first MNAzyme with cleavage activity (a first MNAzyme cleaver), a first cleavable MNAzyme substrate, components for a first MNAzyme with ligase activity (a first MNAzyme ligase), a first MNAzyme ligase assembly facilitator, a first MNAzyme ligase substrate, components for a second MNAzyme cleaver and a second cleavable MNAzyme substrate, wherein association of said first assembly facilitator with said components for a first MNAzyme cleaver under conditions permitting catalytic activity of said first MNAzyme cleaver facilitates the catalytic activity of said first MNAzyme cleaver thereby providing modification of said first cleavable MNAzyme substrate wherein; the modification of the first cleavable MNAzyme substrate results
- At least one of said oligonucleotide components, assembly facilitators or substrates may comprise DNA or an analogue thereof.
- the modification of the first or second cleavable MNAzyme substrate or both may provide a detectable effect.
- the ligation of the substrates may provide a detectable effect.
- the first assembly facilitator may be a target to be identified, detected or quantitated.
- oligonucleotide as a multi-component nucleic acid enzyme (MNAzyme) substrate wherein said substrate is modified by said MNAzyme following the engagement of said substrate by the substrate arms of said MNAzyme.
- MNAzyme multi-component nucleic acid enzyme
- a method for making MNAzymes which incorporate partial sequences from a DNAzyme comprising the following steps: a) selecting positions within said DNAzyme sequence for splitting into partial catalytic core sequences b) incorporating said partial catalytic core sequences into partzymes c) detecting catalytic activity of the partzyme pairs to determine which combination of said partial catalytic core sequences in said partzymes is compatible with the formation of active MNAzymes.
- a method for testing partial catalytic core sequences derived from a DNAzyme catalytic core which, upon incorporation into partzymes, generate functionally active MNAzymes comprising a) splitting the catalytic core sequence of said DNAzyme b) incorporating the resulting partial catalytic core sequences into partzymes: c) detecting MNAzyme activity to determine which combination of said partial catalytic core sequences in said partzymes is compatible with the formation of active MNAzymes.
- a method for identifying which positions within a DNAzyme catalytic core sequence are suitable for splitting into partial catalytic core sequences which, upon incorporation into partzymes, result in functionally active MNAzymes comprising the following steps: a) splitting the catalytic core sequence from said DNAzyme b) incorporating the resulting partial catalytic core sequences into partzymes: c) detecting MNAzyme activity to determine which combination of said partial catalytic core sequences in said partzymes is compatible with the formation of active MNAzymes.
- FIG. 1 Depiction of one exemplary design of a Multi-component nucleic acid (MNAzyme):
- an MNAzyme is comprised of two oligonucleotide components (partzyme A and partzyme B), which self assemble in the presence of an assembly facilitator.
- a catalytically active MNAzyme forms which is capable of modifying, for example cleaving, a substrate.
- the two component partzymes have (i) sensor arms, which bind to the assembly facilitator, (ii) substrate arms, which bind the substrate, and (iii) partial catalytic core sequences.
- an assembly facilitator molecule provides the "input" signal which directs the assembly of partzyme components in a highly specific fashion which is amenable to modulation.
- the assembly facilitator may be, for example, a target nucleic acid sequence present in a test sample.
- the assembly facilitator may be, for example a synthetic oligonucleotide included in the milieu to direct the self- assembly of the partzyme components in the presence of a detectable entity or event. Modification of the substrate by the assembled MNAzyme can provide an "output" signal which may be detected and/or quantified.
- cleavage of the substrate by an active MNAzyme separates the fluorophore and the quencher resulting in a concomitant increase in fluorescence.
- partzyme B has a truncated sensor arm (T), which is insufficient to allow stable MNAzyme assembly in the absence of a second component, referred to as a stabiliser arm component (S).
- T truncated sensor arm
- S stabiliser arm component
- the partzyme arm which is truncated, could be any of the following; the partzyme A sensor arm, the partzyme B sensor arm (as illustrated), the partzyme A substrate arm or the partzyme B substrate arm, or a combination thereof.
- Figure 3 Changes in output signal (fluorescence) over time in the presence of active and inactive multi-component nucleic acid complexes.
- the first partzyme (A) comprises a single component oligonucleotide.
- the second partzyme (B) has one component containing a substrate arm, a partial catalytic core and a truncated sensor arm (T), and a second component which serves as a stabiliser arm (S).
- reaction (i) An increase in fluorescence was observed in reaction (i), which contained all the components of partzymes A and B and an assembly facilitator. This is consistent with the assembly of active MNAzymes and cleavage of the substrate in this reaction.
- the omission of the stabilizer arm portion of partzyme B (reaction (U)) resulted in no increase in signal over time indicating that this component is essential for the assembly of active MNAzymes in this system.
- a control reaction lacking an assembly facilitator (reaction (Ui)) also showed no increase in fluorescence over time.
- reaction (i) represents the active complex ("on" state).
- reaction (U) partzyme stabiliser arm component
- reaction (Ui) assembly facilitator component
- off states are representative of inactive MNA complexes.
- the MNAzyme catalytic activity can be regulated by the presence or absence of various oligonucleotides and/or by the ability of such oligonucleotide components to be functionally active, for example to be capable of hybridizing with other oligonucleotide components to form stable MNAzymes or MNAzyme complexes.
- the truncated arm is designed to be insufficient to allow stable MNAzyme assembly under the reaction conditions, unless accompanied by a stabiliser arm component.
- the stabiliser arm, and the assembly facilitator can function as "on" switches for MNAzyme activity.
- FIG. 4 Depiction of an exemplary design for an inactive multi- component nucleic acid inhibitor (MNAi) complex (left hand side) and an active MNAzyme (right hand side):
- An MNAi complex is formed when partzymes A and B complex with an assembly facilitator component and an activity inhibitor (left hand side).
- the inactive MNAi complex is capable of interacting with, but not catalytically modifying, the substrate.
- the activity inhibitor may further include a labile or cleavable linker (indicated by the dotted arrow), which may separate two or more domains within the activity inhibitor.
- Such domains may include, for example, (i) an activity inhibitor portion which is substantially non-complementary to the partzyme components and which exerts an inhibitory effect by disrupting the secondary structure required for formation of a catalytically active MNAzyme and (ii) an activator assembly facilitator portion, which if separated from the activity inhibitor portion, may function as an assembly facilitator component and direct the assembly of an active MNAzyme.
- Figure 5 Demonstration of catalytic activity from various multi- component nucleic acid complexes. All reactions contained partzyme A, partzyme B, and a substrate labelled with a fluorophore quencher dye pair. In addition, reactions contained either (i) an assembly facilitator F 1/2, (ii) an assembly facilitator comprising portions Fl and F2 (the sequence of which together correspond to that of assembly facilitator F 1/2), (iii) assembly facilitator portion Fl and an activity inhibitor containing two joined domains corresponding to an activity inhibitor portion and portion with the same sequence as F2 or (iv) no assembly facilitator or activity inhibitor.
- FIG. 1 Exemplary structures for variant MNAzyme designs. Some examples of active MNAzyme complexes are shown in Panels A to D. These structures are all capable of forming catalytically active enzymes, which can modify, for example cleave, a substrate (S). Illustrated are schemes for MNAzyme complexes, which include one assembly facilitator Fl (panel A), two assembly facilitator components Fl and F2 (panels B and D) or three assembly facilitator components Fl, F2 and F3 (panel C). Panel A includes sensor arms with self complementary regions within the partzyme sensor arms. MNAzyme complexes may also include one or more stabilising arms (sA) as shown in panel D.
- sA stabilising arms
- MNAzymes The strategy in this system can be used either as (i) a method to control MNAzyme activity using ligands as activator molecules, and/or (ii) a method of detection of nucleic and non-nucleic acid targets using apta-MNAzymes.
- the nucleic acid oligonucleotides included in this exemplary apta- MNAzyme detection strategy include; a) a partzyme; b) an apta-partzyme which is a partzyme with an aptamer incorporated into one of its ends; c) an assembly facilitator which is an oligonucleotide which binds to both the apta-partzyme and the partzyme enabling assembly of an active MNAzyme; d) a substrate e.g.
- a reporter substrate e) an assembly inhibitor oligonucleotide 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 apta-partzyme sequence.
- the assembly inhibitor oligonucleotide In the absence of an activator ligand (left hand panel), the assembly inhibitor oligonucleotide binds to the apta-partzyme thus competing with and blocking binding of the reporter substrate.
- an activator ligand When an activator ligand is present (right hand panel), it binds to the aptamer sequence of the apta-partzyme, blocking the binding of the assembly inhibitor oligonucleotide, and thus allowing assembly of an active apta-MNAzyme complex and subsequent modification, for example cleavage of the substrate (as illustrated) or for example ligation of substrates.
- an active apta-MNAzyme can only form and cause an increase or decrease in signal, for example, a fluorescent signal, in the presence of a ligand that can bind the aptamer portion of the apta-partzyme.
- an aptamer sequence could be attached to, for example, one or more of either a substrate arm and/or a sensor arm of either or both of the partzymes. If the aptamer were attached to a sensor arm then the binding of the assembly facilitator could be blocked by an assembly inhibitor oligonucleotide in the absence of a target ligand.
- the modification can cause for example an increase in fluorescence by cleavage of a dual labelled reporter substrate thus causing separation of a fluorophore/quencher dye pair.
- the modification performed by an apta- MNAzyme could be a modification other than cleavage, for example, ligation of two substrates.
- the signal change could be, for example, a decrease in fluorescence following ligation of two ligatable substrates each labelled with either the fluorophore or the quencher of a fluorophore/quencher dye pair.
- This apta-MNAzyme approach could also be used to develop molecular switches that can turn on and off the catalytic activity of an MNA complex. Alternatively it can also be applied to detection of both nucleic acid and non-nucleic acid target ligands.
- Figure 8 An example of a cleavage/ligation cascade mediated by a DNAzyme with ligase activity and an MNAzyme with cleavage activity:
- a DNAzyme with ligase activity can ligate a first oligonucleotide (oligo 1) to a second oligonucleotide (oligo 2) to create a third ligation product (LP-Oligo 3) with the sequence of oligo 3 and a 2'-5' RNA linkage at the ligation junction, provided oligo 1 and oligo 2 have 2', 3 '-cyclic phosphate and 5'-hydroxyl termini, for example, as shown.
- LP-oligo 3 could be cleaved by an MNAzyme capable of cleaving at a 2 '-5' RNA linkage to create cleavage products (CP), CP-oligo 1 and CP-oligo 2, thus regenerating 2', 3'-cyclic phosphate and 5'-hydroxyl products, which can participate in further rounds of ligation.
- MNAzyme capable of cleaving at a 2 '-5' RNA linkage to create cleavage products (CP), CP-oligo 1 and CP-oligo 2, thus regenerating 2', 3'-cyclic phosphate and 5'-hydroxyl products, which can participate in further rounds of ligation.
- oligo 1 oligonucleotide
- oligo 2 oligonucleotide
- LP-Oligo 3 third ligation product
- oligo 1 and oligo 2 have 2',3'-diol and 5 '-triphosphate termini, for example.
- LP- oligo 3 can be cleaved by an MNAzyme capable of cleaving at a 3'-5' RNA linkage to 00492
- CP cleavage products
- CP-oligo 1 cleavage products
- CP-oligo 2 create cleavage products with a 2', 3 '-cyclic phosphate and 5'-hydroxyl.
- FIG. 9 An example of a cascade reaction which uses two types of nucleic acid enzymes namely MNAzymes, which can cleave substrates, and DNAzyme ligases which can ligate substrates.
- MNAzyme A forms only in the presence of an assembly facilitator (e.g. a target nucleic acid) and then cleaves MNAzyme substrate A releasing a 5' product A with a 2', 3' cyclic phosphate terminus.
- an assembly facilitator e.g. a target nucleic acid
- step (ii) the DNAzyme B with ligase activity ligates the 5' product A from step (i) to a 3' ligation substrate B thus creating a ligation product which can serve as a partzyme component of another MNAzyme C.
- step (iii) the partzyme/ligation product of step (ii) assembles with a second partzyme and an assembly facilitator C to form MNAzyme C which can cleave substrate C into two products, 5' product C and 3' product C.
- an assembly facilitator for example a target nucleic acid, can direct the formation of a first MNAzyme A.
- MNAzyme A can cleave a substrate A thus generating a 5 'cleavage product A which can in turn be used as a substrate for a DNAzyme ligase B.
- a DNAzyme ligase B can ligate the 5' cleavage product A (also referred to as 5' substrate B) generated in step (i) to another oligonucleotide, 3' ligation substrate B, thus creating a new partzyme for an MNAzyme C.
- step (iii) the new partzyme/ligation product generated in step (ii), together with another partzyme, can form a new MNAzyme C in the presence of assembly facilitator C which can cleave a substrate C into two products, 5 'product C and 3' product C.
- a detectable signal can be generated following cleavage of substrate C if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- FIG. 10 Example of a structure of an MNAzyme with ligase activity (an MNAzyme ligase).
- the exemplary MNAzyme ligase is comprised of partzyme sequences which contain three domains including a (i) a sensor arm which hybridizes to an assembly facilitator (e.g. a target nucleic acid), (ii) a domain that constitutes part of a catalytic core and (iii) a substrate binding arm ( Figure 10A).
- assembly facilitator e.g. a target nucleic acid
- partzymes assemble and form active MNAzyme ligases.
- These MNAzymes can ligate a 5' substrate with a 2'3' cyclic phosphate and a 3' substrate with 5' hydroxyl ( Figure 10B) to create ligation products that contain non- native 2 '-5' linkages.
- MNAzymes could ligate two substrates with a 2 '3' diol terminus on the 5' substrate with a 5 '-triphosphate terminus on the 3' substrate to create ligation products that contain native 3'-5' linkage.
- Figure 11 An exemplary strategy for reactions that use MNAzymes with ligase activity and MNAzymes with cleavage activity.
- An MNAzyme with ligase activity could ligate a first oligonucleotide (A) to a second oligonucleotide (B) to create a third ligation product (C) with a 2 '-5' RNA linkage at the ligation junction, provided A and B have 2', 3 '-cyclic phosphate and 5'- hydroxyl termini.
- product C could be cleaved by an MNAzyme capable of cleaving at a 2'-5' RNA linkage to create cleavage products A and B, thus regenerating 2', 3 '-cyclic phosphate and 5 '-hydroxyl products, which can participate in further rounds of ligation.
- an assembly facilitator for example a target nucleic acid, can direct the formation of a first MNAzyme A.
- MNAzyme A can cleave a substrate A thus generating a 5' cleavage product A which can in turn be used as a substrate for an MNAzyme B which has ligase activity.
- an MNAzyme ligase B can ligate the 5' cleavage product A (also called the 5' substrate B) generated in step (i) to another oligonucleotide, 3' substrate B, thus creating a new partzyme for an MNAzyme C.
- step (iii) the new partzyme/ligation product generated in step (ii), together with another partzyme, can form a new MNAzyme C in the presence of assembly facilitator C which can cleave a substrate C into two products, 5' product C and 3' product C.
- a detectable signal can be generated following cleavage of substrate C if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- the strategy allows detection of target analytes using two types of MNAzymes, namely MNAzymes which can ligate substrates (MNAzyme ligases) and MNAzymes which can cleave a substrate (MNAzyme cleavers).
- FIG. 13 Depiction of an exemplary strategy for switching an MNA complex between the "on state" of an active MNAzyme to the "off state” of an MNAi complex using a second MNAzyme with ligase activity.
- An active MNAzyme A which could be capable of modifying (e.g. cleaving or ligating) a substrate(s) A could be formed in the presence of assembly facilitator component 1 (AFC 1) and assembly facilitator component 2 (AFC 2).
- a second MNAzyme B which has ligase activity, could form in the presence of assembly facilitator 3 (AF3) and then could ligate AFC 2 with an activity inhibitor component (AIC) resulting in the formation of an activity inhibitor (AI).
- This AI could bind to the partzyme components for the MNAzyme A resulting in the formation of an inactive MNAi complex.
- the MNAzyme ligase in this example can operate as an off switch to inactivate an MNAzyme A.
- the inactive MNAi complex and the catalytically active MNAzyme represent two alternate states for the assembled components, namely an "off state and the "on" state respectively.
- Figure 14 Depiction of a general method for engineering MNAzymes from uni-molecular DNAzymes.
- DNAzymes have similar basic structures with multiple domains. These DNAzymes have a conserved catalytic domain (catalytic core) flanked by two non- conserved substrate-binding domains ("substrate arms"), which specifically recognize and hybridise to the substrate (top left hand side structure).
- substrate arms two non- conserved substrate-binding domains
- the substrate binding domains can be tailored to any substrate provided the substrate contains a site which can be modified by the DNAzyme.
- positions are identified within the DNAzyme catalytic core at which it can be split, such that each partial portion of the catalytic core can be distributed between two partial sequences such that the two partial cores together constitute an entire catalytic core (top right hand side structure).
- Two oligonucleotides A and B can then be synthesised (bottom left hand side structure).
- An oligonucleotide A can be synthesised to contain (i) one substrate binding arm portion capable of binding to a substrate, (ii) one partial catalytic core portion, and (iii) one sensor arm portion capable of binding to an assembly facilitator molecule.
- a second oligonucleotide B can be synthesised such that it contains (i) one substrate binding arm capable of binding to the same substrate as oligonucleotide A, whereby oligonucleotide B binds the substrate in a position adjacent to that of oligonucleotide A, (ii) one partial catalytic core portion which contains those bases from the entire DNAzyme catalytic core which are not incorporated into oligonucleotide A and (iii) one sensor arm sequence capable of binding to the same assembly facilitator as oligonucleotide A, whereby oligonucleotide B binds the assembly facilitator in a position adjacent to that of oligonucleotide A (bottom left hand side structure).
- Figure 15 A cascade reaction using nucleic acid enzymes which cleave substrates, and enzymes which ligate substrates to form a new assembly facilitator.
- step (1) the MNAzyme cleaver assembles in the presence of an assembly facilitator (e.g. a target nucleic acid) and then cleaves MNAzyme substrate 1 releasing a 5' cleavage product with a 2', 3' cyclic phosphate terminus.
- step (2) the DNAzyme ligase (or MNAzyme ligase) ligates the 5' cleavage product from step (1) to a 3' ligation substrate thus creating a ligation product which can serve as an assembly facilitator for another MNAzyme cleaver 2.
- step (3) the assembly facilitator formed by ligation in step (2) directs the assembly of partzymes which form MNAzyme cleaver 2 which cleaves substrate 2 into two products, a 5' cleavage product with a 2', 3' cyclic phosphate terminus and a 3' cleavage product.
- a detectable signal can be generated following cleavage of substrate 2 when this substrate is labelled, for example, with a fluorophore and quencher dye pair.
- the strategy allows detection of target analytes using multiple types of nucleic acid enzymes, namely enzymes which can ligate substrates and enzymes which can cleave a substrate.
- MNAzyme cleaver 2 could be constantly generating 5' substrate for the ligase which could in turn be ligated by the ligase to create more assembly facilitators for formation of more MNAzyme cleaver 2.
- This strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator such as a target nucleic acid which allows assembly of MNAzyme cleaver 1.
- FIG. 16 A possible schema for a feedback cascade reaction which uses two types of nucleic acid enzymes namely enzymes which can cleave substrates, and enzymes which can ligate substrates.
- the 5' end of oligonucleotides are indicated by a circle.
- the MNAzyme cleaver 1 could assemble only in the presence of an assembly facilitator (e.g. a target nucleic acid) and could then cleave MNAzyme substrate 1 releasing a 5' cleavage product with a 2', 3' cyclic phosphate terminus and a 3 ' waste product.
- an assembly facilitator e.g. a target nucleic acid
- step (2) the DNAzyme ligase (or MNAzyme ligase) could ligate the 5' cleavage product from step (1) to a 3' ligation substrate thus creating a ligation product which could serve as an assembly facilitator for an MNAzyme cleaver 2.
- step (3) the assembly facilitator formed by ligation in step (2) could direct the assembly of partzymes to form MNAzyme cleaver 2 which could cleave substrate 2 into two products, a 5' cleavage product with a 2', 3' cyclic phosphate terminus and 3' cleavage product.
- a detectable signal could be generated following cleavage of substrate 2 if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- MNAzyme cleaver 3 could have different sensor arms than MNAzyme cleaver 1 but could have the same substrate arms as MNAzyme cleaver 1.
- MNAzyme cleaver 3 were assembled in the presence of an assembly facilitator component generated by cleavage by the MNAzyme cleaver 2, and if MNAzyme cleaver 3 cleaved substrate 1 then the 5' cleavage product of MNAzyme cleaver 3 could also serve as a 5' substrate for the DNAzyme ligase (or the assembled MNAzyme ligase) and a feedback amplification cascade reaction could be initiated.
- MNAzyme cleaver 3 could constantly generate 5' cleavage product which in turn could serve as a substrate for ligation by the DNAzyme ligase (or the assembled MNAzyme ligase) to create more assembly facilitators that could direct the assembly of more MNAzyme cleaver 2.
- This strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which allowed assembly of MNAzyme cleaver 1.
- the strategy could allow detection of one or more assembly facilitators (eg target analytes) followed by signal amplification using a DNAzyme or an MNAzyme which can ligate substrates and MNAzymes which can cleave a substrate.
- nucleic acid may be used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide 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, derivatives thereof, amplicons thereof or any combination thereof.
- the source of a nucleic acid may be selected from the group comprising synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, archael sources or any combination thereof.
- oligonucleotide typically denotes a segment of DNA or a DNA- containing nucleic acid molecule, or RNA or RNA-containing molecule, or a combination thereof.
- oligonucleotides include nucleic acid targets; substrates, for example, those which can be modified by a DNAzyme or an MNAzyme with cleavage, ligase or other enzymatic activity; primers such as those used for in vitro target amplification by methods such as PCR; and components of nucleic acid complexes including but not limited to assembly facilitators and assembly inhibitors, activators, activity inhibitors and/or substrates, which in certain embodiments, may comprise oligonucleotides as defined herein. Partzymes as used herein may also comprise oligonucleotides.
- the region(s) of complementarity between the additional oligonucleotide(s) and the first oligonucleotide are separated and replaced by at least one region of complementarity between the displacer oligonucleotide and the first oligonucleotide thus resulting in displacement of the additional oligonucleotide(s).
- the displacer oligonucleotide is required to comprise at least one region which has the same sequence as one or more regions of the additional oligonucleotide(s) within the double stranded region.
- polynucleotide refers to any specified sequence as well as to the sequence complementary thereto, unless otherwise indicated. Oligonucleotides may comprise at least one addition or substitution, including but not limited to the group comprising LNA phosphoramidite, 4- acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2'-O-methylcytidine, 5- carboxymethylaminomethyl thiouridine, dihydrouridine, 2'-O-methylpseudouridine, beta D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1- methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2- dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methyl
- nucleic acid or nucleotide of the present invention includes any functionally equivalent nucleic acids or nucleotides, including any fusion molecules produced integrally (e.g., by recombinant means) or added post-synthesis (e.g., by chemical means).
- 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.
- analogue when used 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 (Warashina et al., 1999; Cairns et al., 2003; Schubert et al., 2004; Sidorov et al., 2004).
- Other analogues could also be compatible with catalytic activity of DNAzymes 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.
- 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.
- nucleic acid enzyme refers herein interchangeably and shall mean a DNA or DNA-containing molecule or complex or an RNA or RNA- containing molecule or complex or a combination thereof, being a DNA-RNA hybrid molecule or complex, which may recognize at least one substrate and catalyse a modification (such as ligation or cleavage) of the at least one substrate.
- the nucleotide residues in the catalytic nucleic acids may include the bases A, C, G, T, and U, as well as derivatives and analogues thereof.
- nucleic acid enzymes which comprise a DNA or DNA-containing complex or an RNA or RNA-containing complex or a combination thereof, being a DNA-RNA hybrid complex which may recognize at least one substrate and catalyse a modification (such as ligation or cleavage) of the at least one substrate.
