NZ622016B2 - Nucleic acid enzyme substrates - Google Patents

Abstract

Discloses an isolated polynucleotide substrate for a catalytic nucleic acid enzyme, said polynucleotide substrate comprising a sequence N1-N2-N3-N4-N5-N6-N7-N8-rR-rY-N9-N10-N11-N12-N13-N14-N15 wherein: rR is a purine ribonucleotide; rY is a pyrimidine ribonucleotide; each of N1-N15 are nucleotides; six or more of N5-N13 are cytosine nucleotides; N9 is a cytosine nucleotide; the pyrimidine ribonucleotide comprises uracil; and less than three of N9-N15 are guanine nucleotides. s; six or more of N5-N13 are cytosine nucleotides; N9 is a cytosine nucleotide; the pyrimidine ribonucleotide comprises uracil; and less than three of N9-N15 are guanine nucleotides.

Description

NUCLEIC ACID ENZYME SUBSTRATES INCORPORATION BY CROSS-REFERENCE This ation 'claims priOrity from Australian Provisional Patent Application No. 2011903686 filed on 9 September 2011, the entire content of which is incorporated herein by reference.

TECHNICAL FIELD The invention relates lly to the field of nucleic acid enzymes. More IO specifically, the invention relates to substrates for nucleic acid enzymes and s ing the substrates.

BACKGROUND OF THE INVENTION A wide variety of nucleic acid molecules with tic or catalytic activity have been discovered in the last '20 years. [RNA enzymes (f‘ribozymes”) occur in nature but can be engineered to specifically recognize and modify a target RNA substrate. In vitro evolution techniques have facilitated the discovery and development of many more catalytic nucleic acids, including deoxyribonucleic acids often referred to as '“deoxyribozymes”, f‘DNA‘ s” or “DNAzymes”. In vitro d DNAzymes and/or ribozymes have been discovered which have the capacity to catalyse a broad range of reactions including cleavage of nucleic acids, on of c acids, porphyrin metallation, and formation of carbon-carbon bonds, ester bonds or amide bonds.

' In particular, DNAzymes and ribozymes have been characterized which specifically cleave distinct nucleic acid sequences after hybridizing via Watson Crick base pairing.

DNAzymes are capable of ng either RNA or DNA molecules. Ribozymes are also able to cleave both RNA and DNA target sequences. The “10-23” and “8-17” DNAzymes are capable of cleaving nucleic acid substrates at specific RNA , phosphodiester bonds to create reaction ts which have 2’, 3’~cyclic phosphate and ’-hydroxyl groups. Examples of deoxyribozymes (DNAzymes), which can ligate 2’, 3’- 3O cyclic phosphate and 5’-hydroxyl products include the “7Z81” and “7248” ligases.

More recently, Multi-component Nucleic Acid enzmes (MNAzymes) have been described which have the capacity to self-assemble from two or more oligonucleotide components (also referred to herein as “partzymes”) in the presence of a MNAzyme assembly facilitator (e. g. a target molecule to be detected).

The versatile nature of catalytic nucleic acids has facilitated their use in many different applications. A key element to the successful use of catalytic nucleic acids is their capacity to modify an appropriate substrate. In general, the substrate is ntially complementary to the hybridizing arms of the catalytic nucleic acid and contains a c sequence or sequence motif at the site of catalytic action. The nature of the interaction between a given catalytic-nucleic acid and its substrate is determinative of how efficiently the enzyme engages and/or catalytically modifies its substrate, and is thus a fundamental consideration in ing any system that utilises catalytic nucleic acids.

Catalytic c acids have in vitro diagnostic applications in the detection of nucleic acids, proteins and small molecules. These applications often involve amplification of either the target or the signal to generate sufficient signal for robust detection of the analyte of interest.

Methods that employ catalytic nucleic acids require substrates that are modified with a sufficient rate of catalytic activity to allow effective discrimination over l5 - ound noise. Different methods may require the use of different on temperatures and so there is a ity for substrates that are efficiently modified (e.g. cleaved) at the required temperatures. Methods such as those utilizing es and DNAzymes permit multiplexed analysis of many targets simultaneously in a single reaction, but the ability to multiplex and distinguish between the multiple targets is 2.0 dependent on the nce of a suitable range of substrates, usually at least one per target.

The number of substrates known in the. art that are modified (e.g. cleaved) with high efficiency is currently insufficient for mass multiplexing.