- nucleic acid enzyme include within their meaning MNAzymes.
- MNAzyme or "multi-component nucleic acid enzyme” as used herein, refers to two or more oligonucleotide sequences (e.g. partzymes) which, only in the presence of an MNAzyme assembly facilitator (for example, a target), form an active nucleic acid enzyme that is capable of catalytically modifying a substrate.
- MNAzymes can catalyse a range of reactions including cleavage of a substrate, ligation of substrates and other enzymatic modifications of a substrate or substrates.
- An exemplary MNAzyme comprising partzyme A and partzyme B which has cleavage activity is depicted in Figure 1.
- MNAzymes with endonuclease or cleavage activity are also known as "MNAzyme cleavers".
- partzymes A and B each bind to an assembly facilitator (e.g. through Watson-Crick base pairing).
- the MNAzyme only forms when the sensor arms of partzymes A and B hybridize adjacent to each other on the assembly facilitator.
- the substrate arms of the MNAzyme engage the substrate, the modification (e.g. cleavage) of which is catalyzed by the catalytic core of the MNAzyme, formed by the interaction of the catalytic domains of partzymes A and B.
- Cleavage of a DNA/RNA chimeric reporter substrate is exemplified in the drawing.
- the MNAzyme cleaves the substrate between a fluorophore and a quencher dye pair, thus generating signal.
- multi-component nucleic acid enzyme and “MNAzyme” are used herein interchangeably and comprise bipartite structures, composed of two molecules, or tripartite structures, composed of three nucleic acid molecules, or other multipartite structures, for example those formed by four or more nucleic acid molecules.
- MNAzyme complex refers to a complex formed by an assembled MNAzyme with at least one of its substrates.
- the MNAzyme or MNAzyme complex may comprise an aptamer and be referred to as an apta-MNAzyme or apta-MNAzyme complex, respectively.
- MNAzyme ligase refers to MNAzymes with ligase activity that comprise two or more oligonucleotide sequences (e.g. partzymes) which, only in the presence of an MNAzyme assembly facilitator which may also be referred to herein as a ligase assembly facilitator or assembly facilitator (for example, a target), form an active nucleic acid enzyme that is capable of ligating two substrates.
- MNAzyme assembly facilitator for example, a target
- An exemplary MNAzyme with ligase activity is illustrated in Figure 10. The MNAzyme ligase ligates the two substrates.
- MNAzyme ligase as used herein comprises bipartite structures, composed of two molecules, or tripartite structures, composed of three nucleic acid molecules, or other multipartite structures, for example those formed by four or more nucleic acid molecules.
- ligate means to join two or more molecules.
- a “ligase” is an enzyme that catalyses the formation (ligation) of a bond between two or more substrate molecules to make a new molecule.
- ligation or “ligase activity” refer to the joining of two or more substrate molecules.
- one or more component partzymes may comprise three regions or domains: a "catalytic” domain, which forms part of the catalytic core that catalyzes a modification; a "sensor arm” domain, which may associate with and/or bind to an assembly facilitator; and a "substrate arm” domain, which may associate with and/or bind to a substrate. Illustrations of these regions or domains are shown in Figure 1 and Figure 10.
- Partzymes may comprise at least one additional component including but not limited to an aptamer, referred to herein as an "
- a partzyme which is composed of more than two molecules is illustrated in Figure 2 (ii).
- a partzyme may comprise multiple components, including but not limited to, a partzyme component with a truncated sensor arm and a stabilizing arm component which stabilises the MNAzyme structure by interacting with either an assembly facilitator (as depicted in Figure 2 (H)) or with a substrate.
- nucleic acid complex refers to catalytically active or inactive complexes which comprise two or more components selected from the group comprising but not limited to, DNAzymes, partzymes, assembly facilitators, substrates, and other IViNA components including stabiliser arms, activity inhibitors, assembly inhibitors, and components or parts thereof.
- nucleic acid complex includes multicomponent nucleic acid complexes (MNA complexes). Additionally, the term “nucleic acid complex” includes uni-molecular nucleic acid enzymes complexed with at least one substrate, for example, a DNAzyme with cleavage activity bound to a substrate or a DNAzyme ligase bound to at least one of its two required substrates. The term “nucleic acid enzyme complex” refers to a DNAzyme complex or an MNAzyme complex.
- DNAzyme complex includes DNAzymes with at least one substrate, for example, a DNAzyme with cleavage activity bound to a substrate or a DNAzyme ligase bound to at least one of its two required substrates.
- multi-component nucleic acid complex refers to complexes which comprise two or more components selected from the group comprising but not limited to, partzymes, assembly facilitators, substrates, and othercomponents including stabiliser arms, activity inhibitors, assembly inhibitors, and components or parts thereof.
- the MNA complex comprises an active MNAzyme.
- the MNA complex is an inactive complex such as an MNAi complex which may also be referred to herein as a multi-component nucleic acid inhibitor (MNAi) complex or MNAi complex.
- MNAi multi-component nucleic acid inhibitor
- the MNA complex is an inactive MNA complex which may lack one or more of the components required for assembly and/or catalysis by an MNAzyme including, but not limited to the assembly facilitator, and the partzymes, or components thereof.
- the MNA complex comprises components for an apta-MNAzyme in the presence or absence of an assembly inhibitor.
- Periodizing formation of a nucleic acid complex which functions as a nucleic acid enzyme and "allowing a nucleic acid complex to function as a nucleic acid enzyme” as used herein refers to the process that occurs when a component allows formation of a nucleic acid enzyme complexed with its substrate(s) such that the enzyme can modify the substrate(s).
- modulator is an entity which can increase or decrease the catalytic activity of an MNA complex.
- Modulators may be “activators”, which activate or switch on the activity of an MNAzyme.
- modulators are “inhibitors” which can switch off or inhibit the activity of the MNAzyme or MNAzyme complex, including but not limited to, "assembly inhibitors” or "activity inhibitors”.
- assembly facilitator molecule refers to entities that can facilitate the self-assembly of component partzymes to form a catalytically active MNAzyme by interaction with the sensor arms of the MNAzyme.
- assembly facilitators may facilitate the assembly of MNAzymes which have cleavage, ligase or other enzymatic activities.
- ligase assembly facilitator refers to an assembly facilitator that facilitates the assembly of an MNAzyme with ligase activity.
- An assembly facilitator for MNAzymes with ligase activity may also be referred to by any of the other terms above.
- an assembly facilitator is required for the self-assembly of an MNAzyme.
- An assembly facilitator may be comprised of one molecule, or may be comprised of two or more "assembly facilitator components" that may pair with, or bind to, the sensor arms of one or more oligonucleotide "partzymes".
- Assembly facilitator portions may be contained within other components, for example within an activity inhibitor molecule, where they are present in a state whereby they can not contribute to active MNAzyme assembly until liberated from the component by, for example, cleavage.
- Assembly facilitators may be comprised of one (e.g. Figure 1, Figure 2(ii)) or more molecules (e.g. Figure 2(i), Figure 4-6).
- the assembly facilitator may be a target.
- the target may be 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.
- the nucleic acid may be amplified.
- the amplification may comprise one or more of: polymerase chain reaction (PCR), strand displacement amplification, loop-mediated isothermal amplification, rolling circle amplification, transcription-mediated amplification, self-sustained sequence replication, ligase chain reaction, nucleic acid sequence based amplification, or reverse transcription polymerase chain reaction (RT-PCR).
- PCR polymerase chain reaction
- strand displacement amplification loop-mediated isothermal amplification
- rolling circle amplification transcription-mediated amplification
- self-sustained sequence replication ligase chain reaction
- nucleic acid sequence based amplification amplification
- RT-PCR reverse transcription polymerase chain reaction
- an "activator” as used herein is any MNA structural or modulator oligonucleotide component, any "ligand”, “target”, or “event” that results in activation of MNAzymes.
- Activator oligonucleotides include, but are not limited to, an assembly facilitator or assembly facilitator component; a displacer oligonucleotide; a partzyme or component thereof, for example those with truncations of the sensor or substrate arm, and partzyme stabiliser arm components.
- activators may activate MNAzymes by removing oligonucleotides that exert an inhibitory effect.
- oligonucleotides which can activate through such a mechanism include modulator oligonucleotides which can displace (remove) inhibitory components, or parts thereof, including but not limited to, an "activity inhibitor” or an "assembly inhibitor”.
- target includes any natural or synthetic entity, constituent or analyte which is sought to be detected, identified or quantitated by a method which uses a particular nucleic acid enzyme such as an MNAzyme(s), with or without an additional amplification step and/or cascade.
- Targets therefore encompass the broadest range of detectable entities, constituents or analytes for which methods of sensitive detection, identification and/or quantification are desirable.
- Some exemplary targets include, but are not limited to, nucleic acid, protein, polypeptide, peptide, glycoproteins, lipids, lipoproteins, entire organisms, cells, viruses, bacteria, archaea, yeast, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, small molecules, polymers, metal ions, metal salts, prions or any derivatives, portions or combinations thereof.
- Other targets are also contemplated for use herein. It will be understood that the target may also be an assembly facilitator or activator.
- the term "detectable event” includes a change in the microenvironment of an MNAzyme or MNAzyme complex and/or inactive MNA complex, for example, an MNAi complex or a complex comprising components for an apta-MNAzyme in the presence of an assembly inhibitor.
- the change may be, for example, physical manipulation, a change in temperature, a change in pH, salt concentration, electric charge, magnetic charge, a change in the concentration of one or more analytes, anions, cations, chelators, or a change in concentration of MNA or modulator components, or any combination thereof.
- a "change in the concentration of includes an increase or a decrease in concentration and also includes the appearance of an entity, including any component(s) for one or more MNA complexes, for example an assembly facilitator or assembly facilitator component, that was previously absent or at undetectable concentration in the microenvironment of the MNA complex, including one or more component(s) for one or more MNA complexes including MNAzymes and apta-MNAzymes, MNAzyme complexes and apta-MNAzyme complexes and/or inactive MNA complexes.
- Entities which represent detectable events may also be used as "activators” or “inhibitors” of the catalytic activity of MNAzymes since changes in the microenvironment can be used to manipulate catalytic activity of MNA complexes. As such, these entities allow the catalytic activity of MNAzymes to be switched "on” or “off, for example by promoting transition from an inactive MNA complex to an active MNAzyme, or vice versa.
- the event or entity promotes assembly and activation of MNAzymes.
- the event or entity promotes disassembly and inactivation of MNAzymes.
- the event or entity may direct the assembly or disassembly of inactive MNA complexes.
- multi-component nucleic acid complexes may be inactive due to the composition of the microenvironment, which may promote either disassembly of MNA complex components or assembly of only a subset of those molecules required for formation of catalytically active MNAzymes.
- the activator assembly facilitator component is the product of a reaction occurring in the microenvironment of the inactive MNA complex and/or MNAzyme.
- a "detectable effect" or “output” signal is an effect that can be detected or quantified as an indication that modification of a substrate(s) has occurred.
- the magnitude of the effect may be indicative of the quantity of an input such as an assembly facilitator (e.g. a target).
- the detectable effect may be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance, mass spectroscopy, 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.
- An "activator" assembly facilitator or assembly facilitator portion is a molecule which can be used to control the assembly of active MNAzymes or facilitate the transition from inactive multi-component nucleic acid complexes to active MNAzymes.
- the said multi-component nucleic acid complexes may be catalytically inactive due to the presence of an activity inhibitor, such as in MNAi complexes.
- an activity inhibitor refers to any entity that can bind to one or more components of an MNA complex and direct assembly of catalytically inactive "MNAi" complex (e.g. Figures 4, Figure 5(iii), Figure 13).
- the inhibition of catalytic activity by the activity inhibitor is mediated by an "activity inhibitor portion", also called an "activity inhibitor component” or an "activity inhibitor domain” which is substantially non-complementary to the partzymes.
- an activity inhibitor may comprise several distinct functional domains, for example, including but not limited to, functional domains in any combination selected from an activity inhibitor domain, an assembly facilitator domain, a substrate domain, and/or a reporter domain. Such distinct functional domains may or may not coincide with several distinct structural domains in an activity inhibitor.
- an activity inhibitor may comprise an activity inhibitor domain which is substantially non-complementary to the partzyme components and which exerts an inhibitory effect by disrupting the secondary structure required for formation of a catalytically active MNAzyme.
- the presence of an activity inhibitor drives the assembly of inactive MNAi complexes that are capable of interacting with, but not catalytically modifying, a substrate.
- an activity inhibitor may comprise an assembly facilitator domain.
- the activity inhibitor may further include a labile or cleavable linker or substrate, which may be located between two or more domains within the activity inhibitor, for example an activity inhibitor domain and an activator assembly facilitator domain. Cleavage at the substrate or linker site may allow separation of an activity inhibitor portion from an activator assembly facilitator portion, which may then function as an assembly facilitator component and direct the assembly of an active MNAzyme.
- an MNA component such as an activity inhibitor may be conjugated to other entities.
- the component e.g. activity inhibitor
- the component could be conjugated to a gold nanoparticle coupled to a radio-frequency magnetic field to allow remote electronic control of hybridisation.
- radio- frequency magnetic fields function as antennas enabling reversible thermal denaturation of specific oligonucleotides, while leaving the surrounding molecules relatively unaffected.
- the component e.g. the activity inhibitor
- the component could be labelled with biotin to facilitate capture and physical isolation of the component.
- an "assembly inhibitor” is a component which inhibits the assembly of an MNAzyme or MNAzyme complex by complementary binding to an essential component of an active MNAzyme or MNAzyme complex, for example by binding to a partzyme component or an assembly facilitator or a substrate.
- the binding of the assembly inhibitor sequence to at least a first MNAzyme or MNAzyme complex component leads to competition between the assembly inhibitor and the said first component for binding to a second MNAzyme or MNAzyme complex component.
- the assembly inhibitor may bind to either a partzyme substrate arm (that binds the substrate) or a partzyme sensor arm (that binds the assembly facilitator).
- the assembly inhibitor When the assembly inhibitor is complementary to (and bound to) the substrate arm, it competes (and blocks) binding of the substrate to the partzyme ( Figure 7) and hence blocks formation of MNAzyme complexes.
- the assembly inhibitor When the assembly inhibitor is complementary to (and bound to) the sensor arm, it competes (and blocks) binding of the assembly facilitator to the partzyme and hence blocks formation of active MNAzymes. In this manner an assembly inhibitor blocks the assembly of MNAzymes and/or MNAzyme complexes.
- the assembly inhibitor molecule can be used to control the assembly of MNAzymes and MNAzyme complexes, and further allow the development of strategies for the detection of both non-nucleic acid and nucleic acid analytes.
- substrate and substrate molecule as used herein include any molecule which is capable of being recognized, acted upon or modified by an enzyme including a nucleic acid enzyme.
- the modification of the substrate(s) provides the "output" signal for monitoring the activity of the MNA system.
- one or more substrate(s) may be recognized and modified by an enzyme.
- one or more substrate(s) may be recognized and modified by a nucleic acid with catalytic activity.
- a substrate or substrates may be recognized and modified by an MNAzyme.
- the modification of a substrate or substrates can be measured by the appearance of, or increase in, a product(s) of the modification reaction, or by the disappearance of, or decrease in, a substrate(s) modified in the reaction(s). It will be understood that reference to a substrate may refer to at least one of two or more substrates when used in the context of a reaction requiring two or more substrates such as a ligation reaction.
- a substrate or substrates may be modified by various enzymatic activities including but not limited to cleavage or ligation.
- a substrate may include a "cleavable substrate” which is amenable to enzymatic cleavage, or a "ligatable substrate” or “ligase substrate” which is amenable to enzymatic ligation.
- a "reporter substrate” as used herein is a substrate (or substrates) that is particularly adapted to facilitate measurement of either the disappearance of a substrate or the appearance of a product in connection with a catalyzed reaction.
- 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, biotin (e.g. biotinylation) or chemiluminescent labels.
- “generic substrates” are substrates, for example reporter ⁇ substrates, that are recognized by and acted on catalytically by a plurality of MNAzymes, each of which can recognize a different assembly facilitator.
- the use of such substrates facilitates development of separate assays for detection, identification or quantification of a wide variety of assembly facilitators using structurally related MNAzymes all of which recognize a universal generic substrate.
- These generic substrates can each be independently labelled with one or more labels.
- independently detectable labels are used to label one or more generic substrates to allow the creation of a convenient system for independently or simultaneously detecting a variety of assembly facilitators using MNAzymes.
- substrates cleaved by MNAzymes could be reconstituted, and hence recycled, using an MNAzyme or DNAzyme ligase.
- substrate(s) cleaved or ligated by MNAzymes can be further used as components or modulators of additional MNAzyme(s) or DNAzyme(s).
- the term "product” refers to the new molecule or molecules that are produced as a result of enzymatic modification of a substrate.
- cleavage product refers to a new molecule produced as a result of cleavage or endonuclease activity by an enzyme.
- ligation product refers to a new molecule produced as a result of the ligation of substrates by an enzyme.
- MNAi complex refers to an MNA complex that is in a catalytically inactive state, wherein catalytic activity is inhibited by an "activity inhibitor" as herein defined.
- the MNAi complex may be catalytically inactive due to binding of an activity inhibitor oligonucleotide, for example, as depicted in Figure 4, which shows an exemplary design for an MNAi complex.
- An MNAi complex can be formed when partzyme A, partzyme B, an assembly facilitator and an activity inhibitor associate to form an inactive complex which can bind a substrate but not modify it.
- an "aptamer” may comprise a structure that has the ability to recognize one or more ligands.
- the recognition may have a high degree of specificity due to higher level structure of the aptamer, such as, a 3 -dimensional binding domain or pocket.
- Aptamers may therefore bind protein, polypeptide, peptide or nucleic acid, 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 derivative, portion or combination thereof, or any other entity.
- Preferred aptamers herein may comprise short single- stranded DNA or RNA oligomers that can be isolated from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification.
- Other preferred aptamers herein may comprise protein, polypeptide, peptide or phylomers or a combination thereof that has the ability to recognize one or more ligands. Aptamers may therefore be generated against almost any target, ranging from small molecules such as amino acids, or antibiotics to protein and nucleic acid structures.
- An aptamer may be used to build molecular switches into MNA complexs containing assembly inhibitors.
- an activator ligand can switch the MNAzyme activity on and the removal of an activator ligand can switch off the activity of an MNAzyme.
- aptamers can be used to facilitate detection of nucleic acid and non-nucleic acid ligands. Detection of a ligand can further be used to trigger an amplification cascade.
- cascade refers to any succession of processes or operations that occur in successive stages, wherein the occurrence of each stage is typically dependent on the occurrence of a preceding stage.
- a cascade may be a "linear cascade” wherein stages (or reaction steps) occur in one direction and the occurrence of each stage (or step) is dependent on the occurrence of a preceding stage or step.
- feedback cascade or “feedback amplification cascade” refers to any succession of processes or operations that occur in successive stages, wherein the occurrence of each stage is typically dependent on the occurrence of a preceding stage and whereby the occurrence of a preceding stage typically depends on processes or operations that occur in subsequent stages.
- the product of any preceding stage could serve as a component or substrate for any subsequent stage and the product of any subsequent stage could serve as a component or substrate for any preceding stage.
- the cascade may involve activation of an MNAi complex via removal of the influence of an activity inhibitor or component thereof.
- a cascade may therefore include, but is not limited to, an enzymatic cascade or any other signal transduction cascade.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme with cleavage activity.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme with ligase activity.
- a cascade may comprise amplification of a signal resulting from catalytic activity of DNAzymes or MNAzymes with ligase activity and DNAzymes or MNAzymes with cleavage activity.
- such an amplification cascade may involve repeated and therefore cyclic amplification of a signal, wherein catalytic activity of a first nucleic acid enzyme creates a molecule required for modification of a substrate(s) by a second nucleic acid enzyme, which can in turn create a molecule required for modification of a substrate(s) by one or more additional nucleic acid enzymes.
- MNA multi-component nucleic acid
- MNA complex multi-component nucleic acid complex
- MNAzyme multi- component nucleic acid enzyme
- MNAi multi-component nucleic acid inhibitor
- DNAzyme deoxyribonucleic acid enzyme
- RT-PCR reverse transcriptase polymerase chain reaction
- LNA locked nucleic acid
- PNA peptide nucleic acid
- F fluorophore
- RNA stRNA small nucleolar RNA stRNA: small temporal RNA smRNA: small modulatory RNA pre-microRNA: precursor microRNA pri-microRNA: primary microRNA
- GTP guanosine 5 '-triphosphate
- CTP cytosine 5 '-triphosphate
- dATP deoxyadenosine 5 '-triphosphate ATP: adenosine 5 '-triphosphate
- LP ligation product
- CP cleavage product oligo: oligonucleotide
- MNA complexes may be active enzymes (MNAzymes) with modifying activity such as ligase or cleavage activity.
- MNAzymes active enzymes
- the invention relates to cascades which may also include one or more DNAzymes.
- the invention also relates to methods which use these cascades for the identification, detection and quantification of assembly facilitators such as targets.
- MNA complexes including MNAzymes and inactive MNA complexes
- MNAzymes The Multi-component Nucleic Acid enzymes (also referred to herein as "MNAzymes") and inhibitors thereof are described in detail in co-pending US applications, serial numbers 11/544,926 filed October 6, 2006 and 11/697,021 filed April 5, 2007 and in co-pending international applications PCT/AU2006/001473 and PCT/AU2007/001517, the contents of which are all incorporated herein by reference.
- MNAzymes are capable of self-assembling from two or more oligonucleotide components, also referred to herein as partzymes.
- the partzyme oligonucleotides self- assemble in the presence of an MNAzyme assembly facilitator to form an MNAzyme.
- MNAzymes are therefore catalytically active nucleic acid enzymes.
- 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.
- a wide variety of assays based on the basic principles outlined above are provided herein.
- Compositions comprising oligonucleotides capable of forming MNAzymes, and MNAzymes of various sequences are also provided herein.
- at least one of the oligonucleotide components, assembly facilitator, activity inhibitor or substrate may comprise an aptamer which is capable of binding to an activator or target.
- the present invention describes new methods, and applications for nucleic acid enzymes and complexes.
- the invention provides oligonucleotide components that can be used to form an MNAzyme with ligase activity which can be useful in detection of analytes, in one step formats or in nucleic acid enzyme cascade reactions.
- MNAzymes with ligase activity or other catalytic modifying activities can be used to manipulate the activity of other MNAzymes by creating new components which could direct the formation of a new active MNAzyme or MNAzyme complex or promote assembly of an alternative structure which lacks catalytic activity.
- MNAzymes with ligase activity or other catalytic activity can create new components for nucleic acid enzymes or nucleic acid enzyme complexes such as new MNAzyme or MNAzyme complex components (e.g. assembly facilitators, partzymes, substrates, stabiliser arms) or DNAzyme complex components (e.g. active DNAzymes or DNAzyme substrates).
- new MNAzyme or MNAzyme complex components e.g. assembly facilitators, partzymes, substrates, stabiliser arms
- DNAzyme complex components e.g. active DNAzymes or DNAzyme substrates.
- MNAzymes can be used to direct formation of inactive MNA complexes, including MNAi complexes.
- MNAi complexes comprise oligonucleotide components, which would be capable of being assembled into active MNAzymes under appropriate conditions, but which when assembled with an "activity inhibitor” result in the formation of complexes which are catalytically inactive.
- Such inactive MNA complexes are defined herein as "MNAi" complex, and such inactivity may result from the exertion of an inhibitory influence by an activity inhibitor.
- MNAi complexes can interact with the substrate via the substrate arms of the partzyme components, but cannot catalytically modify the substrate.
- the activity inhibitor may incorporate additional entities to facilitate removal by physical means which may include, but are not limited to, entities such as attached nucleic acids, nanoparticles, microparticles, proteins, antibodies, RNA, DNA, nucleic acid analogues, biotin groups, glycoproteins, lipoproteins, peptide nucleic acids, locked nucleic acids, peptide-nucleic acid chimeras, radio-frequency moiety, or any combination thereof.
- the MNAi complex represents an "off state, whereas the transition to an MNAzyme represents an "on" state.
- the present invention describes MNAzyme ligases and their use as a mechanism for switching from an active MNAzyme to an inactive MNAi complex ( Figure 13).
- the MNAzyme ligase could be used to ligate an activity inhibitor component to an assembly facilitator component to create an activity inhibitor.
- an activity inhibitor binds and replaces an assembly facilitator component in an active MNAzyme the complex is switched to an inactive MNAi complex.
- MNA complexes may contain some or all of the components of an MNAzyme but are not catalytically active either because some required component(s) have not associated to form an active MNAzyme or the microenvironment is incompatible with catalytic activity of an MNAzyme.
- the catalytic activity may be activated upon the occurrence of a particular event, for example, including but not limited to the presence of an activator component, for example an assembly facilitator, or a change in parameter including but not limited to a change in temperature, wavelength, pressure, concentration of salt, detergent, cations or any other parameter.
- the catalytic activity of one nucleic acid enzyme can create a new activator for a new nucleic acid enzyme or nucleic acid enzyme complex which may include, but is not limited to, an assembly facilitator or a partzyme.
- the MNAzymes are based on one or more DNAzymes and/or ribozymes.
- DNAzymes and/or ribozymes are known which can perform catalytic modification of at least one substrate wherein the modification may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl
- More preferred partzyme components for MNAzymes are based on a particular DNAzyme structure.
- Presently preferred structures for MNAzymes with cleavage activity are based on DNAzymes which cleave, including the 10:23 and 8:17 DNAzymes.
- Presently preferred structures for MNAzymes with ligase activity are based on DNAzyme ligases which ligate substrates and create 2' 5' linkages, such as the "7Z81" and "7Z48" and 7Q10 DNAzymes, as well as DNAzyme ligases which ligate substrates and create 3 '-5' linkages such as the 9DBl ligase.
- the MNAzymes and inactive MNA complexes comprise either or both ribonucleotide bases and deoxyribonucleotide bases.
- an MNAzyme and inactive MNA complex is based at least in part on the structure of a DNAzyme.
- MNAzymes and inactive MNA complexes comprise at least some deoxyribonucleotide bases or analogues thereof.
- the catalytic core portions of partzymes assembled into an MNAzyme comprise one or more deoxyribonucleotide bases or analogues thereof.
- one or more deoxyribonucleotide bases or analogues thereof are involved in the catalysis of a substrate by an MNAzyme.
- at least one deoxyribonucleotide base, or its analogue, in the catalytic core improves catalytic activity of an MNAzyme.
- the present invention discloses MNAzyme ligases which are capable of self- assembling from two or more oligonucleotide components, also referred to herein as partzymes.
- the partzyme oligonucleotides self-assemble in the presence of at least one MNAzyme ligase assembly facilitator to form an MNAzyme ligase.
- MNAzyme ligases are therefore catalytically active nucleic acid ligases.
- FIG. 10 An exemplary MNAzyme with ligase activity comprising a first partzyme and a second partzyme is depicted in Figure 10.
- the first partzyme and the second partzyme each bind to an assembly facilitator (e.g. through Watson-Crick base pairing).
- the MNAzyme ligase only forms when the sensor arms (i) of the first partzyme and the second partzyme hybridize adjacent to each other on the assembly facilitator.
- the substrate arms (iii) of the MNAzyme engage the substrates, the modification (ligation) of which is catalyzed by the catalytic core of the MNAzyme ligase, formed by the interaction of the catalytic domains (ii) of the first partzyme and the second partzyme. Ligation of two nucleic acid substrates is exemplified in the drawing.
- the presence of an MNAzyme with ligase activity can be detected, and is indicative of the presence of a target, because the MNAzyme ligase forms only in the presence of the target, wherein the target comprises the assembly facilitator.
- assays based on the basic principles outlined above are provided herein.
- Compositions comprising oligonucleotides capable of forming MNAzyme ligases, and MNAzyme ligases of various sequences are also provided herein.
- the MNAzyme ligase structures are based on one or more DNAzyme ligases. More preferred are those MNAzyme ligase structures which are based on a particular DNAzyme ligase structure. Presently preferred structures are based on DNAzyme ligases including the 7Q10, 7Z81, 7Z48 and 9DBl DNAzyme ligases.
- An assembly facilitator such as a target nucelcic acid may be detected using an MNAzyme with ligase activity.
- the provision of target nucleic acid is the initiating step which results in the formation of an MNAzyme with ligase activity.
- the initiating target nucleic acid would be the assembly facilitator B and the assembly facilitator A would be a synthetic molecule included to drive the assembly of MNAzyme A.
- MNAzyme A would then have the sole purpose of generating a 5' ligatable substrate B with a 2'3' cylic phosphate terminus in a reaction which could then be added to the reaction which detects the target assembly facilitator B using an MNAzyme B with ligase activity.
- the MNAzyme A partzymes and the assembly facilitator A could be replaced by a DNAzyme with cleavage activity present either in a separate preliminary reaction for ligatable substrate generation or the DNAzyme could be present in the same reaction mix as the MNAzyme with ligase activity.
- Example 11 demonstrates that cleavage and ligation using a combination of an MNAzyme and a DNAzyme can be performed in a single reaction mix.
- MNAzymes and inactive MNA complexes 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 in fact are the basis of allowing tailoring of the molecules to different substrates/assembly facilitators.
- modification of the sensor arms will allow tailoring to different assembly facilitators
- modification of the substrate arms will allow tailoring to different substrates.
- Analysis of multiple substrates allows the simultaneous monitoring of multiple "detectable effects" or "output" signals as an indication of the presence of the assembly of multiple active MNAzymes in response to an input event/s or entity/ies (e.g. provision of assembly facilitator/s) which are then able to modify multiple substrates
- the invention envisages MNAzymes with catalytic activity that are comprised of deoxyribonucleotides or which are derived from such molecules by certain modifications/ substitutions etc.
- replacement of the whole molecule with, for example, ribonucleotides will render the molecule inactive because it relies for its activity on certain key deoxyribonucleotides.
- some ribonucleotides in a ribozyme may be substituted with deoxyribonucleotides but replacement of the whole molecule with, for example, deoxyribonucleotides, will render the molecule inactive.
- MNAzymes with cleavage, ligation or other enzymatic activities, and inactive MNA complexes comprise either deoxyribonucleotides or ribonucleotides, or even both.
- Those MNAzymes and inactive MNA complexes comprising at least one and more preferably, all, deoxyribonucleotide component oligonucleotides are presently preferred.
- those MNAzymes and inactive MNA complexes comprising at least one deoxyribonucleotide base, or its analogue, within at least one of the partial catalytic cores of the partzymes. Even more preferred are those embodiments where such a base is required for catalytic activity of an MNAzyme with cleavage, ligase or other enzymatic activity.
- At least one component of an MNA complex may comprise a region of self complementarity that may under some conditions form a hairpin structure.
- a region of self complementarity may be located in one or both of the partzyme sensor arms.
- the region of self complementarity may be located in one or both of the partzyme substrate arms.
- a region or regions of self complementarity may be present in an assembly facilitator, an assembly inhibitor or an activity inhibitor component, or any combination thereof.
- MNA complexes may bind substrates which may contain regions of self complementarity.
- the catalytic modification of at least one substrate is selected from the group consisting of cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion and phosphoramidate
- MNAzymes comprised of DNA have advantages over uni-molecular or multipartite ribozymes, for example with respect to stability and ease of use. It is also to be appreciated that in some embodiments, MNAzymes offer advantages over uni-molecular nucleic acid enzymes, for example DNAzymes, which can only recognize one molecule which functions as both the target and the substrate. Further, catalytic modification, for example cleavage, can destroy the DNAzyme's target/substrate and as such the target is not available for another round of recognition.
- a single MNAzyme for example an MNAzyme with cleavage activity, can recognize at least two molecules, namely at least one assembly facilitator and at least one substrate.
- an assembly facilitator for example a target nucleic acid present in a sample, is not catalytically modified in the reaction and remains available for further rounds of substrate modification by the MNAzyme.
- a single MNAzyme which can act on two substrates can recognize at least three molecules, namely at least one assembly facilitator and at least two substrates.
- MNAzymes make them adaptable for systems or processes that require the components to be able to "read" an "input” signal, for example detect the presence of an assembly facilitator, and “write” an "output” signal, for example producing a detectable signal indicative of enzymatic modification of a substrate, or by the creation of a new product (e.g. a new assembly facilitator or component) which can be useful as a new input signal.
- Such processes include cascade reactions, logic gates and other processes.
- MNAzymes therefore could provide a mechanism for transduction of information, for example, to receive an input signal and respond with an appropriate output response.
- MNAzyme assembly and disassembly may be controlled by changing the microenvironment. Examples of such changes include, but are not limited to, temperature, divalent cation type and concentration, salt concentration, pH, additives, and the presence or absence of critical components essential for assembly and/or activity of an active MNAzyme.
- the assembly of catalytically active MNAzymes may be regulated in a temperature-dependent manner. In one embodiment the assembly of catalytically active MNAzymes may be regulated in a pH-dependent manner. In one embodiment the assembly of catalytically active MNAzymes may be regulated in a manner dependent on the presence or absence of divalent cations or chelators. In one embodiment the assembly of catalytically active MNAzymes may be regulated in a salt-dependent manner. In one embodiment the assembly of catalytically active MNAzymes may be regulated in a manner dependent on ionic concentration.
- the assembled MNAzyme represents an "on” state
- the disassembled MNA complex components represent an "off state.
- the assembly and disassembly of MNA complexes can be controlled by temperature.
- the "on” state can be induced by switching the temperature to one within the range that is compatible with both assembly and catalytic activity of an MNAzyme or MNAzyme complex.
- the "off states can be induced by switching the temperature to outside the range that is compatible with either assembly and/or catalytic activity of an MNAzyme or MNAzyme complex.
- the melting temperatures of the components of MNA complexes can be adjusted to only allow assembly within a restricted temperature range.
- Oligonucleotides which are particularly useful in this aspect of the invention, include but are not restricted to, stabilizer arm components, partzyme components with truncated sensor arms and components of assembly facilitator and/or modulator oligonucleotides.
- MNA complexes in which the components of the basic design ( Figure 1) have been further t split into smaller component subunits or portions ( Figure 2), the sequence of which can be tailored with respect to the melting temperature, the sequence composition and complementarity, or lack thereof, with other component oligonucleotides.
- the partzyme arm which is truncated could be any of the following: the partzyme A sensor arm, the partzyme B sensor arm, the partzyme A substrate arm, the partzyme B substrate arm, or any combination thereof.
- MNAzyme complexes with the structures as exemplified in Figure 6 could also be used to modify substrates in ways other than cleavage, for example, the MNAzyme structures could also ligate substrates.
- thermo-sensors and rheostats The sensitivity of an MNAzyme to temperature can be exploited to build thermo-sensors and rheostats. If the temperature were either too high, or too low, for the assembly (hybridization) of the component oligonucleotides, and/or for catalytic activity, then the MNAzyme substrate would not be modified (eg cleaved or ligated). If the temperature were permissive for MNAzyme activity then the substrate would be modified and a signal would be generated. A rise or fall in temperature from one that is incompatible with MNAzyme activity, to another which is compatible with MNAzyme activity, would be detected by a signal generated following substrate modification by the MNAzyme.
- MNAzymes can thus provide a device capable of detecting temperature changes.
- a magnetic force can regulate cation concentration and hence provide a switch for modulating MNAzyme activity on and off. Positively charged cations are required for the catalytic activity of some MNAzymes.
- a magnetic force could alternatively switch the MNAzyme activity off by physically separating the negatively charged MNA oligonucleotides, for example the partzymes, assembly facilitators and substrates or components thereof, from the positively charged cations, for example Mg 2+ . The MNAzyme could then be switched back on by allowing the MNA oligonucleotides and cations to come back in contact.
- the active "on” states can be induced using a pH within the range that is compatible with activity.
- an "off state can be induced using a pH outside the range that is compatible with activity. pH may further be used to control activity of MNA complexes by inducing hydrolysis of labile sequences, and thus either creating or destroying a new component for an MNAzyme and/or inactive MNA complex.
- the presence or absence of any component of the MNA complexes can provide either an "on” or “off switch. Changing, for example, the oligonucleotide sequence, the melting temperature and or concentration can achieve finer regulation.
- the broad scope for designs of components which can assemble into MNA complexes for example two-part assembly facilitators and/or two-part partzyme components (e.g. with truncated sensor domains and stabilizer arms), introduces flexibility into systems which can allow tailoring (fine tuning) of conditions compatible with hybridization and hence MNA complex assembly.
- Physical manipulation of components can be achieved, for example, by exploiting either physical properties of attached moieties as molecular "hooks", and/or by exploiting inherent properties of the oligonucleotides, for example, negative charge, or sequence complementarity.
- the attached moiety allows oligonucleotides to be selectively captured, for example using a biotin group.
- the moiety contains a radio-frequency magnetic field radio to facilitate remote electronic control of hybridisation. This approach could be designed to allow the selective removal of component molecules by targeted thermal denaturation of specific oligonucleotides within an MNA complex, thus allowing activation, or inhibition, of enzymatic activity depending on whether the component molecule is itself an activator or an inhibitor sequence.
- the activity inhibitor can be selectively denatured from an MNAi complex, allowing transition to the active MNAzyme state.
- an activity inhibitor could be removed from an MNAi complex by a modulator oligonucleotide such as a displacer oligonucleotide which functions by branch migration.
- a ligase substrate could be removed from an MNAzyme complex with ligase activity by a modulator oligonucleotide such as a displacer oligonucleotide which functions by branch migration.
- An alternate exemplary strategy is designed to facilitate switching an MNA complex between the "on state" of an active MNAzyme to the "off state” of an MNAi complex using a second MNAzyme with ligase activity (or a DNAzyme with ligase activity).
- an active MNAzyme A which could be capable of modifying (eg cleaving or ligating) a substrate(s) A could be formed in the presence of assembly facilitator component 1 (AFC 1) and assembly facilitator component 2 (AFC 2).
- a second MNAzyme B which has ligase activity, could form in the presence of assembly facilitator 3 (AF3) and could then ligate AFC 2 with an activity inhibitor component (AIC) causing the formation of an activity inhibitor (AI).
- AIC activity inhibitor component
- AI activity inhibitor
- This AI could bind to the partzyme components for the MNAzyme A and result in the formation of an MNAi A complex which is inactive.
- the MNAzyme ligase in this example could operate as an off switch to inactivate the MNAzyme A.
- a DNAzyme ligase could replace the MNAzyme B (MNAzyme ligase) and could operate as an off switch to inactivate the MNAzyme A.
- the inactive MNAi complex and the catalytically active MNAzyme would represent two alternate states for the assembled components, namely an "off state and the "on” state respectively.
- the skilled artisan will recognize that the various methods provided herein can generally be used to modulate the assembly or activity of single MNA complexes or of multiple MNA complexes in a single reaction or assay.
- the presence or absence of any component of the multi- component nucleic acid complexes can provide either an "activator” or “on” switch or it may provide an inhibitor or "off switch.
- the presence or addition of a stabilizer arm can provide an "on" switch.
- new stabilizer arms can be generated in the system during a reaction, for example by cleavage of a substrate, which may comprise, for example another MNA complex component.
- the absence, modification or removal of a stabilizer arm can provide an "off switch.
- the presence of an assembly facilitator, or a component thereof can provide an "on" switch.
- new assembly facilitators can be generated by MNAzyme cleavage of MNA complex components, for example, by cleavage of a substrate including, but not limited to, those substrates which can serve as another MNA complex component such as an activity inhibitor prior to modification by cleavage.
- the assembly facilitators can provide specific "input" signal systems, encoded within the sequence.
- the assembly facilitator can be recognized or "read”.
- the partzyme sensor arm can "read" assembly facilitator sequences including those differing by one or more single bases.
- the absence or removal of an assembly facilitator, or a component thereof can provide an "off switch.
- components of MNA complexes may be generated by ligation of components present in the reaction milieu.
- a partzyme is created by ligation of oligonucleotides thereby generating a new partzyme component which can associate to form, for example, an active MNAzyme.
- an assembly facilitator is created by ligation of oligonucleotides thereby generating a new assembly facilitator component which can facilitate assembly of an MNA complex.
- a substrate or other nucleic acid enzyme or component thereof may be generated by ligation of components.
- Transition between states of activation and inactivation can provide a mechanism for creating a molecular switch, which can be regulated by alternating between the active and inactive conformations.
- Such molecular switches may, for example, be applied to the control of nucleic acid replication cascades, or to the regulation of autonomous therapeutic, diagnostic and computational molecular scale devices.
- compositions comprising the components for self-assembling MNAi complexes that self-assemble in the presence of one or more MNAzyme assembly facilitator molecules and one or more assembly inhibitor molecules to form catalytically inactive MNAi complexes.
- Figure 1 depicts an example of a basic method for assembling an MNAzyme using an assembly facilitator. More specifically, partzyme A and partzyme B are shown in Figure 1, each comprising a (i) sensor arm portion, (ii) a substrate arm portion, and (iii) a catalytic core portion.
- the sensor arm portions of partzyme A and partzyme B can hybridize to, and base pair with complementary portions of the assembly facilitator, for example a DNA or RNA target sequence.
- the MNAzyme self-assembles forming a catalytic core which can modify a substrate which is bound by the substrate arms.
- the presence of the MNAzyme is detected through the detection or measurement of its catalytic activity.
- the substrate arms of the assembled MNAzyme can engage a substrate, for example the reporter substrate shown in Figure 1 , through the interaction of the complementary sequences on the substrate arms and the substrate.
- the catalytic core can promote the modification (eg. cleavage) of the substrate, which can in turn be measured or detected, directly or indirectly.
- the MNAzyme can be alternatively assembled (switched on) and dissembled (switched off) using various methods.
- the assembly facilitator may comprise synthetic oligonucleotides which are added to the mix to drive the assembly of MNA complexes.
- FIG. 2 With reference to Figure 2, additional exemplary designs for active MNAzyme complexes are shown.
- the exemplary structure for one MNAzyme complex is depicted in panel (i) where multiple assembly facilitator components are required for formation of an MNAzyme.
- one component (Fl) of the assembly facilitator is complementary to regions of the sensor arms of both partzyme A and B, whereas a second assembly facilitator component (F2) has complementarity with either partzyme B only (as per Figure 2(i)), or partzyme A only.
- the two assembly facilitator components together direct the assembly of an active MNAzyme which can modify (eg cleave) a substrate.
- Panel (ii) depicts an exemplary design of an MNAzyme complex where the assembly of partzyme A with a bi-partite partzyme B component in the presence of assembly facilitator produces an active MNAzyme capable of modifying (eg cleaving) a substrate.
- partzyme B has a truncated sensor arm (T), which is insufficient to allow stable MNAzyme assembly in the absence of a second component, referred to herein as a stabiliser arm component (S).
- T truncated sensor arm
- S stabiliser arm component
- a stabiliser arm component hybridises to the assembly facilitator in a location adjacent to that where the truncated sensor arm of the partzyme binds, this allows assembly into an active MNAzyme.
- the truncated arm is designed to be insufficient to allow stable MNAzyme assembly under the reaction conditions, unless accompanied by a stabiliser arm component.
- the partzymes, stabiliser arm component, and the assembly facilitator can thus function as "on" switches for MNAzyme activity.
- an active MNAzyme complex (right hand side) can be derived from the components of the MNAi complex, following modification of the activity inhibitor such as to cleave or bisect the molecule and separate the (i) activity inhibitor portion and the (ii) activator assembly facilitator portion.
- the released activator assembly facilitator portion is then able to function as a second assembly facilitator component, which in concert with a first assembly facilitator component, can direct the assembly of partzyme components A and B into an active MNAzyme capable of catalytically modifying a substrate.
- An active MNAzyme A which could be capable of modifying (eg cleaving or ligating) a substrate(s) A could be formed in the presence of assembly facilitator component 1 (AFC 1) and assembly facilitator component 2 (AFC 2).
- a second MNAzyme B which has ligase activity, could form in the presence of assembly facilitator 3 (AF3) and could then ligate AFC 2 with an activity inhibitor component (AIC) resulting in the formation of an activity inhibitor (AI).
- This AI can bind to the partzyme components for MNAzyme A resulting in the formation of an MNAi A complex which is inactive.
- the MNAzyme ligase in this example can operate as an off switch to inactivate an MNAzyme A.
- the inactive MNAi complex and the catalytically active MNAzyme represent two alternate states for the assembled components, namely an "off state and the "on" state respectively.
- the methods are applicable in solution, or combined with an insoluble support or solid support on which one or more of any MNA complex component(s), including the substrate or portion thereof, may be attached.
- additional enzymes or components thereof present in an MNAzyme cascade including DNAzymes with for example,either cleavage and/or ligase activity may be attached to an insoluble support or solid support. Accordingly, at least one of a DNAzyme, a partzyme component, a substrate, an assembly inhibitor, an assembly facilitator or assembly facilitator component, a stabilizer arm and/or an inhibitor such as an activity inhibitor or assembly inhibitor may be bound, attached or tethered.
- a portion of at least one of a partzyme component, a substrate or substrates, an assembly inhibitor, an assembly facilitator or assembly facilitator component, a stabilizer arm and/or an inhibitor such as an activity inhibitor or an assembly inhibitor may be bound, attached or tethered.
- Embodiments of the present invention encompass an insoluble support.
- the insoluble support could be in the form of a "chip", otherwise known as an array or microarray, which typically comprise a plurality of oligonucleotides coupled, tethered or otherwise attached to the chip.
- the oligonucleotides comprise nucleic acid.
- the plurality of oligonucleotides could comprise a plurality of components, or portions thereof, for MNA complexes which include but are not limited to, substrates, and/or partzymes, and/or assembly facilitators, and/or assembly inhibitors and/or activity inhibitors and/or any combination thereof.
- the plurality of oligonucleotides could comprise a plurality of oligonucleotides which are complementary to a domain contained within, and/or appended to, any of the components comprising the plurality of components, or portions thereof, for MNA complexes.
- the plurality of components, or portions thereof, for MNA complexes could comprise oligonucleotides with more than one functional domain which may comprise a substrate, and/or a partzyme, and/or an assembly facilitator, and/or an assembly inhibitor and/or an activity inhibitor and/or any combination therof.
- a plurality of oligonucleotides may be positioned upon a solid support, for example a chip, bead, plate or array by any suitable method known in the art, for example, by pipette, ink-jet printing, contact printing or photolithography.
- the solid support may be comprised of at least one element, with each element comprising at least one nucleic acid oligonucleotide.
- the at least one element may be comprised of a plurality of oligonucleotides of the same sequence.
- the number of elements comprising the solid support may be any number, and where a plurality of elements is positioned on a solid support, the elements may be spaced apart at a uniform or a variable distance, or a combination thereof.
- the elements may be positioned randomly, with the respective location of each element then determined.
- the size and shape of the elements will depend upon the particular application of the present invention, and different sized and shaped elements may be combined into a single solid support.
- the surface of the solid support may be substantially planar or may have features such as depressions or protuberances, and the elements may be positioned either into the depressions or onto the protuberances.
- Such depressions may provide a reservoir for solutions into, which the elements are immersed, or such protuberances may facilitate drying of the elements.
- elements may be placed in each well of a 96 well plate.
- the solid support may include unique identifiers such as indicia, radio frequency tags, integrated devices such as microprocessors, barcodes or other markings in order to identify each of the elements.
- the unique identifiers may additionally or alternatively comprise the depressions or protuberances on the surface of the array. Furthermore, the unique identifiers can provide for correct orientation or identification of the chip. The unique identifiers may be read directly by a data capture device or by an optical scanner or detector.