The DNAzyme and MNAzyme substrates previously known in the art were derived by screening multiple le substrates to cally determine those that were cleaved most efficiently. Often this screening was performed using large numbers of DNAzymes targeted to cleave theoretically possible cleavage sites within full length mRNA. This screening was y performed under logical conditions (temperature and ionic strength, composition and pH of buffers). This bias towards finding ntly cleaved sequences of mRNA at physiological conditions exists because such studies were focused on therapeutic uses of es as inhibitors of RNA expression in vivo. Such studies provide a range of laborious protocols for empirical measurement of a large number of putative substrates tofind the few that are cleaved efficiently (see for example Cairns et al., 1999 Nat Biotech 172480486). These studies resulted in a limited set of design guidelines for the selection of ntly cleaved substrates, and in many cases the guidelines focused on the design of the DNAzyme rather than the substrate as the DNAzyme can be easily adjusted and the mRNA cannot. One common guideline generated from these studies is that the exact sequence of the R-Y ribonucleotide motif at the cleavage site of the substrate is important with cleavage efficiency being in the following order: GU ≥ AU > GC >>> The efficiency of cleavage of a full length mRNA under in vitro conditions is not an absolute measure of the cleavage efficiency in a cellular environment as the latter includes ribonuclear proteins and other confounding factors that cannot be easily mimicked in vitro.

The design guidelines ted in the past have some use in selection of sites within a long mRNA molecule that may be efficiently cleaved by DNAzymes and MNAzymes under physiological conditions, but have limited ability to predict which substrates will be d with ient efficiency for utility in in vitro diagnostic applications. In vitro diagnostic applications may require conditions very different from the physiological conditions generally screened and used to establish the limited substrate design guidelines that exist in the art.

There is a need for a set of guidelines, or sequence motifs, for ate sequences that predict with r certainty if a substrate will be efficiently cleaved by a MNAzyme or DNAzyme in conditions le for in vitro diagnostic ations. There is also a need for catalytic nucleic acid ates with properties that facilitate improved catalytic nucleic acid function. These properties may include, for example, an ability to facilitate improved catalytic nucleic acid function over a range of conditions and/or a capacity to extend the number of targets that can be simultaneously ed in a multiplex reaction.

Y OF THE INVENTION While many have attempted to establish a sequence motif or set of design guidelines which consistently produces efficient substrate sequences, to date no ive sequence or set of guidelines has been fied. The present invention provides a series of principles which has facilitated the development of new efficiently d substrates. The present invention thus provides catalytic nucleic acid enzyme substrates with properties that enhance catalytic nucleic acid function thereby addressing a need existing in the art.

The present invention provides an ed polynucleotide substrate for a catalytic nucleic acid enzyme, said polynucleotide ate comprising a sequence N1-N2-N3-N4-N5-N6-N7-N8- rR-rY-N9-N10-N11-N12-N13-N14-N15 wherein: rR is a purine ribonucleotide; rY is a pyrimidine ribonucleotide; each of N1-N15 are nucleotides; six or more of N5-N13 are ne nucleotides; 11013114_1 N9 is a cytosine nucleotide; the pyrimidine ribonucleotide comprises uracil; and less than three of N9-N15 are guanine nucleotides In a first aspect, the present invention provides an isolated cleotide substrate for a catalytic nucleic acid enzyme, said polynucleotide substrate comprising a sequence N3- N4-N5-N6-N7-N8-rR-rY-N9-N10-N11-N12-N13-N14-N15 wherein: rR is a purine ribonucleotide; 11013114_1 rY is a pyrimidine ribonucleotide; each of Nl-le are nucleotides; six or more of N5-N13 are cytosine nucleotides; and less than three of N9-N15 are e nucleotides.

In one embodiment of the first aspect, the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 25-27, 29-30, 33, 72-90, or 172-175. ‘ In one embodiment of the first aspect, seven or more, or eight or more of N5-N13 are cytosine nucleotides.

' In one embodiment of the first aspect, seven or more of N5-N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by anyone of SEQ ID N03: 29, 73, 76—80, 82-83, 85-90, or 172-175.

In one embodiment of the first aspect, eight of Ns-NB are cytosine nucleotides and the polynucleotide ate comprises or ts of a sequence defined by any one of SEQ ID N03: 76, 77, 80, 83 or 87.

In one embodiment of the first , seven or more, or eight or more of N4-Ni3 are ‘ cytosine nucleotides.

In one embodiment of the first aspect, seven or more of N4-N13 are cytosine nucleotides and the polynucleotide ate-comprises ‘of a sequence defined by any one of SEQ ID NOS: 27, 29, 73, 76-83, 85-90, or 172-175.

In one embodiment of the first aspect, eight of N4-N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 76, 77, 79-80, 82-83, 87, 88 or 90.

In one embodiment of the first aspect, six or more, seven ormore, or eight or more Nn are cytosine nucleotides.

In one embodiment of the first aspect, seven or more of N4-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID N05: 27, 29, 73, 76, 77, 79-83, 85-88, 90 or 172-175.

In one ment of the first aspect, eight of N4-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consiSts of a sequence defined by any one of SEQ ID N03: 76, 77, 80, 83, 87, or 88.

In one embodiment of the first aspect, six or more, seven or more, or eight or more ofN5-N12 are cytosine nucleotidesf ' In one embodiment of the" first. aspect, seven or more of N5-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 29,73, 76, 77, 80, 83, 85-88 or 172-175.