- 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 assembly facilitators.
- a generic substrate sequence or sequences is provided and the same substrate or substrates can therefore be incorporated in systems for various diverse applications. Further, the same substrate(s) can be incorporated into the methods in various embodiments herein, including assays where the substrate(s) is free in solution or is tethered or attached to a support.
- a series of generic substrates can be used in a multiplex reaction allowing simultaneous detection of multiple assembly facilitators.
- substrates which have been cleaved by an MNAzyme or DNAzyme with endonuclease or cleavage activity can be reconstituted and hence recycled using a DNAzyme ligase or a MNAzyme ligase.
- Substrates which have been ligated by a DNAzyme or MNAzyme with ligase activity can be recycled as substrates for a DNAzyme or MNAzyme with cleavage activity provided the DNAzyme or MNAzyme cleaver can cleave the link created by ligation by the DNAzyme or MNAzyme ligase.
- an MNAzyme with cleavage activity may cleave at a site within a ligation product which is not at the junction of the two ligatable substrates provided the cleavage site requirements are met at this site.
- MNA complexes have an advantageous property in certain embodiments of being able to utilize a universal or generic substrate or substrates.
- a substrate is shown in Figure 1 in a presently preferred configuration wherein the substrate for an MNAzyme with cleavage activity comprises both a detectable portion and a quencher portion.
- the quencher portion is adapted to diminish or eliminate a detectable signal from the detectable portion of the substrate until the substrate is cleaved by an MNAzyme.
- the quencher portion may comprise "Black Hole Quencher 1" (BHQl) or "Black Hole Quencher 2" (BHQ2).
- the MNAzyme cleaves the substrate between the detectable portion and the quencher portion allowing the two portions to separate in solution, thereby allowing the detectable signal to appear or increase as the quencher portion is distanced from, or effectively removed from the local environment of the detectable portion.
- substrate(s) for an MNAzyme with ligase activity can be designed such that one substrate comprises a detectable portion and one substrate comprises a quencher portion.
- the quencher portion is adapted to diminish or eliminate a detectable signal from the detectable portion when the substrates are ligated into a ligation product thus bringing the detectable and quencher portions into close proximity.
- the quencher portion may comprise "Black Hole Quencher 1" (BHQl) or "Black Hole Quencher 2" (BHQ2).
- an MNAzyme with ligase activity or a DNAzyme with ligase activity could ligate substrates bringing the detectable portion of one substrate in close proximity with the quencher portion of the second substrate, thereby allowing the detectable signal to decrease.
- the 5' ligation substrate for example, were labelled with a detectable moiety, for example a fluorophore, and the 3' ligation substrate were attached to a discrete location, for example on a solid support such as a chip or a bead, then ligation would result in the appearance of a signal at the discrete location in the presence of target analyte.
- a detectable moiety for example a fluorophore
- reporter substrate for an MNAzyme cleaver, or a single pair of ligatable substrates for an MNAzyme ligase, can be used for detection of a single assembly facilitator or other input event using an MNAzyme, and/or detection of multiple assembly facilitators in a series of systems using altered MNAzymes.
- a plurality of generic cleavable reporter substrates, or generic ligatable substrate pairs, allows multiplexed monitoring of multiple MNAzymes within one system.
- the MNAzyme cleaver 3 could have different sensor arms than MNAzyme cleaver 1 but it may have one substrate arm (on one of the partzymes) that is the same as the sequence of one substrate arm of the partzyme of MNAzyme cleaver 1 which interacts with that portion of the sequence on substrate 1 (here, the 5' portion of the substrate) which is subsequently ligated by the ligase.
- MNAzyme cleaver 3 would cleave another substrate (referred to as substrate 3) which has the same sequence at the 5' portion but which has a different portion at the 3' end (or vice versa) with respect to cleavable substrate 1.
- substrate 3 Another substrate which has the same sequence at the 5' portion but which has a different portion at the 3' end (or vice versa) with respect to cleavable substrate 1.
- the cleavage site of substrate 3 would lie at the junction of those sequences which are shared between the cleavable substrates 1 and 3 and those which differ.
- MNAzyme cleaver 3 when MNAzyme cleaver 3 was assembled in the presence of an assembly facilitator component generated by cleavage by the MNAzyme cleaver 2, and if MNAzyme cleaver 3 cleaved substrate 3 then the 5' cleavage product of MNAzyme cleaver 3 could also serve as a 5' substrate for the DNAzyme ligase (or the assembled MNAzyme ligase) and a feedback amplification cascade reaction could be initiated.
- the substrates may incorporate additional entities such as labelled nucleic acids, nanoparticles, microparticles, proteins, antibodies, RNA, DNA, nucleic acid analogues, biotin group, glycoproteins, lipoproteins, peptide nucleic acids, locked nucleic acids, peptide-nucleic acid chimeras, moiety for radio-frequency magnetic field, or any combination thereof.
- Substrates can be modified by an MNAzyme thereby providing a "detectable effect" or "output” signal.
- the substrate modification by an MNAzyme may involve, for example, cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide
- a detectable effect may be generated and the magnitude of the effect may therefore be indicative of the quantity of the input signal.
- the detectable effect may be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance, mass spectroscopy, 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 UV, visible light or infra red spectroscopy, enzymatic methods or any combination thereof.
- the determination of catalytic activity may be performed in a manner permitting the catalytic activity to be quantified.
- the magnitude of the detectable event may be determined from the quantified catalytic activity.
- a DNAzyme substrate sequence within the bridging oligonucleotide can provide a mechanism for reversing the aggregation of the gold particles (Liu and Lu, 2004). Activation of the lead-dependent DNAzyme by the addition of lead, caused cleavage of the bridging oligonucleotide, dissociation of the gold particles and a change in colour from blue to red. [ 00276 ] Simple detectors for monitoring changes could be developed using this principle and an MNAzyme with endonuclease or ligase activity.
- the temperature at which such methods may be performed may be in the range of about 2O 0 C to about 96 0 C, about 2O 0 C to about 75 0 C, about 2O 0 C to about 6O 0 C or about 20 0 C to about 55 0 C.
- the temperature may be constant or may be cycled between one temperature which is compatible with assembly of an MNA complex and/or catalytic activity of an MNAzyme and one temperature which is incompatible with assembly of an MNA complex and/or catalytic activity of an MNAzyme.
- a variation in a parameter, and/or the microenvironment may be used to trigger activation of the MNA complex from an inactive to an active state.
- a parameter in the microenvironment may comprise an "activator" as herein defined, including but not limited to events such as change in temperature, wavelength, concentration of salts, detergents, cations, and concentration of structural and modulator components which include but are not limited to assembly facilitators or assembly facilitator components, partzymes or partzyme components, substrates, assembly inhibitors, activity inhibitors and activator oligonucleotide components. Accordingly, such optimization of parameters and/or microenvironment may be undertaken in order to achieve use of the MNA complexes as elements of cascade reactions or molecular switches.
- 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 may be influenced in a concentration-dependent fashion by the concentration of divalent cations.
- Preferred optimized reactions are optimized for one or more Of Ba 2+ , Sr 2+ , Mg + , Ca 2+ , Ni 2+ , Co 2+ , Mn 2+ , Zn 2+ , and Pb 2+ .
- a method is illustrated whereby an activator ligand can be used to switch "on” or “off the activity of the apta-MNAzyme.
- the method uses an assembly inhibitor to block activity of apta-MNAzymes in the absence of an activator as illustrated.
- aptamers which may comprise a nucleic acid, protein, polypeptide, phylomer, peptide or combination thereof that has the ability to recognize one or more ligands.
- Aptamers may bind, 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 (Lee et al, 2004).
- Preferred aptamers herein may comprise short single-stranded DNA or RNA oligomers that can be isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery, and reamplification.
- Other preferred aptamers may comprise protein, polypeptide, peptide or phylomers or a combination thereof that has the ability to recognize one or more ligands.
- Aptamers may be generated which may recognize and bind almost any target, ranging from small molecules such as amino acids or antibiotics, to protein and nucleic acid structures.
- 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.
- At least one component of the MNA complex may contain at least one aptamer or portion thereof wherein said aptamer or portion thereof binds a ligand selected from the group comprising nucleic acids, polypeptide, peptide, entire organisms, proteins, glycoproteins, lipids, lipoproteins, cells, viruses, bacteria, archaea, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, small molecules, polymers, metal ions, metal salts, prions or any derivatives, portions or combinations thereof, or any other entity.
- a ligand selected from the group comprising nucleic acids, polypeptide, peptide, entire organisms, proteins, glycoproteins, lipids, lipoproteins, cells, viruses, bacteria, archaea, fungi, antibodies, metabolites, pathogens, toxins, contaminants, poisons, small molecules, polymers, metal ions, metal salts, prions or any derivatives, portions or combinations thereof, or any other entity.
- An apta-MNAzyme for example, with either cleavage or ligase activity may incorporate one or more aptamers.
- the assembly of the apta-MNAzyme may therefore be directed by the presence or absence of one or more analytes which bind the aptamer and cause dissociation of an assembly inhibitor.
- aptamers may be incorporated into either end of the assembly facilitator molecule or molecules, and or the activity inhibitor. Further it will be appreciated that multiple aptamers could be incorporated into one or more of the partzyme oligonucleotide components. Still further it will be appreciated that aptamers could also be incorporated into at least one of the partzyme oligonucleotide components.
- the assembly facilitator in the strategies illustrated in Figure 7 can comprise DNA, RNA, LNA, PNA or a sequence containing one or more nucleotide base analogues.
- an aptamer sequence is incorporated at the end of a partzyme (apta-partzyme) in a configuration whereby an active apta-MNAzyme is only formed in the presence of the activator.
- aptamers could be incorporated into any of the oligonucleotide components, including the partzymes, the assembly facilitator, activity inhibitor or the substrate. Further the aptamer could be incorporated into either end of any one of these oligonucleotides.
- the strategy illustrated in Figure 7 can be used either (i) to provide a method to control apta-MNAzyme activity using ligands as activator molecules, and/or (ii) to provide a method for detection of non-nucleic acid, and nucleic acid targets using apta- MNAzymes.
- the apta-MNAzyme could be the first nucleic acid enzyme in a cascade reaction designed to detect a range of ligand targets.
- the nucleic acid oligonucleotides required for one schema for apta-MNAzyme detection strategy include; a) a partzyme; b) an apta-partzyme which is a partzyme with an aptamer incorporated into one of its ends; c) an assembly facilitator which is an oligonucleotide which binds to both the apta- partzyme and the partzyme enabling assembly of an active MNAzyme; d) a substrate e.g.
- a reporter substrate e) an assembly inhibitor oligonucleotide 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 apta-partzyme sequence.
- the system could be used to simultaneously detect a combination of nucleic acid and non- nucleic acid molecules.
- the apta-MNAzyme may be used to detect the presence of (i) a nucleic acid target, which functions as an assembly facilitator and directs the formation of the apta-MNA complex, and (ii) a target activator ligand (e.g. a protein or small molecule) which could displace the assembly inhibitor oligonucleotide and allow assembly of an active apta-MNAzyme complex and subsequent substrate modification.
- a target activator ligand e.g. a protein or small molecule
- apta-MNAzyme with ligase activity at least two ligatable substrates could be present in the reaction, one of which could be labelled with a fluorophore and the other of which could be labelled with a quencher.
- an activator ligand When an activator ligand is present, it would bind to the aptamer sequence of the apta-partzyme, blocking the binding of the assembly inhibitor oligonucleotide, and thus allowing binding and ligation of the substrates.
- apta-MNAzyme complexes could only form and cause a decrease in fluorescent signal in the presence of activators that can bind aptamers.
- modulation of MNAzyme activity can be achieved using either a nucleic acid or a non-nucleic acid target activator ligand as a switch mechanism.
- the assembly inhibitor molecule is manipulated by other means so as to modulate activity.
- the assembly inhibitor could be removed by several strategies including selective thermal denaturation or methods that use oligonucleotides to compete for binding and/or displacer olignucleotides that use branch migration to displace oligonucleotide components of MNA complexes.
- the aptamer sequences for an apta-MNAzyme with modifying activity could be appended to either partzyme A or partzyme B or both.
- the assembly inhibitor oligonucleotide bound to portions of the aptamer and the substrate arm of at least one of the partzymes.
- the aptamer sequences for an apta-MNAzyme could be appended to the sensor arms of at least one of either partzyme A or partzyme B or both.
- the assembly inhibitor oligonucleotide would bind to portions of the aptamer and the sensor arm. In the absence of an activator ligand, the assembly inhibitor oligonucleotide binds to the apta-partzyme thus competing with and blocking binding of an assembly facilitator, or assembly facilitator component. When an activator ligand was present, it would bind to the aptamer sequence of the apta-partzyme, blocking the binding of the assembly inhibitor oligonucleotide, and thus allowing binding of the assembly facilitator or assembly facilitator component and directing assembly of an active apta-MNAzyme capable of modifying, for example cleaving or ligating, the substrate or substrates. As such, apta- MNAzymes could only form and cause a change in fluorescent signal, for example an increase or decrease in fluorescence, in the presence of activators that can bind aptamers.
- the modification can cause for example an increase in fluorescence by cleavage of a dual labelled reporter substrate which results in separation of a fluorophore/quencher dye pair.
- the modification performed by an apta- MNAzyme could be a modification other than cleavage, for example, ligation of two substrates.
- the signal change could be, for example, a decrease in fluorescence following ligation of two ligatable substrates each labelled with either the fluorophore or the quencher of a fluorophore/quencher dye pair.
- This apta-MNAzyme approach could also be used to develop target detection methods or molecular switches that can turn on and off the catalytic activity of the MNA complex. Further, the apta-MNAzyme approach may comprise the first step in a linear nucleic acid enzyme cascade or a feedback nucleic acid enzyme amplification cascade. The apta-MNAzyme could also be applied to simultaneous detection of both nucleic acid targets such as assembly facilitator and non-nucleic acid target ligands such as those which can bind to aptamers. 8. Cascades
- Cascades are any succession of processes or operations that occur in successive stages, wherein the occurrence of each stage is typically dependent on the occurrence of a preceding stage.
- stages or reaction steps
- the occurrence of each stage or step
- feedback cascades or feedback amplification cascades there may be a succession of processes or operations that occur in successive stages, wherein the occurrence of each stage is typically dependent on the occurrence of a preceding stage and whereby the occurrence of a preceding stage further depends on processes or operations that occur in subesequent stages.
- the product of any preceding stage could serve as a component or substrate for any subsequent stage and the product of any subsequent stage could serve as a component or substrate for any preceding stage.
- a cascade may therefore include, but is not limited to, an enzymatic cascade or any other signal transduction cascade.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme with cleavage activity.
- a cascade may comprise amplification of a signal resulting from catalytic activity of an MNAzyme with ligase activity.
- a cascade may comprise amplification of a signal resulting from catalytic activity of DNAzymes or MNAzymes with ligase activity and DNAzymes or MNAzymes with cleavage activity.
- such an amplification cascade may involve repeated and therefore cyclic amplification of a signal, wherein catalytic activity of a first nucleic acid enzyme creates a molecule required for modification of a substrate(s) by a second nucleic acid enzyme, which can in turn create a molecule required for modification of a substrate(s) by one or more additional nucleic acid enzymes.
- the required molecule may include but is not limited to a partzyme, a nucleic acid enzyme, for example a DNAzyme, an assembly facilitator, an assembly facilitator component, a substrate, a stabiliser arm, a portion or component thereof or a combination thereof.
- a cascade may therefore involve production of a cumulative effect, and thus detect a target of low abundance by generating a signal to a level at which it may be detected.
- Cascades may be employed to amplify a signal, for example, in applications where an input event is of low intensity, for example when a target is in low abundance, and may not otherwise provide for a output signal that is detectable. In other embodiments, more than two catalytic stages may be employed.
- the cascade may be linear. In a preferred embodiment, the cascade may be exponential.
- each step in the cascade employs a nucleic acid enzyme, some or all of which may be undergoing multiple turnover whereby a single nucleic acid enzyme (e.g. a DNAzyme or MNAzyme) may perform multiple cleavages or ligations or other modifications at one or more steps in the cascade. Multiple turnover may facilitate amplification at one or more steps in the cascade resulting in amplification of the signal detected during the detection step(s).
- a nucleic acid enzyme e.g. a DNAzyme or MNAzyme
- an extension of the linear cascade could result in a feedback amplification cascade. If, for example, the sequence of a product of a third nucleic acid enzyme was the same as a product of a first nucleic acid enzyme then the said product of the third nucleic acid enzyme could also serve as a molecule permitting a second nucleic acid complex to function as a second nucleic acid enzyme and a feedback amplification cascade could be initiated.
- the third nucleic acid enzyme would constantly generate a product which could in turn serve as a molecule permitting a second nucleic acid complex to function as a second nucleic acid enzyme thus allowing the creation of more product by the second nucleic acid enzyme that could permit the third nucleic acid complex to function as a third nucleic acid enzyme. It is envisaged this strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which could allow assembly of a first nucleic acid enzyme.
- an assembly facilitator eg a target analyte
- the strategy could allow detection of assembly facilitators (eg target analytes) followed by signal amplification using for example, a DNAzyme (or MNAzyme) which could ligate substrates and MNAzymes which could cleave a substrate.
- assembly facilitators eg target analytes
- MNAzyme MNAzyme
- FIG 16. One possible feedback cascade is illustrated in Figure 16. Other variations of this schema are described throughout the specification. [ 00301 ] Persons skilled in the art will appreciate that the methods described herein may be used to perform a cascade as herein defined. It will be also be appreciated by one skilled in the art that other strategies based on Figures 9, 12, 15 and 16 could involve modification of at least one substrate by at least one MNAzyme by a variety of means including, for example cleavage or ligation.
- linear and feedback nucleic acid enzyme (DNAzyme and MNAzyme) cascades allow amplification in formats that require no protein enzymes to facilitate the process. This provides a major advantage over these commonly used target amplification protocols. Further, the ability to control and regulate the catalytic activity of MNAzymes using several oligonucleotide components, such as stabiliser arm components or assembly facilitator components allows the assembly of MNAzymes to be tightly regulated by conditions and components in the microenvironment.
- the cascades described herein may be initiated by formation of an active MNAzyme or apta- MNAzyme as herein defined.
- Displacer oligonucleotides can bind to single stranded regions within a DNA complex which has both single stranded and double stranded regions.
- an oligonucleotide e.g. Oligo 1
- Oligo 1 e.g. cOligol
- the displacer has the same sequence as the cOligo 1 within the double stranded regions. This can result in a new double stranded structure being formed between the displacer oligonucleotide and Oligo 1 and can cause the release of cOligo 1 as a single stranded oligonucleotide.
- a ligated product bound to an MNAzyme ligase has several non paired bases (a single stranded region) between the double stranded regions formed by complementarity between the substrate arms of the partzymes and the substrates.
- a displacer oligonucleotide with complementarity to the substrate (Oligo 1) binds to the single stranded region of the substrate it could displace the complementary partzyme sensor arms (e.g. cOligola and cOligo Ib) by branch migration and separate the partzymes from the ligated product. As such, this could increase the turnover of the ligase enzyme.
- the process would result in a double stranded molecule comprising the displacer and the ligated product.
- the ligated product were required for a subsequent step in the reaction, for example, if it were to act as an assembly facilitator for a subsequent MNAzyme, then it would be necessary to separate the ligated product from the displacer. If the displacer oligonucleotide was shorter than the ligated product then this would result in single stranded regions at the termini of the duplex formed by the displacer oligonucleotide and the ligation product.
- the partzyme sensor arm could bind to these single stranded regions and remove the displacer oligonucleotide by branch migration thus resulting in a new MNAzyme assembled with partzymes (which themselves would have functioned as displacer oligonucleotides in this instance) and ligated product (now functioning as an assembly facilitator).
- a displacer could be used to remove the activity inhibitor by binding to the activity inhibitor domain of the activity inhibitor and displace it by branch migration such that the partzyme sensor arm would be rendered single stranded and thus available for binding of an assembly facilitator component.
- MNAzyme cascade reactions could be applied to a range of biotechnological applications, especially in diagnostics, but also in other areas such as medical, veterinary, agricultural, food technology, imaging and anti-bioterrorism applications. They could allow detection of proteins and nucleic acids for disease diagnosis, especially so if they facilitate signal amplification. Catalytic nucleic acids and/or cascade reactions can be used for applications other than diagnostics, for example, within the field of computation analysis and biomolecular engineering of nano-scale devices and molecular switches which may be used in therapeutics.
- MNAzymes offer advantages over DNAzymes for use in various cascades.
- DNAzymes can only recognize one molecule which is both its target and the substrate. While the DNAzyme target/substrate(s) can be considered to provide the "input” and the enzyme product (modified target/substrate(s)) can be considered the "output” the DNAzyme is limited in its ability to transduce more complex processes in cascades since both the input and output are merely alternate (modified and unmodified) forms of the same target substrate(s) molecule(s). Further, catalytic modification, for example cleavage, can destroy the DNAzyme's target/substrate and as such the target is not available for another round of recognition.
- a single MNAzyme for example an MNAzyme with cleavage activity, can recognize at least two molecules, namely at least one assembly facilitator and at least one substrate.
- An assembly facilitator for example a target nucleic acid present in a sample, would not be catalytically modified in the reaction and would remain available for further rounds of substrate modification by the MNAzyme.
- a single MNAzyme which can act on two substrates e.g. an MNAzyme with ligase activity
- a molecule which permits a nucleic acid complex to function as a nucleic acid enzyme and output (e.g. a molecule which is produced and permits a subsequent nucleic acid complex to function as a nucleic acid enzyme).
- output e.g. a molecule which is produced and permits a subsequent nucleic acid complex to function as a nucleic acid enzyme.
- MNAzymes and MNAzyme complexes are useful in one step processes (e.g. detection of a target) and also in, for example, systems employing cascades, logic gates and other processes.
- MNAzyme complexes provide a mechanism for transduction of complex information, for example, to receive an input signal and respond with an appropriate output response.
- DNAzymes may still be useful in cascades. Some examples include production of a DNAzyme or a substrate for a DNAzyme complex by a preceding nucleic acid enzyme such as an MNAzyme.
- MNAzyme ligases are particularly useful in making, for example, new DNAzymes.
- the "output" information of one nucleic acid enzyme generated by, for example ligation or cleavage by an MNAzyme can provide an "input" for the next nucleic acid enzyme complex for example by creating a new DNAzyme or substrate, which can participate in the formation of a DNAzyme complex useful in a following, and /or preceding reaction stage.
- FIG. 9 An exemplary mechanism for generating a linear cascade using an MNAzyme and a nucleic acid enzyme with ligase activity in a format designed for target detection is depicted in Figure 9.
- This figure also describes a general method for target detection. This method relies on a DNAzyme with ligase activity (DNAzyme ligase) to control the assembly of a partzyme for one MNAzyme from at least one of the products of the catalytic activity of one other MNAzyme.
- DNAzyme ligase DNAzyme with ligase activity
- a DNAzyme ligase B can ligate the 5' cleavage product A (also referred to as 5' substrate B) generated in step (i) to another oligonucleotide, 3' ligation substrate B, thus creating a new partzyme for an MNAzyme C.
- the new partzyme/ligation product generated in step (ii) together with another partzyme can form a new MNAzyme C in the presence of assembly facilitator C which can cleave a substrate C into two products, 5 'product C and 3' product C.
- a detectable signal can be generated following cleavage of substrate C if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- This cascade provides a mechanism for signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which allows assembly of MNAzyme A.
- assembly facilitator eg a target analyte
- each step employs a nucleic acid enzyme, some or all of which may be undergoing multiple turnover whereby a single nucleic acid enzyme (e.g. a DNAzyme or MNAzyme) may perform multiple cleavages, ligations or other modifications at one or more steps in the cascade.
- FIG. 12 Another mechanism for generating an MNAzyme cascade in a format designed for target detection is depicted in Figure 12. This figure also describes a general method for target detection. This method relies on a combination of MNAzymes with ligase and cleavage activity. The MNAzyme ligase controls the assembly of a partzyme for one MNAzyme cleaver from at least one of the products of the catalytic activity of one other MNAzyme cleaver.
- an assembly facilitator A for example a target nucleic acid, can direct the formation of a first MNAzyme A.
- MNAzyme A can cleave a substrate A thus generating a 5 'cleavage product A which can in turn be used as a substrate for a MNAzyme B which has ligase activity.
- an MNAzyme ligase B (formed by the partzyme components of MNAzyme B in the presence of assembly facilitator B) can ligate the 5' cleavage product A (also referred to as 5' substrate B) generated in step (i) to another oligonucleotide, 3' substrate B, thus creating a new partzyme for an MNAzyme C.
- the new partzyme/ligation product generated in step (ii) together with another partzyme can form a new MNAzyme C in the presence of assembly facilitator C which can cleave a substrate C into two products, 5 'product C and 3' product C.
- a detectable signal can be generated following cleavage of substrate C if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- nucleic acid ligases could then make various elements for the MNAzyme C reaction, such as an assembly facilitator C, a partzyme A, B, or components thereof. So, for example, one ligase reaction driven by the product of one initial MNAzyme could make an assembly facilitator C for MNAzyme C and another ligase reaction driven by the product of another initial MNAzyme could make a partzyme for MNAzyme C.
- the assay could be arranged such that only when both targets are present to drive both of the ligase reactions and hence make both the assembly facilitator and the partzyme, then MNAzyme C is assembled and cataytically active. This would be a mechanism for detection of multiple targets. Only in the presence of the required number of targets would all the required elements of MNAzyme C be available to modify the substrate C. In this example, the added elements of the reaction mix would be tailored according to how many MNAzyme C elements are made by virtue of recognition of the original targets.
- one or more assembly facilitators e.g. targets
- one initial reaction could provide the 5' ligase substrate and another initial reaction could produce the 3' ligase substrate.
- one initial MNAzyme cleaver could produce the 5' ligase substrate for MNAzyme B and one initial MNAzyme ligase could create a partzyme for MNAzyme ligase B. Only when both products are available to participate in the formation of a MNAzyme complex will the ligase substrates be modified and the cascade reaction continue.
- the different assembly facilitators e.g. targets
- the different assembly facilitators may comprise different molecules or different sequences within one molecule, or a combination. For example, three different SNPs within one molecule could be detected by three different MNAzymes.
- one MNAzyme directed by one target could provide a molecule required for the next reaction and another MNAzyme directed by another target could provide a product required for the next or other following reaction. It would be obvious that other variations may also apply. It would be obvious that this strategy can also be applied to enzymes which effect different modifications (e.g cleavage or ligation) as described above. In addition various combinations of products made by a varying number of nucleic acid enzymes responding to various numbers of targets are possible, which include but are not limited to, partzymes, assembly facilitators and other MNA complex components.
- a number of nucleic acid enzymes responding to a number of targets may produce one or more components of an additional nucleic acid enzyme or nucleic acid enzyme complex or may produce all the components of an additional nucleic acid enzyme or nucleic acid enzyme complex.
- FIG 15. Another example of a cascade reaction which uses nucleic acid enzymes which can cleave substrates, together with nucleic acid enzymes which can ligate substrates is illustrated in Figure 15. In this example the process of ligation results in the generation of a new assembly facilitator.
- the MNAzyme cleaver 1 assembles only in the presence of an assembly facilitator (e.g.
- a DNAzyme ligase (or an MNAzyme ligase) ligates the 5' cleavage product from step (1) to a 3' ligation substrate thus creating a ligation product which can serve as an assembly facilitator for another MNAzyme cleaver 2.
- step (3) the assembly facilitator formed by ligation in step (2) directs the assembly of partzymes which form MNAzyme cleaver 2 which can cleave substrate 2 into two products, a 5' cleavage product with a 2', 3' cyclic phosphate terminus and 3' cleavage product.
- a detectable signal can be generated following cleavage of substrate 2 if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- each step in the cascade in Figure 15 employs a nucleic acid enzyme, some or all of which may be undergoing multiple turnover whereby a single nucleic acid enzyme (e.g.
- a DNAzyme or MNAzyme may perform multiple cleavage or ligation or other modifications at one or more steps in the cascade. Multiple turnover may facilitate amplification at one or more steps in the cascade resulting in amplification of the signal detected during one or more step(s).
- an extension of the linear cascade illustrated in Figure 15 could result in a feedback amplification cascade. If the sequence of the 5' cleavage product of MNAzyme cleaver 2 were the same as the 5' cleavage product of MNAzyme cleaver 1 then the 5' cleavage product of MNAzyme cleaver 2 could also serve as a 5' substrate for the DNAzyme ligase (or MNAzyme ligase) and a feedback amplification cascade reaction could be initiated.
- MNAzyme cleaver 2 would constantly generate 5' cleavage products which could in turn serve as a substrate for ligation by the DNAzyme ligase (or MNAzyme ligase) thus allowing the creation of more assembly facilitators that could direct the assembly of more MNAzyme cleaver 2. It is envisaged this strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which could allow assembly of MNAzyme cleaver 1.
- an assembly facilitator eg a target analyte
- step (1) the MNAzyme cleaver 1 assembles only in the presence of an assembly facilitator (e.g. a target nucleic acid) and then cleaves MNAzyme substrate 1 releasing a 5' cleavage product with a 2', 3' cyclic phosphate terminus and a 3' waste product.
- an assembly facilitator e.g. a target nucleic acid
- step (2) the DNAzyme ligase (or MNAzyme ligase) ligates the 5' cleavage product from step (1) to a 3' ligation substrate thus creating a ligation product which can serve as an assembly facilitator for an MNAzyme cleaver 2.
- step (3) the assembly facilitator formed by ligation in step (2) directs the assembly of partzymes to form MNAzyme cleaver 2 which can cleave substrate 2 into two products, a 5' cleavage product with a 2', 3' cyclic phosphate terminus and 3' cleavage product.
- a detectable signal can be generated following cleavage of substrate 2 if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- MNAzyme cleaver 3 may have different sensor arms than MNAzyme cleaver 1 but would require at least one substrate arm in common with MNAzyme cleaver 1 in order to provide at least the same 5' ligation substrate to participate in the ligase reaction of step (ii).
- an MNAzyme cleaver 3 could assemble in the presence of an assembly facilitator component generated by cleavage by the MNAzyme cleaver 2. Further, MNAzyme cleaver 3 could cleave a substrate and generate a 5' cleavage product which could be the same as the 5' cleavage product produced by cleavage of substrate 1 by MNAzyme cleaver 1.
- this product could also serve as a 5' substrate for the DNAzyme (or MNAzyme) ligase and a feedback amplification cascade reaction could be initiated.
- MNAzyme cleaver 3 would be constantly generating 5' cleavage product which in turn could serve as a substrate for ligation by the DNAzyme (or MNAzyme) ligase to create more assembly facilitators that could direct the assembly of more MNAzyme cleaver 2.
- MNAzyme cleaver 2 could then produce more assembly facilitator components for more MNAzyme cleaver 3 and a feedback cascade would be created.
- This strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which directs assembly of MNAzyme cleaver 1.
- the strategy would allow detection of assembly facilitators (eg target analytes) followed by signal amplification using a DNAzyme (or MNAzyme) which could ligate substrates and MNAzymes which could cleave substrates.
- MNAzymes with cleavage activity can be selected based on the linkage to be cleaved.
- the DNAzyme (or MNAzyme) with ligase activity could ligate fragments and form a DNAzyme capable of cleaving substrate 2 to create an assembly facilitator or other component for another MNAzyme (labelled as MNAzyme cleaver 3 in Figure 16).
- MNAzyme cleaver 2 could be replaced with a DNAzyme cleaver.
- the DNAzyme (or MNAzyme) ligase could ligate fragments and create a new partzyme capable of associating with another partzyme and an assembly facilitator present in the reaction thus forming an MNAzyme cleaver 2 which could cleave substrate 2 to create an assembly facilitator component for an MNAzyme 3.
- MNAzyme complexes are possible and may be used in cascades. Several of these designs are illustrated in Figure 6 where some examples of active MNAzyme complexes are shown in Panels A to D.
- MNAzymes which include one assembly facilitator Fl (panel A), two assembly facilitator components Fl and F2 (panels B and D) or three assembly facilitator components Fl, F2 and F3 (panel C).
- panel A includes sensor arms with self complementary regions within the partzyme sensor arms.
- MNAzymes may also include one or more stabilising arms (sA) as shown in panel D. Variations in one panel could be used in combination with a variation in another panel.
- Steps can be performed sequentially in individual reaction vessels, various steps can be combined or all steps could be performed in a single reaction vessel with one or more chambers. Each variation could comprise additional steps, including steps which feedback into other parts of the cascade.
- Detection of catalytic activity of 1 or more of the 1st, 2 nd , 3rd or any additional NA enzymes could be indicative of the presence of a target [ 00323 ]
- All reactions can use a combination of nucleic acid enzymes, namely multiple MNAzymes or a mix of MNAzymes and DNAzyme(s).
- the modifications by these enzymes involve a combination of various numbers of cleavage and/or ligation steps.
- the product of one step serves as a substrate, an enzyme or an enzyme component for the subsequent step.
- Nucleic acid enzymes which cleave can be used to create, for example, a new substrate for ligation or a new assembly facilitator component for a new MNAzyme.
- Nucleic acid enzymes which ligate can be used to create, for example, a new substrate for cleavage in a next step by either a DNAzyme or an MNAzyme enzyme. Further, nucleic acid enzymes which ligate can be used to create, for example, a new DNAzyme or a new component for an MNAzyme, such as an assembly facilitator, a new partzyme or a new stabiliser arm.
- a new DNAzyme or a new component for an MNAzyme such as an assembly facilitator, a new partzyme or a new stabiliser arm.
- nucleic acid enzyme [ 00324 ] It will be obvious to one skilled in the art that multiple products produced by a single nucleic acid enzyme could be used to permit one or more nucleic acid complex(es) to function as one or more nucleic acid enzyme(s) in one or more subsequent steps.
- step 1 of Table 3 an MNAzyme could create a product that is useful as an assembly facilitator, rather than as a substrate, for assembly of a nucleic acid enzyme complex.
- a signal for example a fluorescent signal can be generated at a step where a catalytic modification occurs.
- a fluorescent signal could be generated at any step where there is cleavage by a DNAzyme or MNAzyme one could separate a fluorophore quencher dye pair and cause an increase in fluorescence.
- a fluorescent signal could be generated at any step where there is, for example, ligation by a DNAzyme or MNAzyme one could bring a fluorophore quencher dye pair into close proximity and cause a decrease in fluorescence.
- a fluorescent signal could be generated at multiple steps within a cascade reaction.
- a cascade could be initiated by nucleic acid targets (DNA/RNA) in the presence of components for one or more MNAzyme(s), or a cascade could be initiated by other target analytes (proteins, small molecules etc) in the presence of components for one or more apta-MNAzyme(s)s. Since the reaction would only be initiated in the presence of target analytes, it could provide a technique for detection and/or identification of target analytes. The method is based on the transition between states of inactivation and activation of nucleic acid complexes.
- cascades involving involving amplification using MNAzyme(s) require no protein enzymes to facilitate the process. Further, the ability to control and regulate the catalytic activity of MNAzyme(s) using several oligonucleotide components, such as stabiliser arm components or assembly facilitator components allows the assembly of MNAzymes to be tightly regulated by conditions and components in the microenvironment. [ 00329 ] In relation to cascades initiated by non-nucleic acid target analytes aptamers are useful.
- Aptamers are DNA, RNA or peptide sequences that have the ability to recognize one or more ligands with great affinity and specificity due to their high level structure, for example, a 3-D binding domain or pocket.
- Many aptamers have been evolved in vitro for their capacity to bind to ligands, including for example, nucleic acids, proteins, prions, small organic compounds, and/or entire organisms.
- "Aptazymes” are sequences which have been previously described which have sequences comprised of both aptamer and nucleic acid enzyme sequences, for example an aptamer appended to a DNAzyme or ribozyme.
- Apta-MNAzymes are MNAzymes which have an aptamer appended to one of the components in an apta- MNAcomplex.
- an aptamer may be appended to a partzyme, an assembly facilitator, an MNAzyme substrate or any other component of an MNA complex.
- Association of a non-nucleic acid target analyte with the aptamer may facilitate activity of the nucleic acid enzyme.
- a cascade may be initiated by a non-nucleic acid target analyte associating with an apta-MNAzyme, for example, as the first step in a cascade.
- the product of a reaction catalysed by a nucleic acid enzyme in that cascade may associate with an aptamer present on a component of a nucleic acid enzyme other than the initiating MNAzyme or apta-MNAzyme, thereby facilitating activity of that nucleic acid enzyme.
- MNAzyme and/or apta-MNAzyme mediated amplification cascades could be applied to a range of biotechnology applications, especially in diagnostics, but also in other medical, veterinary, agricultural, food technology, imaging and anti-bioterrorism applications. They could allow detection of proteins, nucleic acids or other molecules for disease diagnosis by facilitating signal amplification. Catalytic nucleic acids and/or cascade reactions can be used for applications other than diagnostics, for example, within the field of computational analysis and biomolecular engineering of nanoscale devices and switches which may be used in therapeutics. 9. Method which allows switching between active and inactive states of MNA complexes by removal or creation of components such as the activity inhibitor or the assembly facilitator.
- a transition between active MNAzymes and inactive MNA complexes, or vice versa can be achieved by the provision or removal of MNA complex components, including but not limited to, one or more activity inhibitors, assembly facilitators, assembly inhibitors, partzymes or stabiliser arms, or parts thereof.
- the activity inhibitor may further include a labile or cleavable linker or substrate, which may be located between two or more domains within the activity inhibitor, for example an activity inhibitor domain and an activator assembly facilitator domain. Cleavage at the linker site may allow separation of an activity inhibitor domain from an activator assembly inhibitor domain, which may then function as an assembly facilitator component and direct the assembly of an active MNAzyme. Cleavage of the linker could be achieved by several methods, including but not limited to, MNAzyme cleavage, protein enzyme cleavage, or hydrolysis induced by changes in the pH and or temperature.
- the assembly inhibitor and/or assembly facilitator could be selectively removed using a process involving branch migration and/or complementarity to component oligonucleotides.
- Modulator oligonucleotides which function through complementarity may do so by altering the secondary structure of oligonucleotides to which they bind. In some embodiments this may result in an alternate conformation where an activator sequence is now able to assemble with other components to form active MNAzymes.
- such a modulator oligonucleotide may cause the disruption of intramolecular structures such as hairpins which constrain activator molecules in non- functional conformations.
- An alternate exemplary strategy is designed to facilitate switching an MNA complex between the "on state" of an active MNAzyme to the "off state” of an MNAi complex using a second MNAzyme with ligase activity (or a DNAzyme with ligase activity).
- an active MNAzyme A which could be capable of modifying (eg cleaving or ligating) a substrate(s) A could be formed in the presence of assembly facilitator component 1 (AFC 1) and assembly facilitator component 2 (AFC 2).
- a second MNAzyme B which has ligase activity, could form in the presence of assembly facilitator 3 (AF3) and could then ligate AFC 2 with an activity inhibitor component (AIC) causing the formation of an activity inhibitor (AI).
- This AI could bind to the partzyme components for MNAzyme A and result in the formation of an MNAi A complex which is inactive.
- the MNAzyme ligase in this example could operate as an off switch to inactivate an MNAzyme A.
- a DNAzyme ligase could replace the MNAzyme ligase and be used to operate as an off switch to inactivate an MNAzyme A.
- the inactive MNAi complex and the catalytically active MNAzyme would represent two alternate states for the assembled components, namely an "off state and the "on" state respectively.
- kits for practising the methods disclosed herein contain all the necessary reagents to carry out the method.
- kits may comprise at least a first container containing a first nucleic acid complex (e.g. an MNA complex), wherein formation of a first nucleic acid enzyme requires the addition of a sample putatively containing a target to be detected, identified and/or quantitated. Formation of the enzyme may lead to the production of a component, for example a substrate, suitable for participation in a subsequent reaction. In the subsequent reaction the component allows a further nucleic acid complex to function as a nucleic acid enzyme. Reagents to allow formation of further nucleic acid complexes which form nucleic acid enzyme complexes when contacted by a component produced by a nucleic acid enzyme of a previous reaction may also be provided.
- a first nucleic acid complex e.g. an MNA complex
- components for each of the nucleic acid complexes which can allow formation of active nucleic acid enzyme complexes may be present in the same or separate containers.
- components for each of the nucleic acid complexes which can allow formation of active nucleic acid enzyme complexes may be present in separate containers.
- a kit may comprise one or more required components for each of the nucleic acid complexes in separate containers.
- kits of the present invention may include DNAzymes or parts thereof.
- kits of the present invention will also comprise one or more other containers, containing for example, wash reagents, and/or other reagents as required in the performance of the methods of the invention.
- kits in which reagents are contained 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.
- 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.
- 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.
- kits of the invention find application in any circumstance in which it is desirable to detect, identify or quantitate any entity or event. 11. Methods of making MNAzymes
- the present invention also provides methods for engineering MNAzymes from uni-molecular DNAzymes.
- DNAzymes and/or ribozymes are known which are capable of catalytic modification of at least one substrate wherein the modification may be selected from the group comprising cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thy
- MNAzymes described herein are applicable to a plurality of nucleic acid enzymes such as those above and including but not limited to those described in Hobartner and Silverman (2007) and those listed in the table below.
- Table 4 Some uni-molecular nucleic acids enzymes which may be used to form an MNAzyme.
- DNAzymes have similar basic structures with multiple domains. These DNAzymes have a conserved catalytic domain (catalytic core) flanked by two non-conserved substrate-binding domains ("substrate arms"), which specifically recognize and hybridise to the substrate.
- the substrate binding domain can be tailored to any substrate provided the substrate contains a site which can be modified by the DNAzyme.
- FIG. 14 a method for engineering an MNAzyme from a uni-molecular DNAzyme (top left hand side structure) is illustrated whereby in the first step, positions are identified within the DNAzyme catalytic core at which it can be split, such that each partial portion of the catalytic core can be distributed between two partial sequences such that the two partial cores together constitute an entire catalytic core (top right hand side structure). Two oligonucleotides A and B (candidate partzymes) can then be synthesised (bottom left hand side structure).
- An oligonucleotide A can be synthesised to contain (i) one substrate binding arm portion capable of binding to a substrate, (ii) one partial catalytic core portion, and (iii) one sensor arm portion capable of binding to an assembly facilitator molecule.
- a second oligonucleotide B can be synthesised such that it contains (i) one substrate binding arm capable of binding to the same substrate as oligonucleotide A, whereby oligonucleotide B binds the substrate in a position adjacent to that of oligonucleotide A, (ii) one partial catalytic core portion which contains those bases from the entire DNAzyme catalytic core which are not incorporated into oligonucleotide A and (iii) one sensor arm sequence capable of binding to the same assembly facilitator as oligonucleotide A, whereby oligonucleotide B binds the assembly facilitator in a position adjacent to that of oligonucleotide A.
- This process can be repeated thus making a series of pairs of oligonucleotides A and B which incorporate the structure and domains of partzymes, but may or may not have catalytic activity in the presence of a substrate and an assembly facilitator.
- Some or all of the candidate partzyme pairs can then be incubated in the presence of the assembly facilitator complementary to the sensor arm portions of the partzymes and a substrate which is complementary to the substrate arm portions of the partzymes. Incubation is performed under conditions which are compatible with modification of a substrate by the DNAzyme from which the partial catalytic core sequences were derived. Pairs of oligonucleotides A and B, which can perform the same type of modification (e.g.
- cleavage to the substrate as the DNAzyme from which partial sequences were derived, are useful as partzymes which can be assembled into MNAzymes and MNAzyme complexes (bottom right hand side structure).
- the sequences of the partial catalytic cores are suitable for incorporation into other partzymes of new MNAzymes.
- New partzymes can be synthesised which are tailored to new substrates (by changing the substrate binding domains of each partzyme) and/or to new assembly facilitators (by changing the sensor binding domains of each partzyme).
- the method for engineering an MNAzyme that acts on a first and second substrate e.g. a MNAzyme ligase starting from a uni-molecular nucleic acid enzyme (e.g.DNAzyme ligase) requires the similar steps whereby in the first step, positions are identified within the DNAzyme catalytic core at which it can be split, such that each partial portion of the catalytic core can be distributed between two partial sequences such that the two partial cores together constitute an entire catalytic core. Two oligonucleotides A and B (candidate partzymes) can then be synthesised.
- An oligonucleotide A can be synthesised to contain (i) one substrate binding arm portion capable of binding to a first substrate, (ii) one partial catalytic core portion, and (iii) one sensor arm portion capable of binding to an assembly facilitator molecule.
- a second oligonucleotide B can be synthesised such that it contains (i) one substrate binding arm capable of binding to a second substrate, (ii) one partial catalytic core portion which contains those bases from the entire DNAzyme catalytic core which are not incorporated into oligonucleotide A and (iii) one sensor arm sequence capable of binding to the same assembly facilitator as oligonucleotide A, whereby oligonucleotide B binds the assembly facilitator in a position adjacent to that of oligonucleotide A.
- This process can be repeated thus making a series of pairs of oligonucleotides A and B which incorporate the structure and domains of partzymes, but may or may not have catalytic activity in the presence of the substrates and an assembly facilitator.
- Some or all of the candidate partzyme pairs can then be incubated in the presence of the assembly facilitator complementary to the sensor arm portions of the partzymes and the two substrates which are complementary to the substrate arm portions of the partzymes. Incubation is performed under conditions which are compatible with modification (e.g. ligation) of the substrates by the DNAzyme from which the partial catalytic core sequences were derived. Pairs of oligonucleotides A and B which can perform the same type of modification (e.g.
- partzymes which can be assembled into MNAzymes and MNAzyme complexes.
- the sequences of the partial catalytic cores are suitable for incorporation into other partzymes for other MNAzymes.
- New partzymes can be synthesised which are tailored to new substrates (by changing the substrate binding domains of each partzyme) and/or to new assembly facilitators (by changing the sensor binding domains of each partzyme).
- a similar method can be used to examine tolerance to changes in the partial sequence derived from splitting the DNAzyme catalytic core that can be incorporated into component partzymes.
- modifications to the partial catalytic cores incorporated into partzymes can be made which include, by way of example, insertions, deletions, substitution with alternate DNA bases, or substitution with DNA analogues such as ribonucleotides, inosine or other analogue bases.
- Candidate partzyme pairs containing one or more modification(s) can then be assayed as above to determine whether or not they contain a pair of partial catalytic core sequences which are suitable for incorporation into partzymes that can assemble into catalytically active MNAzymes.
- Example 1 Example of an MNAzyme where partzyme B comprises two molecules.
- oligonucleotides required for this strategy are described below: a) a partzyme A; b) a partzyme B comprising a first component which contains a substrate arm, a partial catalytic core and truncated sensor arm; and a second stabiliser arm component, which hybridizes to the assembly' facilitator, adjacent to the truncated sensor arm of the partzyme.
- This stabiliser arm is designed to facilitate MNAzyme assembly when the truncated sensor arm of the partzyme is hybridized to the assembly facilitator; and c) a substrate, for example a reporter substrate.
- oligonucleotides useful in this strategy are a partzyme B; a partzyme A comprising a first component which contains a substrate arm, a partial catalytic core and truncated sensor arm; and a second stabiliser arm component, which can hybridize to the assembly facilitator, adjacent to the truncated sensor arm of the partzyme.
- Active MNAzyme assembly also requires the presence of an assembly facilitator.
- partzyme B was only 5 nucleotides in length.
- sequences of partzymes A and the two partzyme B components are shown below (5' to 3')- In the following sequences the bases in bold hybridize with the assembly facilitator, bases underlined form part of the catalytic core of the assembled MNAzyme, and bases in italics hybridize to the substrate.
- the "-P" indicates 3' phosphorylation of the oligonucleotide.
- the reporter substrate used in this example was SubBi-2 end-labelled with a 6-FAM moiety at the 5' end, a BHQl moiety at the 3' end and designated SubBi-2-FB. Cleavage of SubBi-2-FB was monitored at 520 nm (FAM emission wavelength) with excitation at 490 nm (FAM excitation wavelength).
- the sequence of SubBi-2-FB is listed below (5' to 3'); the lower case bases represent RNA and the upper case bases represent DNA.
- Detection of the assembly facilitator was measured by an increase in fluorescent signal caused by cleavage of the reporter substrate by the catalytically active MNAzyme. Reactions were initiated by the addition of substrate and the total volume of all reactions was 50 ⁇ L. Reactions were conducted at 55°C on a FLUOstar OPTIMA (BMG Biotech). Fluorescence for each reaction was read every 2 seconds for a total of 5 minutes. All reactions contained 200 nM SubBl-2-FB, 200 nM XdA4/2-P, 200 nM XdB5/2-P, 1 x PCR Buffer II (Applied Biosystem) and 25 raM MgCl 2 . In addition, reactions contained oligonucleotides as listed in Table 5.
- reaction (i): Figure 3 When both the assembly facilitator, and a partzyme stabiliser arm component were included in the reaction (Reaction (i): Figure 3), active MNAzymes assembled and cleaved the substrate, resulting in an increase in fluorescence over time. In contrast, there was no increase in fluorescence in the absence of the assembly facilitator (Reaction (iii): Figure 3). Further, the presence of the stabiliser arm was shown to be essential for formation of active MNAzymes or MNAzyme complexes. A reaction containing all reaction components including the assembly facilitator, but which lacked the stabiliser arm component, gave no increase in fluorescence over time (Reaction (ii): Figure 3).
- the 5 bases of the sensor arm of partzyme B were insufficient to form a stable MNAzyme or MNAzyme complex but the presence of a stabiliser arm component was shown to be capable of compensating for the short length (truncation) of the partzyme sensor arm and allowing stable MNAzyme formation under stringent temperature conditions (55 0 C in this example).
- the stabiliser arm component is thus an essential oligonucleotide for assembly of active MNAzymes in this system, which uses a partzyme with a truncated sensor arm.
- MNA complexs which contain multiple oligonucleotide components, the sequences of which can be tailored with respect to the melting temperature, the sequence composition and complementarity or lack thereof with other component oligonucleotides. Shorter sequences, including but not limited to, partzyme components with truncated arms, stabilizer arms, and assembly facilitator components are particularly useful in this aspect of the invention. While this example uses an MNAzyme with cleavage activity, it would be appreciated that similar structural designs for MNAzymes or MNAzyme complexes could be used in MNAzymes which cause modifications other than cleavage, for example, MNAzyme which could ligate.
- Example 2 Detection of target analytes using MNAzymes with ligase activity.
- An example of an MNAzyme which can modify at least two substrates could be used for detection of a nucleic acid target.
- An MNAzyme with ligase activity could be assembled from two partzymes which comprise active partial catalytic core pairs derived from, for example, the 7Q10 DNAzyme or the 7Z81 DNAzyme as exemplified in example 7 and 9 respectively.
- the partzymes could have sensor arms complemenary to the target nucleic acid which could act as an assembly facilitator for an active MNAzyme ligase.
- the substrate arms could be complementary to two substrate molecules which contain ligatable sequences with appropriate termini (e.g.Table 4).
- the strategy could also be used for detection of other ligands if the MNAzyme were an apta-MNAzyme with ligase activity which contained an aptamer sequence capable of specifically binding the target ligand.
- Example 3 Mechanisms for facilitating and inhibiting the assembly of active MNAzymes or MNAi complexes .
- An MNAzyme is composed of partzymes, which assemble in the presence of one or more assembly facilitators, to form an active enzyme (e.g. Figures 1, 2, 4 (right hand panel) and Figure 5 (i and U)).
- the assembly facilitator(s) which binds to partzyme sensor arms, can be a target analyte, or can be a synthetic nucleic acid molecule(s) added to the reaction mix to drive MNAzyme assembly.
- partzymes can assemble into an inactive, non- catalytic, MNAi complex when they hybridise with an "activity inhibitor" molecule ( Figure 4 (left hand panel) and Figure 5 (iii).
- Various alternative oligonucleotide sequences were tested for their capacity to regulate the assembly of either active MNAzyme or inactive MNAi complexes.
- MNAzyme assembly was examined in the presence of (i) a single molecule (assembly facilitator F 1/2), or (ii) two molecules (assembly facilitator component Fl and assembly facilitator component F2), whose sequences together comprise that present in facilitator F 1/2.
- Facilitator F2 binds to the whole sensor arm of one partzyme and overlaps to bind 4 base pairs of the sensor arm of the second partzyme.
- an "activity inhibitor" molecule which has a sequence that includes that of facilitator F2 plus an additional activity inhibitor domain, was tested for its ability to drive the reaction towards assembly of inactive MNAi complexes.
- partzymes A and partzyme B are shown below (5' to 3'). In the following sequences the bases in bold hybridize with the assembly facilitator, bases underlined form part of the catalytic core of the assembled MNAzyme, and bases in italics hybridize to the substrate. The "-P" indicates 3' phosphorylation of the oligonucleotide.
- SEQ ID NO 6 Partzyme A RO5A4/2-P:
- the reporter substrate for this example was SubBi-2-FB with the sequence, 5' to 3', as below.
- SubBi-2-FB was end-labelled with a 6-FAM moiety at the 5' end and a BHQl moiety at the 3' end.
- Cleavage of SubBi-2-FB was monitored at 530 run (FAM emission wavelength) with excitation at 485 nm (FAM excitation wavelength).
- the lower case bases represent RNA and the upper case bases represent DNA.
- SEQ ID NO 8 Assembly Facilitator Fl/2: GCAAGTGGGAAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTG
- an MNAzyme designed to form in the presence of an assembly facilitator F 1/2, generated fluorescence.
- the example demonstrated that the assembly facilitator F 1/2 could be split into two parts (assembly facilitator components Fl and F2) and retain the capacity to direct the assembly of catalytic active MNAzymes. Together the two assembly facilitator components can stabilise active MNAzyme formation and cause fluorescence, provided they bind adjacent to each other on the partzyme sensor arm.
- this example demonstrates the assembly of partzymes into active MN Azymes can require the presence of multiple component assembly facilitators. When multi-component assembly facilitators are required, the presence or absence of one or more of these components can be used to control the assembly of active MNAzymes and as such switch them on and off.
- an activity inhibitory molecule could prevent MNAzyme assembly by hybridising to a partzyme and disrupting the secondary structure at the junction of the two assembly facilitator component on a partzyme sensor arm which is required for enzymatic activity (see Figures 4 and 5 as examples). It would be appreciated by one skilled in the art, that an inhibitory molecule could be designed to hybridise to either partzyme A or B, and to either the sensor or substrate arm of partzyme.
- Example 4 Application of MNAzymes to detect non-nucleic acid analytes including small molecules such as adenosine 5 '-triphosphate.
- Aptamers are single-stranded DNA or RNA molecules evolved in vitro from large pools of random-sequence oligonucleotides for their capacity to bind target analytes with high affinity and specificity. Aptamers have been selected for their ability to bind specifically to many types of analytes including proteins, carbohydrates, lipids, nucleotides, whole cells and viruses.
- an aptamer sequence was incorporated at the end of a partzyme (apta-partzyme) in a configuration whereby an active apta-MNAzyme was only formed in the presence of the activator ligand.
- apta-partzyme a partzyme
- nucleic acid oligonucleotides included in this exemplary apta-MNAzyme detection strategy are illustrated in Figure 7 and include; a) a partzyme; b) an apta-partzyme which is a partzyme with an aptamer incorporated into one of its ends; c) an assembly facilitator which binds to both the apta-partzyme and the partzyme enabling assembly of an active MNAzyme; d) a substrate e.g.
- a reporter substrate e) an assembly inhibitor oligonucleotide 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.
- the assembly inhibitor oligonucleotide binds to the apta-partzyme thus preventing it from binding to the substrate.
- the activator ligand can interact with the aptamer sequence of the apta-partzyme, thus preventing binding of the assembly inhibitor and allowing an active apta-MNAzyme to assemble, then bind to and cleave the substrate.
- apta-MNAzymes can only form and cause fluorescent signal generation in the presence of an activator ligand.
- ATP aptamer sequence was adjoined to the substrate arm of a partzyme, to produce an apta-partzyme molecule ( Figure 7).
- the sensor arms of the apta-partzyme and another partzyme were designed to bind to a synthetic assembly facilitator, included in the reaction to drive the assembly of MNAzymes when targets or regulatory analytes are present.
- the sequences of apta-partzyme AtpA2/l and partzyme AtpB3/l are shown below (5' to 3'). In the following sequences the bases in bold hybridize with the assembly facilitator, bases underlined form part of the catalytic core of the assembled MNAzyme, and bases in italics hybridize to the substrate.
- bases in plain text in partzyme AtpA2/l indicate a DNA aptamer sequence that can bind to ATP or dATP.
- [ 00386 ] Apta-MNAzyme activity was monitored by cleavage of a dual-labelled nucleic acid reporter substrate.
- the reporter substrate for this example is SubBi-1-FB with the sequence, 5' to 3', as below.
- the lower case bases represent RNA and the upper case bases represent DNA.
- the underlined bases indicate the position of a 6-FAM moiety at the 5' end and a BHQl moiety at the 3' end. Changes in fluorescence due to cleavage of SubBi-1-FB at the ribonucleotide between the FAM and BHQl were monitored at 520 nm (FAM emission wavelength) with excitation at 490 nm (FAM excitation wavelength).
- the activator ligands used for this example were adenosine 5 '-triphosphate (ATP) and deoxyadenosine 5 '-triphosphate (dATP). Guanosine 5 '-triphosphate (GTP) and cytosine 5 '-triphosphate (CTP) were used as negative control molecules. All molecules were purchased from Bioline. Nuclease-free water was used as a no analyte control.
- Each reaction contained a final concentration of 200 nM AtpA2/l, 200 nM AtpB3/l, 200 nM AtpC/1, 200 nM AtpR/1, 200 nM SubBi-1-FB, 25 mM MgCl 2 , 50 mM Tris HCl pH 7.5 and 2 mM of either ATP, or dATP, or GTP, or CTP or no analyte (water control).
- MNAzymes can be coupled to aptamers for the detection of analytes in an approach that is highly specific for the target analyte.
- an assembly inhibitor molecule can be used to control the assembly of apta-MNAzymes, and that a molecule e.g. ATP can serve as a activator ligand or molecular regulator in this system.
- the aptamer can be incorporated, for example, into either end (5' or 3') of either of the two partzymes containing partial catalytic core sequences.
- the assembly inhibitor can bind to the aptamer region and to either the substrate arm (that binds the substrate) or the sensor arm (that binds the assembly facilitator).
- the inhibitor blocks binding of the substrate.
- the inhibitor would prevent binding of the assembly facilitator with the apta-partzyme and therefore would prevent assembly of active MNAzymes.
- the literature contains sequences for a large number of aptamers capable of detecting many types of analytes. These include proteins, carbohydrates, lipids, prions, nucleotides, whole cells and viruses. Aptamers to all these types of analytes could be linked to partzymes to detect a very diverse range of molecules. Reaction conditions (buffer, temperature, divalent cation concentration etc), which are compatible with both binding of analytes to aptamers (or apta-partzymes) and cleavage of a reporter substrate by an MNAzyme, can be determined by empirical testing.
- MNAzyme activity can be modulated by the removal or addition of the assembly inhibitor. Changing the oligonucleotide sequence, the melting temperature and or concentration can achieve finer regulation.
- the hybridization of the assembly inhibitor within an MNA is affected by many factors, including but not limited to, salt concentration, cation concentration, temperature and the presence or absence of additives (e.g. DMSO). As such, entities that affect hybridization can provide a tool for controlling the assembly and disassembly of MNAzymes,
- the assembly facilitator can be removed by physical manipulation of components and can be achieved, for example, by exploiting either physical properties of attached moieties as molecular "hooks", and/or by exploiting inherent properties of the oligonucleotides, for example, negative charge, or sequence complementarity.
- MNAzymes and apta-MNAzymes can be used for detection of target analytes including nucleic acid targets and/or non-nucleic acid ligands. Further they can be used to detect analytes in a direct detection format (as demonstrated in this example) or they can be used in nucleic acid enzyme cascade reactions. In one embodiment an MNAzyme and/or an apta-MNAzyme could facilitate the first step in the cascade. In one embodiment the nucleic acid cascade could use a combination of MNAzymes, or a combination of MNAzyme(s) and DNAzyme(s) which could modify one or more substrate(s) by, for example, cleavage and/or ligation.
- Example 5 A cascade reaction which uses a nucleic acid enzyme with ligase activity and a nucleic acid enzyme with cleavage activity.
- a reaction useful for inclusion in cascade reactions and/or molecular switches exploiting the catalytic activities of two nucleic acid enzymes is outlined in Figure 8.
- One reaction could be mediated by a DNAzyme ligase, which can ligate a 5' substrate with a 2', 3 '-cyclic phosphate and a 3' substrate with a 5' hydroxl to form a ligation product with a non-native 2'-5' linkage at the junction of the two substrates.
- DNAzyme ligases are known in the art (see summary Table 4).
- the other reaction could be mediated by an MNAzyme cleaver which could use the ligation product as a substrate and could cleave it at the non-native 2'-5' linkage created by ligation.
- Cleavage could produce two cleavage products, a 5' product which would have a 2', 3 '-cyclic phosphate terminus and a 3' product which would have a 5' -hydroxy 1 terminus.
- one or both of the cleavage products could serve as a substrate or substrates for the DNAzyme ligase.
- a reaction could be mediated by a DNAzyme ligase, which can ligate a 5' substrate with a 2',3'-diol and a 3' substrate with a 5 '-triphosphate terminus to form a ligation product with a native 3'-5' linkage at the junction of the two substrates.
- DNAzyme ligases are known in the art (see summary Table 4).
- the other reaction could be mediated by an MNAzyme cleaver which could use the ligation product as a substrate and could cleave it at the native 3 '-5' linkage created by ligation.
- Cleavage could produce two cleavage products, a 5' product which would have a 2', 3'- cyclic phosphate terminus and a 3' product which would have a 5'-hydroxyl terminus.
- one or both of the cleavage products could serve as a substrate or substrates for a DNAzyme ligase that can ligate such termini.
- an MNAzyme with cleavage activity may cleave at a site within a ligation product which is not at the junction of the two ligatable substrates provided the cleavage site requirement are met at this site.
- a DNAzyme ligase could ligate a first oligonucleotide (oligo 1) to a second oligonucleotide (oligo 2) to create a third ligation product (LP) oligonucleotide (oligo 3), provided oligo 1 and oligo 2 have 2', 3'-cyclic phosphate and 5'-hydroxyl termini.
- LP-oligo 3 could be cleaved by an MNAzyme into cleavage products (CP), CP-oligo 1 and CP-oligo 2, thus regenerating oligonucleotides with 2', 3 '-cyclic phosphate and 5'-hydroxyl termini, which could participate in further rounds of ligation.
- oligonucleotides for example four oligonucleotide, each with 2',3'-cyclic phosphate and a 5'-hydroxyl termini, could be ligated by a series of DNAzyme ligases into 16 new unique LP sequences (i.e. each combination of oligonucleotides 1, 2, 3 and 4).
- a series of MNAzymes could then cleave LP-oligo 1 through to LP-oligo 16 back into component oligonucleotides, now called CP- oligo 1, CP-oligo 2, CP-oligo 3 and CP-oligo 4.
- an MNAzyme with cleavage activity may cleave at a site within a LP which is not at the junction of the two ligatable substrates provided the cleavage site requirements are met at this site.
- complex mixtures of oligonucleotide sequences could be created, shuffled and/or rearranged.
- a set of MNAzymes could use a constant assembly facilitator (as illustrated) in Figure 8, and/or the MNAzyme could use the LP sequences from previous ligation rounds as its assembly facilitator.
- new information (input) data produced by the ligation of oligonucleotides, could be recognized "read” by the MNAzyme.
- the MNAzyme could then cleave "write” to produce new output products, and/or information.
- Systems where MNAzymes could read the input LP, and then cleave them into CP-oligos, other than those originally in the pool of starting molecules, could be used to rewrite or recode new output sequences.
- ligation by a DNAzyme could "write" input data, for example, by making new assembly facilitators, or components thereof.
- An MNAzyme could "read” the data, by interrogating the information encoded within the assembly facilitator using the partzyme sensor arms.
- An MNAzyme could then "write” data, for example, a new sequence (e.g. a cleavage product) thus creating new output data which could then be "read” by a DNAzyme ligase (e.g. by ascertaining the suitability of MNAzyme cleavage products to serve as ligation substrates for the DNAzyme ligase).
- cleavage by an MNAzyme could "write" input data, for example, by creating new ligatable substrates.
- a DNAzyme ligase could "read” the data, by interrogating the information encoded within the substrates using the substrate binding arms.
- a DNAzyme ligase can then "write” data, for example, by making a new ligation product sequence.
- the new ligation product could serve as a component necessary for formation of an active MNAzyme cleaver, for example it could serve as a new assembly facilitator or component thereof.
- the ligation product could thus provide new output data which could then be "read” by an MNAzyme cleaver (by ascertaining the suitability of DNAzyme ligation product to serve as an assembly facilitator or assembly facilitator component for the MNAzyme cleaver).
- this MNAzyme/DNAzyme ligase cascade could form an automaton.
- Such devices are capable of converting information from one form into another, according to a defined procedure. In this case the procedures would be encoded and directed by the substrate arms and/or the sensor arms of the MNAzymes and DNAzyme ligases.
- the automaton proceeded via a cascade of restriction endonuclease cleavage, hybridization and ligation cycles, producing a detectable output molecule that encoded the automaton's final state and thus the computational result (Benenson et al, 2001).
- the DNAzyme ligase/MNAzyme cascade could be used in a similar manner to the cascade used by Benenson et al (2001) and thus provide a device capable of solving computational problems. Unlike Benenson's device, a DNAzyme ligase/ MNAzyme cascade, requires no protein enzymes to achieve the same results. While Benenson's device was programmed by double stranded DNA, a DNAzyme ligase/MNAzyme cascade, would be encoded by the various sequences, including for example, the initial input oligonucleotide(s), the substrate arms and/or the sensor arms of the MNAzymes and the substrate arms of DNAzyme ligases.
- DNAzyme ligase/MNAzyme cascade could also be used to "shuffle" oligonucleotide sequences as a method of constructing, and/or increasing the diversity of, molecular libraries.
- a DNAzyme ligase could be used to create or destroy components of MNAzyme and or inactive MNA.
- a DNA ligase could attach an "activity inhibitor” to an "assembly facilitator component” resulting in the assembly of MNAi complex ("off state).
- a DNAzyme ligase could attach a sensor or substrate arm to a partzyme component to create an "on" switch for MNAzymes by promoting assembly.
- the DNAzyme ligase could be attached to a sequence labeled with a moiety that allows oligonucleotides to be selectively captured, for example using a biotin group or the moiety could contain a radio-frequency magnetic field radio to facilitate remote electronic control of hybridisation.
- This approach could allow selective removal of component molecules allowing activation or inhibition of enzymatic activity.
- the activity inhibitor could be selectively denatured from an MNAi complex resulting in transition to the active MNAzyme state.
- the DNAzyme ligase used in this example (as illustrated in Figure 8) could be replaced by an MNAzyme ligase (as illustrated in Figure 11).
- Example 6 Demonstration of a reaction which exploits the activity of multiple nucleic acid enzymes, in this example specifically MNAzymes which cleave nucleic acid substrates and DNAzymes which ligate nucleic acid substrates, whereby the cleavage or ligation products created by one type of nucleic acid enzyme forms a new substrate or enzyme component which participates in the next nucleic acid enzyme reaction.
- FIG. 9 The strategy used in this example is illustrated in Figure 9.
- This example demonstrates (i) using target nucleic acid to direct the formation of an MNAzyme (MNAzyme A) which can cleave a substrate (MNAzyme substrate A) thus generating a 5 'cleavage product A which can in turn be used as a substrate for a DNAzyme B with ligase activity; (ii) using a DNAzyme ligase B to ligate the 5' cleavage product A generated in step (i) to another oligonucleotide ligation substrate thus creating a new partzyme; and (iii) using the new partzyme/ligation product generated in step (ii) to form a new MNAzyme (MNAzyme C) which can cleave a substrate (MNAzyme substrate C).
- MNAzyme substrate C the labelling of MNAzyme substrate C with a fluorophore and quencher dye pair allowed fluorescent monitoring of the reaction.
- the DNAzyme ligase B used in this example has previously been reported to ligate RNA through the formation of a 2'-5' phosphodiester linkage from a 2', 3'-cyclic phosphate and a 5'-hydroxyl group (Prior et al, 2004). Apart from the requirement of a 5' substrate with a 2', 3'-cyclic phosphate end, the DNAzyme ligase used in this example also requires a specific sequence motif at the ligation junction, being ua*g(A or G) (where lower case denotes RNA bases and uppercase notes an RNA or DNA base and * denotes the ligation site).
- MNAzyme A forms in the presence of target nucleic acid and is used to cleave MNAzyme substrate A (preSub5) thus generating a cleavage fragment which has a 2', 3' - cyclic phosphate end.
- the DNAzyme ligase was then used to ligate the 5' product A (preSub5 cleavage fragment) with another oligonucleotide ligation substrate (preSub3).
- preSub3 oligonucleotide ligation substrate
- the ligated product functions as a new partzyme, which together with a second partzyme and an assembly facilitator can form MNAzyme C and cleave a fluorescently labelled substrate C (SubBi- 2-FB).
- DNAzyme ligase 7Z81-10/10 CCTCTCGTTGACGGCGGAGTGATTGGGAGGTTAGCTCTAGTGAGTGC
- SEQ ID NO: 18 Partzyme A2/MNAzyme A miR20A2/l: TACCTGCACTACGGTCGAA ⁇ TAGTGA GT
- the lower case base represents RNA and the uppercase bases represent DNA.
- the 3' DNAzyme ligase substrate was synthesised to have the sequence below and a 5' hydroxyl group:
- SEQ ID NO: 22 preSub3: gGAACAACGAGAGGAAACCTT
- the reporter substrate used in this example was SubBi-2.
- SubBi-2 was end-labelled with a 6-FAM moiety at the 5' end, a BHQl moiety at the 3' end and designated SubBi-2-FB.
- Cleavage of SubBi-2-FB was monitored at 520 nm (FAM emission wavelength) with excitation at 490 nm (FAM excitation wavelength).
- the sequence of SubBi-2-FB is listed below (5' to 3'); the lower case bases represent RNA and the upper case bases represent DNA.
- the target sequence recognised by MNAzyme A was a synthetic DNA oligonucleotide homologous to the human microRNA miR-20 RNA sequence. It had the following sequence:
- the assembly facilitator required for MNAzyme C formation was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 24 Assembly Facilitator for MNAzyme C: CGACGACCACTCCGACGACAGTCCTATAGTGAGTGCTACAG 6.8 Reaction Conditions
- MNAzyme A cleaved MNAzyme substrate A in the presence of specific target (D-20) producing two fragments, one of which is the 5' fragment with the sequence CTGTAGCACTCACTAua (SEQ ID NO: 40) that had a 2', 3'-cyclic phosphate terminus.
- this 5' fragment was ligated to a second oligonucleotide ligase substrate present in the reaction mix of step (ii) and this resulted in the formation of a new oligonucleotide (ligation product) with the sequence of CTGTAGC ACTC ACTAuagGAACAACGAGAGGAAACCTT (SEQ ID NO: 51) (where upper case represent DNA bases and lower case represent RNA bases).
- This ligation product in turn functioned as a partzyme component for MNAzyme C.
- MNAzyme C cleaved MNAzyme substrate C (SubBi-2-FB) resulting in separation of a fluorophore/quencher dye pair thus causing an increase in fluorescence.
- the "no MNAzyme A cleavage" control (Reaction 2) demonstrated that cleavage and generation of the 5' fragment with the 2', 3 '-cyclic phosphate terminus was essential for subsequent ligation and formation of one of the partzymes required for MNAzyme C.
- the "no ligation” control (Reaction 3) demonstrated that the ligation of the 5' cleavage product A with 2', 3 '-cyclic phosphate terminus to a second 3' ligase substrate was essential for the formation of one of the components of MNAzyme C.
- DNAzyme ligases DNAzymes which can ligate substrates
- MNAzyme cleavers MNAzyme cleavers
- Example 7 Sequences and conditions which allow the formation ofMNAzymes with ligase activity and testing of these MNAzymes with ligase activity in a format useful in cascade reactions.
- DNAzyme ligase 7Q10 was used as a basis for generating candidate MNAzyme ligase partzyme sequences in this example.
- each ligase partzyme was comprised of three sequence domains, including (i) a sensor arm which hybridizes to the assembly facilitator (eg a target nucleic acid), (ii) a domain that constitutes part of a catalytic core and (iii) a substrate binding arm.
- the partial core sequences were designed to incorporate portions of the catalytic region of the 7Q10 DNAzyme ligase which has previously been reported to ligate RNA through the formation of a 2'-5' phosphodiester linkage from a 2', 3'-cyclic phosphate and a 5'- hydroxyl group (Flynn-Charlebois et al, 2003).
- a multi-step assay was used to assess the enzymatic activity of combinations of candidate partzyme sequences.
- the general strategy is outlined in Figure 12 and can also form the basis of a signal amplification technique.
- MNAzyme A forms in the presence of assembly facilitator A and is used to cleave MNAzyme substrate A thus generating a 5' cleavage product A which has a 2', 3'- cyclic phosphate end.
- active MNAzyme B with ligase activity assembles in the presence of assembly facilitator B and then ligates the 5' product A (5' substrate B) to a 3' substrate B.
- the ligated product B functions as a new partzyme, which together with another partzyme and an assembly facilitator C can form MNAzyme C and then cleave a substrate C which may be, for example, fluorescently labelled.
- the catalytic activity of several ligase partzyme pairs was assayed.
- SEQ ID NO: 26 Split 1 Partzyme B - RO5LS1B: rcrCG7TG7TACGTGGAGGTGGTGGAGACGGATTACACCTT
- SEQ ID NO: 29 Split 3 Partzyme A - RO5LS3A: AACGAGTCCTGGCCTTGTCTTGGGCTCL4 GTGAGTGC
- SEQ ID NO: 34 Split 5 Partzyme B - RO5LS5B: rCrCG7TG7TACGTGTGGAGACGGATTACACCTT
- SEQ ID NO: 38 Split 7 Partzyme B - RO5LS7B: rCrCG7TG7TACGTGGTGGAGACGGATTACACCTT
- the lower case bases represent RNA and the uppercase bases represent DNA.
- the sequence of the 3' substrate for ligation used in this experiment is listed below (5' to 3')-
- the oligonucleotide has a 5' hydroxyl group.
- the lower case bases represent RNA and the upper case represent DNA.
- the reporter substrate used in this example was SubBi-2.
- SubBi-2 was end-labelled with a 6-FAM moiety at the 5' end, a BHQl moiety at the 3' end and designated SubBi-2-FB.
- Cleavage of SubBi-2-FB was monitored at 520 nm (FAM emission wavelength) with excitation at 490 nm (FAM excitation wavelength).
- the sequence of SubBi-2-FB is listed below (5' to 3'); the lower case bases represent RNA and the upper case bases represent DNA.
- the assembly facilitator A recognized by MNAzyme A sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 42 AF-MzCA: CGTCGTGAGATGAGGAAGAGATGGATGGGCAC
- the assembly facilitator B recognized by MNAzyme B sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 43 AF-MzLB: AAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTG
- the assembly facilitator C recognized by MNAzyme C sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 24 AF-MzCC: CGACGACCACTCCGACGACAGTCCTATAGTGAGTGCTACAG
- SEQ ID NO: 46 Partzyme B/MNAzyme A - 4SyntB3/l(16): CATCTCTTCTCCGAGCTTCCTCATCTCACGACG
- Reactions contained 10 ⁇ l of previous ligation reaction mix (ii-a or ii-b or ii-c or ii-d or ii-e or ii-f or ii-g or ii-h or ii-i or "no-ligation substrate" control), 200 nM of partzyme B for MNAzyme C (STB5/2(21)), 200 nM of the assembly facilitator for MNAzyme C (AF-MzCC), 50 mM Tris HCl (pH 9.0 at 25°C), 25 mM MgCl 2 and 200 nM of the substrate SubBi-2-FB in a total reaction volume of 25 ⁇ l. The increase in fluorescence due to cleavage of MNAzyme substrate C (SubBi-2-FB) was monitored over time for reactions.
- Previous ligation reaction mix ii-a or ii-b or ii-c or ii-d or ii-e or ii-
- This control reaction demonstrated the uni-molecular DNAzyme ligase 7Q10-10/10 ligated 5LSubB and 3LSubB and formed a new oligonucleotide (ligation product) with the sequence of CTGTAGCACTC ACTAuagGAAC AACGAGAGGAAACCTT (SEQ ID NO: 51) (where upper case represents DNA bases and lower case represents RNA bases).
- This ligation product in turn functioned as a partzyme component of MNAzyme C which cleaved substrate C (SubBi-2-FB) resulting in an increase in fluorescence. This increase in fluorescence acted as a surrogate marker of ligase activity.
- the example has generated a number of partzyme sequences that can be used to assemble MNAcomplexes, which are inactive or have various levels of enzymatic activity depending on the specific sequences and combinations thereof.
- the general design for candidate partzyme sequences tested are summarized in Table 7.
- N is any nucleotide
- Nx is any number of nucleotides which bind to a substrate sequence (substrate domain)
- N)y is any number of nucleotides which bind to an assembly facilitator (sensor
- MNAzyme ligases could be used to detect target analytes.
- MNAzyme ligases can be designed to assemble only in the presence of an analyte such as an RNA or DNA target sequence (the assembly facilitator). If the 5'ligation substrate and the 3' ligation substrate were each labelled with either a fluorophore and quencher dye respectively (or vice versa) then ligation would result in a decrease in fluorescence which could serve as an indicator of the presence of the target analyte.
- the 5' ligation substrate were labelled with a detectable moiety, for example a fluorophore, and the 3' ligation substrate were attached to a discrete location, for example on a chip or bead, then ligation would result in the appearance of a signal at the discrete location in the presence of target analyte.
- a detectable moiety for example a fluorophore
- FIG. 12 The strategy used to assay for ligase activity in this example is illustrated in Figure 12. Once active partzymes have been identified the same cascade reaction can then be used to detect the presence of an assembly facilitator, for example a target analyte.
- the cascade reaction allowing detection uses two types of nucleic acid enzymes, namely MNAzymes which can ligate substrates and MNAzymes which can cleave substrates. Multiple turnover at each enzymatic step in the cascade could allow signal amplification.
- this example can also be considered as an example of a reaction where the initiating step results in the formation of an MNAzyme with ligase activity.
- the initiating target nucleic acid would be the assembly facilitator B (AF-MzLB) and the assembly facilitator A (AF-MzCA) would be a synthetic molecule included to drive the assembly of MNAzyme A.
- MNAzyme A would then have the sole purpose of generating a 5' ligatable substrate B with 2 '3' cylic phosphate termini in a reaction which could then be added to the reaction which detects the target assembly facilitator B using an MNAzyme B with ligase activity.
- MNAzyme A partzymes and the assembly facilitator A could be replaced by a DNAzyme with cleavage activity present either in a separate preliminary reaction for ligatable substrate generation or the DNAzyme could be present in the same reaction mix as the MNAzyme with ligase activity.
- Example 11 demonstrates that cleavage and ligation using a combination of an MNAzyme and a DNAzyme can be performed in a single reaction mix. [ 00452 ] One skilled in the art would appreciate that there are many variants on this basic cascade strategy.
- the MNAzyme B could be used to create ligation products which serve as assembly facilitators for another MNAzyme with cleavage, ligase or other enzymatic activity.
- MNAzyme C could modify a substrate by means other than cleavage. Further embodiments are included throughout the specification.
- Example 8 An exemplary strategy for switching an MNA complex between the "on state” of an active MNAzyme to the "off state” of an MNAi complex using a second
- Figure 13 illustrates an exemplary strategy for switching an MNA complex between the "on state" of an active MNAzyme to the "off state” of an MNAi complex using a second MNAzyme with ligase activity.
- an active MNAzyme A which could be capable of modifying (e.g. cleaving or ligating) a substrate(s) A could be formed in the presence of assembly facilitator component 1 (AFC 1) and assembly facilitator component 2 (AFC 2).
- a second MNAzyme B which has ligase activity, could form in the presence of, for example, an assembly facilitator 3 (AF3) which could comprise one or more components, and could then ligate AFC 2 with an activity inhibitor component (AIC) causing the formation of an activity inhibitor (AI).
- AF3 assembly facilitator 3
- AIC activity inhibitor component
- AI activity inhibitor
- This AI could bind to the partzyme components for the MNAzyme A and result in the formation of an MNAi A complex which is inactive.
- the MNAzyme ligase in this example could operate as an off switch to inactivate an MNAzyme A.
- the inactive MNAi complex and the catalytically active MNAzyme would represent two alternate states for the assembled components, namely an "off state and the "on" state respectively.
- Example 9 Sequences and conditions which allow the formation ofMNAzymes with ligase activity and testing of these MNAzymes with ligase activity in a format useful in a signal amplification cascade.
- DNAzyme ligase 7Z81 was used as a basis for generating candidate MNAzyme ligase partzyme sequences in this example.
- each ligase partzyme was comprised of three sequence domains, including (i) a sensor arm which hybridizes to the assembly facilitator (e.g. a target nucleic acid), (ii) a domain that constitutes part of a catalytic core and (iii) a substrate binding arm.
- the partial core sequences were designed to incorporate portions of the catalytic region of the 7Z81 DNAzyme ligase which has previously been reported to ligate RNA through the formation of a 2'-5' phosphodiester linkage from a 2', 3'-cyclic phosphate and a 5'- hydroxyl group (Prior et al, 2004).
- a multi-step assay was used to assess the enzymatic activity of combinations of candidate partzyme sequences.
- the general strategy is outlined in Figure 12 and can also form the basis of a signal amplification technique.
- MNAzyme A forms in the presence of assembly facilitator A and is used to cleave MNAzyme substrate A thus generating a 5' cleavage product A which has a 2', 3'- cyclic phosphate end.
- active MNAzyme B with ligase activity assembles in the presence of assembly facilitator B and then ligates the 5' product A (5' substrate B) to a 3' substrate B.
- the ligated product B functions as a new partzyme, which together with another partzyme and an assembly facilitator C can form MNAzyme C and then cleave substrate C, which may be, for example, fluorescently labelled.
- cleave substrate C which may be, for example, fluorescently labelled.
- the DNAzyme ligase used in this example has previously been reported to ligate RNA and form a 2 '-5' phosphodiester linkage using a 5' ligation substrate with a 2', 3 '-cyclic phosphate and a 3' ligation substrate with a 5'-hydroxyl group (Prior et al, 2004). Apart from the requirement for specific termini on the substrates, the DNAzyme ligase used in this example also requires a specific sequence motif at the ligation junction. [ 00458 ] A DNAzyme ligase 7Z81 with substrate arms designed to hybridize to the substrate sequences 5LSubB and 3LSubB was used in this experiment and the sequence is listed below (5' to 3'). The bases underlined form the catalytic core and bases in italics hybridize to the substrates.
- Substrate A (Pre5LSubB2-FB) is below. It is internally labelled 5' of the ribobases with a 6-FAM moiety and labelled 3' of the ribobases with BHQl moiety (underlined bases).
- the lower case bases represent RNA and the upper case bases represent DNA.
- the sequence of the 3' substrate for ligation used in this experiment is listed below (5' to 3')-
- the oligonucleotide has a 5' hydroxyl group.
- the lower case bases represent RNA and the upper case bases represent DNA.
- the assembly facilitator A recognized by MNAzyme A sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 43 AF-MzLB: AAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTG
- the assembly facilitator C recognized by MNAzyme C sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 24 AF-MzCC: CGACGACCACTCCGACGACAGTCCTATAGTGAGTGCTACAG
- step (iii) Fluorescent detection due to MNAzyme C cleavage mediated by a partzyme formed by ligation in step (ii) was performed isothermally at 55°C for 2 hours on the Cepheid Smartcycler with data collection points at 72 second intervals. Reactions were initiated by the addition of the fluorescently labelled substrate SubBi-2-Q6B2.
- Reactions contained 10 ⁇ l of previous ligation reaction mix (ii-a or ii-b or ii-c or ii-d or ii-e or ii-f or ii-g or ii-h, 500 nM of partzyme B for MNAzyme C (STB5/2(21)), 100 nM of the assembly facilitator for MNAzyme C (AF-MzCC), PCRII Buffer (Applied Biosystems), 50 mM MgCl 2 and 1000 nM of the substrate SubBi-2-Q6B2 in a total reaction volume of 25 ⁇ l.
- the increase in fluorescence due to cleavage of MNAzyme substrate C was monitored over time for each reaction.
- MNAzyme A formed and cleaved Pre5LSubB2-FB to generate a product (5LSubB) which has a 2', 3 '-cyclic phosphate which makes it suitable for use as a substrate by the DNAzyme ligase.
- the sequence of the cleavage product 5LSubB is CTGTAGCACTCACTAua (SEQ ID NO: 40) where upper case represents DNA bases and lower case represents RNA bases.
- This control reaction demonstrated the uni- molecular DNAzyme ligase 7Z81-10/10 ligated 5LSubB and 3LSubB and formed a new oligonucleotide (ligation product) with the sequence of
- CTGT AGC ACTC ACTAuagGAACAACGAGAGGAAACCTT (SEQ ID NO: 51) (where upper case represents DNA bases and lower case represents RNA bases).
- This ligation product in turn functioned as a partzyme component of MNAzyme C, which cleaved substrate C (SubBi-2-Q6B2) resulting in an increase in fluorescence.
- This increase in fluorescence acted as a surrogate marker of ligase activity.
- the example has generated a number of partzyme sequences that can be used to assemble MNAzymes, which have various levels of activity depending on the specific sequences and combinations thereof.
- the general design for the candidate partzymes sequences which were tested are summarized in Table 9. Table 9. General design for partzymes sequences tested.
- MNAzyme ligases could be used to detect target analytes.
- MNAzyme ligases can be designed to assemble only in the presence of an analyte such as an RNA or DNA target sequence (the assembly facilitator).
- an analyte such as an RNA or DNA target sequence (the assembly facilitator).
- an analyte such as an RNA or DNA target sequence (the assembly facilitator).
- the 5' ligation substrate and the 3' ligation substrate were each labelled with either a fluorophore or quencher dye respectively (or vice versa) then ligation would result in a decrease in fluorescence, which could serve as an indicator of the presence of the target analyte.
- the 5' ligation substrate were labelled with a detectable moiety, for example a fluorophore, and the 3' ligation substrate were attached to a discrete location, for example on a chip or bead, then ligation would result in the appearance of a signal at the discrete location in the presence of target analyte.
- a detectable moiety for example a fluorophore
- the strategy used to assay for ligase activity in this example, as illustrated in Figure 12 constitutes a linear cascade which could be used to create a feedback signal amplification cascade. If the sequence of 5' product C were the same as that of 5' product A then this product could also serve as a 5' substrate B and a feedback cascade reaction could be initiated.
- MNAzyme C could constantly be generating a 5' substrate B which could in turn be ligated by MNAzyme ligase B to create more partzymes for formation of more MNAzyme C.
- This strategy could provide a mechanism for feedback amplification following initiation of a reaction by an assembly facilitator (e.g. target analyte) which could direct assembly of MNAzyme A.
- the strategy could allow detection of assembly facilitators (e.g. target analytes) followed by signal amplification using two types of MNAzymes, namely MNAzymes which could ligate substrates (MNAzyme ligases) and MNAzymes which could cleave a substrate (MNAzyme cleavers).
- MNAzyme B could be used to create ligation products, which serve as assembly facilitators for another MNAzyme with cleavage, ligase or other enzymatic activity.
- MNAzyme C could modify a substrate by means other than cleavage. Further embodiments are included throughout the specification.
- DNAzymes have similar basic structures with multiple domains. These DNAzymes comprise a conserved catalytic domain (catalytic core) flanked by two non- conserved substrate-binding domains ("arms"), which specifically recognize and hybridise to the substrate (for example, for DNAzyme cleavers or those that act on one substrate) or at least two substrates (for example, for DNAzyme ligases or those that act on at least two substrates).
- the substrate binding domains can be tailored to any substrate provided the substrate or substrates contains a site(s) which can be modified by the DNAzyme.
- This example describes a general strategy for engineering new MNAzymes based on any known DNAzyme. This general strategy is illustrated schematically in Figure 14.
- the method is described firstly for deriving MNAzymes from DNAzymes with modifiying activity, for example cleavage activity.
- positions within the DNAzyme catalytic core at which it can be split are identified, in order that each partial portion of the catalytic core can be distributed between two sequences such that the two partial cores together constitute an entire catalytic core.
- An oligonucleotide A can then be synthesised to contain (i) one substrate binding arm portion capable of binding to a substrate, (ii) one partial catalytic core portion, and (iii) one sensor arm portion capable of binding to an assembly facilitator molecule.
- a second oligonucleotide B is synthesised such that it contains (i) one substrate binding arm portion capable of binding to the same substrate as oligonucleotide A, whereby oligonucleotide B binds the substrate in a position adjacent to that of oligonucleotide A, (ii) one partial catalytic core portion which contains those bases from the entire DNAzyme catalytic core which are not incorporated into oligonucleotide A and (iii) one sensor arm portion capable of binding to the same assembly facilitator as oligonucleotide A, whereby oligonucleotide B binds the assembly facilitator in a position adjacent to that of oligonucleotide A.
- This process can be repeated thus making a series of pairs of oligonucleotides A and B which incorporate the structure and domains of partzymes, but may or may not have catalytic activity in the presence of a substrate and an assembly facilitator.
- Some or all of the candidate partzyme pairs can then be incubated in the presence of the assembly facilitator complementary to the sensor arm portions of the partzymes and a substrate which is complementary to the substrate arm portions of the partzymes. Incubation is performed under conditions which are compatible with modification of a substrate by the DNAzyme from which the partial catalytic core sequences were derived. Pairs of oligonucleotides A and B which can perform the same type of modification to the substrate as the DNAzyme from which partial sequences were derived are useful as part2ymes which can be assembled into MNAzymes. The sequences of the partial catalytic cores are suitable for incorporation into other partzymes. New partzymes can be synthesised which are tailored to new substrates (by changing the substrate binding domains of each partzyme) and/or to new assembly facilitators (by changing the sensor binding domains of each partzyme).
- the method for engineering an MNAzyme that acts on two substrates (e.g. an MNAzyme ligase) from a uni-molecular nucleic acid enzyme (e.g. a DNAzyme ligase) requires similar steps whereby in the first step, positions are identified within the DNAzyme catalytic core at which it can be split, such that each partial portion of the catalytic core can be distributed between two partial sequences such that the two partial cores together constitute an entire catalytic core. Two oligonucleotides A and B (candidate partzymes) can then be synthesised.
- An oligonucleotide A can be synthesised to contain (i) one substrate binding arm portion capable of binding to a first substrate (e.g. ligatable substrate), (ii) one partial catalytic core portion, and (iii) one sensor arm portion capable of binding to an assembly facilitator molecule.
- a second oligonucleotide B can be synthesised such that it contains (i) one substrate binding arm capable of binding to a second substrate (e.g.
- ligatable substrate (ii) one partial catalytic core portion which contains those bases from the entire DNAzyme catalytic core which are not incorporated into oligonucleotide A and (iii) one sensor arm sequence capable of binding to the same assembly facilitator as oligonucleotide A, whereby oligonucleotide B binds the assembly facilitator in a position adjacent to that of oligonucleotide A.
- This process can be repeated thus making a series of pairs of oligonucleotides A and B which incorporate the structure and domains of partzymes, but may or may not have catalytic activity in the presence of the substrates and an assembly facilitator.
- Some or all of the candidate partzyme pairs can then be incubated in the presence of the assembly facilitator complementary to the sensor arm portions of the partzymes and substrates (e.g. ligatable substrates) which are complementary to the substrate arm portions of the partzymes. Incubation is performed under conditions which are compatible with modification of the substrates (e.g. by ligation) by the DNAzyme from which the partial catalytic core sequences were derived. Pairs of oligonucleotides A and B which can perform the same modification (e.g.
- partzymes which can be assembled into MNAzymes.
- the sequences of the partial catalytic cores are suitable for incorporation into other partzymes.
- New partzymes can be synthesised which are tailored to new substrates (by changing the substrate binding domains of each partzyme) and/or to new assembly facilitators (by changing the sensor binding domains of each partzyme).
- a similar method can be used to examine tolerance to changes in the partial sequence derived from splitting the DNAzyme catalytic core that can be incorporated into component partzymes.
- modifications to the partial catalytic cores incorporated into partzymes can be made which include, by way of example, insertions, deletions, substitution with alternate DNA bases, or substitution with DNA base analogues such as ribonucleotides, inosine bases or other analogues.
- Candidate partzyme pairs containing one or more modification(s) can then be assayed as above to determine whether or not they are suitable as partzymes that can assemble into catalytically active MNAzymes.
- Example 11 A cascade reaction which exploits the activity of multiple nucleic acid enzymes.
- a DNAzyme ligase was used to ligate the 5' cleavage product to another oligonucleotide ligation substrate B thus creating a ligation product that functioned as a new MNAzyme component (e.g. a partzyme).
- the partzyme ligation product generated in the first step participates in the formation of a new MNAzyme (MNAzyme C), which can cleave a substrate (MNAzyme substrate C).
- MNAzyme C new MNAzyme
- MNAzyme substrate C the labelling of MNAzyme substrate C with a fluorophore and quencher dye pair allowed fluorescent monitoring of the reaction.
- MNAzyme A formed in the presence of an assembly facilitator (AF-MzCA2) and was used to cleave MNAzyme substrate A (Pre5LSubB2- FB).
- assembly facilitator A-MzCA2
- Pre5LSubB2- FB MNAzyme substrate A
- the sequences of the MNAzyme A partzymes (RO4A2/1(11) and RO4B3/1(12)) are listed below.
- the bases underlined form part of the catalytic core, bases in bold hybridize with the assembly facilitator and bases in italics hybridize to the substrates.
- the DNAzyme ligase used in this example has previously been reported to ligate RNA through the formation of a 2'-5' phosphodiester linkage from a 2', 3'-cyclic phosphate and a 5'-hydroxyl group (Prior et al, 2004). Apart from the requirement of a 5' substrate with a 2', 3'-cyclic phosphate end, the DNAzyme ligase used in this example also requires a specific sequence motif at the ligation junction.
- DNAzyme ligase 7Z81 (7Z81-10/10) with substrate arms designed to hybridize to the substrate sequences 5LSubB and 3LSubB was used in this experiment and the sequence is listed below (5' to 3').
- the bases underlined form the catalytic core and bases in italics hybridize to the substrates.
- SEQ ID NO: 17 DNAzyme ligase - 7Z81-10/10: CCTCTCGTTGACGGCGGAGTGATTGGGAGGTTAGCTCTAGTGAGTGC
- the ligated product functioned as a new partzyme, which together with a partzyme B (STB5/2(21)) and assembly facilitator (AF-MzCC), formed MNAzyme C and cleaved a fluorescently labelled substrate C (SubBi-2-Q6B2).
- the control partzyme A (LS817A4/2), which was the same sequence as the ligated product also formed MNAzyme C with partzyme B (STB5/2(21)).
- the following sequences correspond to the partzymes, which form part of MNAzyme C.
- the bases underlined form part of the catalytic core, bases in bold hybridize with the assembly facilitator and bases in italics hybridize to the substrates.
- MNAzyme substrate A was cleaved by the MNAzyme cleaver A.
- the resultant 5' fragment denoted 5LSubB was then ligated to 3LSubB by the DNAzyme ligase.
- the sequence of substrate A (Pre5LSubB2-FB) is below. It is internally labelled 5' of the ribobases with a 6-FAM moiety and labelled 3' of the ribobases with BHQl moiety (underlined bases).
- the lower case bases represent RNA and the upper case bases represent DNA.
- Substrate A - Pre5LSubB2-FB CTGTAGCACTCACTAuaGGAAGAGATGAG 11.5 DNAzyme Ligase substrate B
- the sequence of the 3' substrate for ligation used in this experiment is listed below (5' to 3').
- the oligonucleotide has a 5' hydroxyl group.
- the lower case bases represent RNA and the upper case bases represent DNA.
- the reporter substrate used in this example was SubBi-2.
- SubBi-2 was end-labelled with a Quasar 670 moiety at the 5' end, a BHQ2 moiety at the 3' end and designated SubBi-2-Q6B2.
- Cleavage of SubBi-2-Q6B2 was monitored at 665 nm with excitation at 635 nm.
- the sequence of SubBi-2-Q6B2 is listed below (5' to 3'); the lower case bases represent RNA and the upper case bases represent DNA.
- the assembly facilitator A recognized by MNAzyme A sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 68 AF-MzCA2 GCCATTGTCGAACACCTGCTGGATGACCAGC
- the assembly facilitator C recognized by MNAzyme C sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 24 AF-MzCC: CGACGACCACTCCGACGACAGTCCTATAGTGAGTGCTACAG
- the reactions contained, 70 mM Tris HCl (pH 9.0 at 25°C), 50 mM MgCl 2 , 150 mM NaCl, 2mM KCl, 40 nM RO4B3/1(12), 40 nM RO4A2/1(11), 200 nM Pre5LSubB2-FB and either: a) 40 nM AF-MzCA2, 200 nM DNA Ligase 7Z81-10/10 and 100 nM 3LSubB; or b) 40 nM AF-MzCA2 and 100 nM 3LSubB; or c) 40 nM AF-MzCA2 and 200 nM DNA Ligase 7Z81 - 10/ 10; or d) 200 nM DNA Ligase 7Z81-10/10 and 100 nM 3LSubB; or e) 100 nM 3LSubB only; or f) 20O nM DNA Ligase 7Z81-10/10 only
- Reaction 2 ( 1 -b), which lacked the DNAzyme ligase (7Z81-10/10) oligonucleotide for step (1) showed no increase in fluorescence over time.
- Reaction 3 (1- c), which lacked ligase substrate B (3LSubB) but contained the DNAzyme ligase from step (1) showed no increase in fluorescence over time.
- Reaction 4 (1-d) which lacked assembly facilitator A (AF-MzC A2) from step (1) showed a minimal increase in fluorescence over time above the background drift observed of approximately 40 arbitrary units over the course of 1 hour of fluorescence monitoring.
- Reaction 5 (1-e) which lacked both the assembly facilitator A and the DNAzyme ligase from step (1) showed no increase in fluorescence over time.
- Reaction 6 (1-f) which lacked both the ligase substrate B and the assembly facilitator A showed no increase in fluorescence over time.
- Reaction 7 (1-g) was the positive control that contained the control MNAzyme C partzyme A (LS817A4/2), and showed an increase in fluorescence over time whereby the reaction was completed in less than 1 minute. Together these reactions indicate that the following events have occurred in Reaction 1 (1-a).
- MNAzyme A cleaves Substrate A (Pre5LSubB2-FB) in the presence of specific target assembly facilitator A (AF-MzCA2) producing two fragments, one of which was the 5' fragment 5LSubB (CTGT AGC ACTCACTAua) (SEQ ID NO: 40) that has a 2', 3 '-cyclic phosphate terminus.
- 5LSubB was ligated to a second DNAzyme oligonucleotide ligase substrate B (3LSubB) present in the reaction mix of step (1) and this resulted in the formation of a new oligonucleotide (ligation product) with the sequence of CTGTAGCACTCACTAuagGAACAACGAGAGGAAACCTT (SEQ ID NO: 51) (where upper case represent DNA bases and lower case represent RNA bases).
- This ligation product in turn functioned as a partzyme for MNAzyme C.
- MNAzyme C cleaved MNAzyme substrate C (SubBi-2-Q6B2) resulting in separation of a fluorophore /quencher dye pair thus causing an increase in fluorescence.
- the cascade reaction in this example allowed the detection of the presence of an assembly facilitator, in this example a target nucleic acid sequence.
- the cascade reaction allowing detection uses two types of nucleic acid enzymes, namely DNAzymes which can ligate substrates and MNAzymes which can cleave substrates. Multiple turnover at each enzymatic step in the cascade could allow signal amplification.
- the DNAzyme ligase could ligate two fragments and create several possible enzymes, enzyme components or substrates useful in this cascade which include but are not limited to, a) a partzyme for a second MNAzyme (e.g. an MNAzyme cleaver as shown in this example and labelled as MNAzyme C in Figure 9); or b) a new assembly facilitator for a second MNAzyme as described in example 12, and labelled as MNAzyme Cleaver 2 in Figure 15; or c) a new DNAzyme capable of modifying a substrate or substrates (e.g.
- step 2 Figure 9 (Ui)
- step 2 Figure 9 (Ui)
- step 2 Figure 9 (Ui)
- step 2 Figure 9 (Ui)
- step 2 Figure 9 (Ui)
- Example 12 An example of a cascade reaction which exploits the activity of multiple nucleic acid enzymes.
- an MNAzyme (MNAzyme cleaver 1) forms in the presence of assembly facilitator (e.g. a target nucleic acid) and is used to cleave a substrate (MNAzyme substrate 1) thus generating a 5' cleavage product which has a 2', 3'- cyclic phosphate end.
- assembly facilitator e.g. a target nucleic acid
- MNAzyme substrate 1 a substrate
- a DNAzyme ligase is used to ligate the cleavage product generated in step one to another oligonucleotide ligation substrate thus creating a ligation product that functions as an assembly facilitator.
- the assembly facilitator ligation product generated in step two participates in the formation of a new MNAzyme (MNAzyme cleaver 2), which can cleave a substrate (MNAzyme substrate 2).
- MNAzyme substrate 2 the labelling of MNAzyme substrate 2 with a fluorophore and quencher dye pair allowed fluorescent monitoring of the reaction.
- MNAzyme cleaver 1 formed in the presence of an assembly facilitator (AF-MzCA2) and was used to cleave MNAzyme substrate 1 (Pre5LSubB2- FB).
- assembly facilitator AMF-MzCA2
- Pre5LSubB2- FB MNAzyme substrate 1
- the sequences of the MNAzyme cleaver 1 partzymes are listed below.
- the bases underlined form part of the catalytic core, bases in bold hybridize with the assembly facilitator and bases in italics hybridize to the substrates.
- the DNAzyme ligase used in this example has previously been reported to ligate RNA through the formation of a 2'-5' phosphodiester linkage from a 2', 3'-cyclic phosphate and a 5'-hydroxyl group (Prior et al, 2004). Apart from the requirement of a 5' substrate with a 2', 3 '-cyclic phosphate end, the DNAzyme ligase used in this example also requires a specific sequence motif at the ligation junction.
- the ligated product functions as a new assembly facilitator, which together with a pair of partzymes can form MNAzyme cleaver 3 and cleave a fluorescently labelled substrate 3 (SubBi-3-TxB2).
- the following sequences correspond to the partzymes, which form components of MNAzyme cleaver 2.
- the bases underlined form part of the catalytic core, bases in bold hybridize with the assembly facilitator and bases in italics hybridize to the substrates.
- MNAzyme substrate 1 substrate for MNAzyme cleaver 1
- MNAzyme substrate 1 was cleaved by the MNAzyme cleaver 1.
- the resultant 5' fragment denoted 5LSubB was then ligated to 3LSubB by the DNAzyme ligase.
- the sequence of substrate 1 (Pre5LSubB2-FB) is below. It is internally labelled 5' of the ribobases with a 6-FAM moiety and labelled 3' of the ribobases with BHQl moiety (underlined bases).
- the lower case bases represent RNA and the upper case bases represent DNA.
- Substrate 1 - Pre5LSubB2-FB CTGTAGCACTCACTAuaGGAAGAGATGAG 12.5 DNA ⁇ yme Ligase substrate
- the sequence of the 3' substrate for ligation used in this experiment is listed below (5' to 3').
- the oligonucleotide has a 5' hydroxyl group.
- the lower case bases represent RNA and the upper case bases represent DNA.
- the reporter substrate used in this example was SubBi-3.
- SubBi-3 was end-labelled with a Texas Red moiety at the 5' end, a BHQ2 moiety at the 3' end and designated SubBi-3-TxB2.
- Cleavage of SubBi-3-TxB2 was monitored at 610 nm (TxB2 emission wavelength) with excitation at 585 nm (TxB2 excitation wavelength).
- the sequence of SubBi-3-TxB2 is listed below (5' to 3'); the lower case bases represent RNA and the upper case bases represent DNA.
- the assembly facilitator recognized by MNAzyme cleaver 1 sensor domains was a synthetic DNA oligonucleotide with the following sequence:
- SEQ ID NO: 68 AF-MzCA2 GCCATTGTCGAACACCTGCTGGATGACCAGC
- the reactions contained 50 mM Tris HCl (pH 9.0 at 25°C), 50 mM MgCl 2, 150 mM NaCl, 2 mM KCl, 50 nM RO4B3/1(12), 50 nM RO4A2/1(11) and either: a) 1000 nM Pre5LSubB2-FB, 50 nM AF-MzCA2; or b) 1000 nM Pre5LSubB2-FB; or c) 50 nM AF-MzCA2.
- Duplicate reactions contained 10 ⁇ l of one of previous ligation reaction mixes ((i- a/ii-a) or (i-a/ii-b) or (i-b/ii-a) or (i-b/ii-b) or (i-c/ii-a) or (i-c/ii-b)) and 200 nM of partzyme A (LIGF ACA5/3) and 200 nM partzyme B (LIGFACB6/3) for MNAzyme cleaver 2, and 400 nM of the substrate SubBi-3-TxB2 in a total reaction volume of 25 ⁇ l.
- Reaction 4 (i-b/ii-b), which lacked both the assembly facilitator 1 (AF-MzCA2) and the DNAzyme ligase from step (ii) showed no increase in fluorescence over time.
- Reaction 5 (i-c/ii-a), which lacked the Substrate 1 (Pre5LSubB2-FB) but contained the DNAzyme ligase from step (ii) showed no increase in fluorescence over time.
- Reaction 6 (i-c/ii-b), which lacked both the Substrate 1 (Pre5LSubB2-FB) and the DNAzyme ligase showed no increase in fluorescence over time. Together these reactions indicate that the following events have occurred in reaction 1.
- MNAzyme cleaver 1 cleaves Substrate 1 (Pre5LSubB2-FB) in the presence of specific assembly facilitator 1 (AF-MzCA2) producing two fragments, one of which was the 5' fragment 5LSubB (CTGTAGC ACTC ACTAua) (SEQ ID NO: 40) that has a 2', 3 '-cyclic phosphate terminus.
- 5LSubB was ligated to a second DNAzyme oligonucleotide ligase substrate (3LSubB) present in the reaction mix of step (ii) and this resulted in the formation of a new oligonucleotide (ligation product) with the sequence of CTGTAGCACTCACTAuagGAACAACGAGAGGAAACCTT (SEQ ID NO: 51) (where upper case represent DNA bases and lower case represent RNA bases).
- This ligation product in turn functioned as an assembly facilitator for MNAzyme cleaver 2.
- This newly ligated assembly facilitator associated with the partzymes to create MNAzyme cleaver 2.
- MNAzyme cleaver 2 cleaved MNAzyme substrate 2 (SubBi-3- TxB2) resulting in separation of a fluorophore/quencher dye pair thus causing an increase in fluorescence.
- DNAzyme ligase could ligate two fragments and create several possible enzymes or enzyme components useful in this cascade which include but are not limited to, a) a new assembly facilitator for a second MNAzyme cleaver (as shown in this example) or b) a new partzyme for a second MNAzyme as shown in examples 6, 7, 9, and 11, or c) a new DNAzyme capable of modifying a substrate or substrates (e.g. by cleavage, ligation etc).
- DNAzyme with ligase activity could be substituted with an MNAzyme with ligase activity as demonstrated in example 7 and 9.
- the strategy is versatile and could be performed as either a sequential cascade of the first, second and third steps or as a feedback cascade reaction where step 3 creates a component for step 2 and step 2 creates components for step 3. Additionally, step 3 could create a component for a further subsequent reaction.
- Example 13 A cascade reaction with possible feedback
- FIG 16. A schematic representation of one variation of a feedback amplification cascade is illustrated in Figure 16.
- the initial steps of a cascade involving MNAzyme cleavage, DNAzyme ligation and MNAzyme cleavage are illustrated in Figure 15 and demonstrated in example 6, 7, 9, 11 and 12.
- the schema described in this example and illustrated in Figure 16 extends the strategy of the cascade in example 6, 7, 9, 11 and 12 and describes a strategy for a feedback amplification cascade.
- an MNAzyme cleaver 1 could assemble only in the presence of an assembly facilitator (e.g. a target nucleic acid) and then cleave MNAzyme substrate 1 to release a 5' cleavage product with a 2', 3' cyclic phosphate terminus.
- an assembly facilitator e.g. a target nucleic acid
- a DNAzyme ligase or an assembled MNAzyme ligase
- an assembly facilitator formed by ligation in the second step could direct the assembly of partzymes which could form an MNAzyme cleaver 2 which could cleave substrate 2 into two products, a 5' product cleavage with a 2', 3' cyclic phosphate terminus and 3' cleavage product.
- a detectable signal could be generated following cleavage of substrate 2 if this substrate were labelled, for example, with a fluorophore and quencher dye pair.
- MNAzyme cleaver 3 could have different sensor arms than MNAzyme cleaver 1 but could have the same substrate arms as MNAzyme cleaver 1.
- MNAzyme cleaver 3 were assembled in the presence of an assembly facilitator component generated by cleavage by the MNAzyme cleaver 2, and if MNAzyme cleaver 3 cleaved substrate 1 then the 5' cleavage product of MNAzyme cleaver 3 could also serve as a 5' substrate for the DNAzyme ligase (or the assembled MNAzyme ligase) and a feedback amplification cascade reaction could be initiated.
- MNAzyme cleaver 3 would constantly generate 5' cleavage product which in turn could serve as a substrate for ligation by the DNAzyme ligase (or the assembled MNAzyme ligase) to create more assembly facilitators that could direct the assembly of more MNAzyme cleaver 2.
- This strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which allowed assembly of MNAzyme cleaver 1.
- the strategy could allow detection of one or more assembly facilitators (eg target analytes) followed by signal amplification using a DNAzyme or an MNAzyme which can ligate substrates and MNAzymes which can cleave a substrate.
- the assembly facilitator component generated by cleavage (or ligation) in step 2 or step 3 by an MNAzyme or a DNAzyme could be any one of numerous designs. By way of example, they could resemble any the configurations for assembly facilitator and/or partzyme sensor arms as illustrated in Figure 6.
- MNAzyme cleaver 3 could have a modified structure.
- MNAzyme cleaver 3 could have different sensor arms than MNAzyme cleaver 1 but it may have one substrate arm (on one of the partzymes) for MNAzyme 3 that is the same as the sequence of the partzyme of MNAzyme cleaver 1 which interact with that portion of the sequence on substrate 1 (the 5' portion of the substrate) which is subsequently ligated by the ligase.
- MNAzyme cleaver 3 would cleave a substrate (cleavable substrate 3) which has the same sequence at the 5' portion but which has a different portion at the 3' end or vice versa with respect to cleavable substrate 1.
- the cleavage site of substrate 3 would lie at the junction of those sequences which are shared between the cleavable substrates 1 and 3 and those which differ.
- MNAzyme cleaver 3 when MNAzyme cleaver 3 was assembled in the presence of an assembly facilitator component generated by cleavage by the MNAzyme cleaver 2, and if MNAzyme cleaver 3 cleaved substrate 3 then the 5' cleavage product of MNAzyme cleaver 3 could also serve as a 5' substrate for the DNAzyme ligase (or the assembled MNAzyme ligase) and a feedback amplification cascade reaction could be initiated.
- MNAzyme cleaver 3 would constantly generate 5' cleavage product which in turn could serve as a substrate for ligation by the DNAzyme ligase (or the assembled MNAzyme ligase) to create more components, for example, an assembly facilitator that could direct the assembly of more MNAzyme cleaver 2.
- This strategy could provide a mechanism for feedback signal amplification following initiation of a reaction by an assembly facilitator (eg a target analyte) which allowed assembly of MNAzyme cleaver 1.
- the strategy could allow detection of an assembly facilitator (eg a target analyte) followed by signal amplification using a DNAzyme or an MNAzyme which can ligate substrates and MNAzymes which can cleave a substrate.
- nucleic acid enzymes used in the cascade could create different components that could be used in a subsequent reaction such as in a feedback situation.
- an MNAzyme cleaver 2 could create a stabiliser arm for MNAzyme cleaver 3, instead of the assembly facilitator component as illustrated.
- MNAzyme cleaver 2 could be replaced by an MNAzyme ligase that could create, for example a partzyme or assembly facilitator for MNAzyme cleaver 3, or a DNAzyme cleaver as opposed to the illustration in Figure 16 whereby an MNAzyme cleaver 2 creates an assembly facilitator component.
- DNAzyme (or MNAzyme) ligase could ligate fragments and create a new partzyme capable of associating with another partzyme and an assembly facilitator present in the reaction thus forming an MNAzyme cleaver 2 which could cleave substrate 2 to create an assembly facilitator component for an MNAzyme 3.
- the nucleic acid enzyme cascades allow signal generation following detection of target analytes.
- the first MNAzyme in the cascade may be an MNAzyme or apta-MNAzyme thus MNAzyme systems can be designed for detection of nucleic acid targets and non-nucleic acid analytes, for example proteins or lipids.
- feedback cascades could further amplify the output or detectable signal.
- Signal amplification cascades using nucleic acid enzymes have advantages over target amplification and detection technologies, for example real time PCR. While target amplification technologies are powerful tools which have been widely used in research and/or in clinical diagnostics, each has inherent disadvantages. They all require the use of protein enzymes (e.g. DNA polymerase, RNA polymerase, reverse transcriptase, and/ or ligase). The inclusion of protein enzymes increases the complexity and cost of reagent manufacture and decreases the shelf life of kits containing reagents. Other associated technical challenges include contamination by replicons (target amplicons) from previous reactions leading to false positive signal, and/or background signal caused by replication of primer sequences (primer-dimers) or background caused by target-independent ligation.
- Example 14 Testing MNAzyme activity from a series of partzyme pairs which contain variant partial catalytic core sequences derived from the 10:23 catalytic core
- Multi-component nucleic acid enzymes can be made which incorporate partial sequences from a variety of in vitro evolved DNAzymes. Active MNAzymes, based on partial sequences from the 8: 17 and 10:23 DNAzymes, have been demonstrated. Further, several alternative partzyme designs based on the 8:17 and 10:23 DNAzymes have been shown to either have or lack cleavage activity (PCT/AU2006/001473, Johnson & Johnson Research Pty Limited). This example identifies both active and inactive partzyme sequences based on partial catalytic core sequences from the 10:23 DNAzyme which can cleave a substrate. Further, the example provides a general protocol for the steps necessary to identify the optimal place(s) to split a catalytic core sequence such that, when the partial catalytic core sequences are incorporated into partzymes, functional active MNAzymes are generated.
- Table 12 Bases and position in the 10:23 DNAzyme and in a series of variant partzyme pairs where the bases at positions 1 to 15 of the core have been distributed differently between two partzymes A and B.
- the bases underlined form part of the catalytic core of the assembled MNAzyme, bases in bold hybridise with the nucleic acid target and bases in italics hybridise to the substrate.
- the "-P" indicates 3' phosphorylation of the oligonucleotide.
- the reporter substrate for this example is SubBi-2 with the sequence, 5' to 3', as below.
- SubBi-2 was end-labelled with a 6-FAM moiety at the 5' end and a BHQl moiety at the 3' end and was designated SubBi-2-FB.
- Cleavage of SubBi-2-FB was monitored at 530nm (FAM emission wavelength) with excitation at 485nm (FAM excitation wavelength).
- the lower case bases represent RNA and the upper case bases represent DNA.
- Each reaction contained 0.04 ⁇ M 5RO5/2 and 0.2 ⁇ M of 3RO5/2, 10 mM MgCl 2 , 50 ⁇ M each dNTP (dATP, dCTP, dGTP, dTTP), Ix Immobuffer (Bioline), 0.2 ⁇ M SubBi-2-FB, Ix Rox reference dye (Invitrogen), 10 Units of Rnasin (Progema) and 1 Unit of Immolase Polymerase (Bioline) and 100 ng of genomic DNA.
- each reaction contained a pair of partzymes 0.2 ⁇ M of partzyme A and 0.2 ⁇ M of partzyme B (RPLPO Partzyme Pair A4:B5 or A5:B6 or A6:B7 or A7:B8 or A8:B9 or A9:B10).
- the Ct values are averaged from triplicate reactions, when the threshold fluorescence level was set at 0.2 and the baseline background fluorescence was subtracted between cycles 1 and 14.
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WO2013033792A1 (en) | 2011-09-09 | 2013-03-14 | Speedx Pty Ltd | Nucleic acid enzyme substrates |
WO2013123552A1 (en) * | 2012-02-20 | 2013-08-29 | Speedx Pty Ltd | Detection of nucleic acids |
WO2014200905A2 (en) | 2013-06-10 | 2014-12-18 | President And Fellows Of Harvard College | Early developmental genomic assay for characterizing pluripotent stem cell utility and safety |
US8945836B2 (en) | 2005-10-07 | 2015-02-03 | Speedx Pty Ltd | Multicomponent nucleic acid enzymes and methods for their use |
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Also Published As
Publication number | Publication date |
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US8962238B2 (en) | 2015-02-24 |
EP2142673B1 (en) | 2013-05-22 |
EP2142673A1 (en) | 2010-01-13 |
US20110143338A1 (en) | 2011-06-16 |
CA2682612A1 (en) | 2008-10-16 |
CA2682612C (en) | 2018-08-21 |
AU2008235256B2 (en) | 2014-05-15 |
JP2010523088A (en) | 2010-07-15 |
BRPI0809964A2 (en) | 2014-10-07 |
ES2425066T3 (en) | 2013-10-11 |
AU2008235256A1 (en) | 2008-10-16 |
CN101802216A (en) | 2010-08-11 |
MX2009010710A (en) | 2009-12-08 |
CN101802216B (en) | 2014-12-10 |
KR101583834B1 (en) | 2016-01-19 |
TW200902726A (en) | 2009-01-16 |
KR20100019429A (en) | 2010-02-18 |
NZ580129A (en) | 2012-05-25 |
HK1133680A1 (en) | 2010-04-01 |
IL201420A0 (en) | 2010-05-31 |
EP2142673A4 (en) | 2010-10-27 |
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