In one embodiment of the first aspect, eight of Ns-le are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by anyone of SEQ ID N05: 76, 77, 80, 83, or 87.

In one embodiment of the first , any one or more of N1, N2, N3 and/or N9 is a cytosine nucleotide.

In one embodiment of the first aspect, N3 and N9 are cytosine nucleotides, and the polynucleotide substrate ses or ts of a sequence defined by any one of SEQ ID NOs: 25-26, 2930, 72-90, or 172-175.

In one embodiment of the first aspect, two, one or none of N9—N15 are guanine nucleotides.

In one embodiment of the first aspect, one or none of Ng-le are guanine IS nucleotides and the polynucleotide ate comprises or consists of a sequence defined by any one of SEQ ID NOS: 25-27, 29-30, 33, 72—80, 82-90, or 172-175.

In one embodiment of the first aspect, none of Ng-le are guanine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of - SEQ ID N08: 25, 26, .30, 33, 72, 75, 77-80, 84-85, or 89.

In one embodiment of the first aspect, more than ten, more than , more than twelve, or more than thirteen ofNI-le are pyrimidine tides.

In one embodiment of the first , eleven, twelve, or more than twelve of Ni- N15 are pyrimidine nucleotides and the cleotide substrate comprises or consists of a sequence defined by any one SEQ ID NOS: 25-27, 29, 33, 73-90 or 173-175.

‘ In one embodiment of the first aspect, thirteen or fourteen of Nl-Nl's are pyrimidine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one SEQ ID N05: 75, 77-80, 82-85 or 88—89.

In one embodiment of thefirst aspect, more than eight, more than nine, more than ten, or eleven ofNl-NM are cytosine nucleotides.

In one embodiment of the first aspect, ten or eleven of Nl-NM are cytosine nucleotides and the polynucleotide ate comprises or consists of a sequence defined by any one SEQ ID NOS: 3'3, 76-80, 82-83, 85, 87, 88, or 89.

In one embodiment of the first aspect, eleven of N1'-N14 are ne nucleotides and the polynucleotide ate comprises or consists of a sequence defined by any one SEQ ID NOS: 77-79.

In one embodiment of the first , the polynucleotide ate further comprises a detectable label for detecting the polynucleotide substrate.

In one embodiment of the first aspect, the polynucleotide substrate further comprising a detectable portion and a quencher portion, wherein a detectable effect provided by the detectable portion is increased or decreased upon modification of the polynucleotide ate by said catalytic nucleic acid enzyme.

In one embodiment ofthe first aspect, the purine ribonucleotide comprises guanine.

In one embodiment ofthe first aspect, the pyrimidine ribonucleotide comprises uracil.

In one embodiment of the first aspect, a portion of the polynucleotide substrate that binds to said catalytic nucleic acid enzyme has a melting temperature (Tm) of between 50°C and 90°C, n 50°C and 65°C, betyVeen 50°C and 60°C, between 52°C and, 58°C, between 66°C and 76°C, between 68°C and 76°C, between 64°C and 70°C, between 70°C and 76°C, between 70°C and 75°C, between 72°C and 76°C, 52°C, 58°C, 64°C, 66°C, I5 68°C, 70°C, 72°C, or 76°C.

' In one embodiment of the first aspect, the catalytic nucleic acid enzyme is: (i) a multi-component nucleic acid enzyme me) and said portion binds to at least one ate arm of said MNAzyme; or (ii) a DNAzyme.

In one ment of the first aspect, the polynucleotide substrate is e of catalytic ation by an MNAzyme.

In one embodiment of the first aspect, the polynucleotide substrate is capable of catalytic ation by a DNAzyme.» In one embodiment of the first aspect, the polynucleotide substrate comprises a able label for detection by fluorescence spectroscopy, surface plasmon resonance, mass spectroscopy, NMR, electron spin resonance, polarization fluorescence spectroscopy, circular 'dichroism, immunoassay, chromatography, radiometry, electrochemical, etry, scintigraphy, onic methods, UV, visible light or infra- red spectroscopy, enzymatic methods, or any combination thereof.

In one embodiment of the first aspect, the polynucleotide-substrate comprises a detectable label for ion by fluorescence spectroscopy.

In one embodiment of the first aspect, the polynucleotide substrate comprises a detectable label for detection by Fluorescence Resonance Energy Transfer (FRET) spectroscopy.

In a second aspect, the present invention provides an ed polynucleotide substrate for a catalytic nucleic acid enzyme, said polynucleotide substrate comprising or ting of a sequence defined by SEQ ID NO: 28.

In one embodiment of the second aspect, the catalytic nucleic acid enzyme is an MNAzyme comprising a pair of oligonucleotide partzymes, said pair comprising or consisting of SEQ ID N03: 15 and 8, SEQ ID N05: 93 and 94, or SEQ ID N03: 114 and 115. ' In one ment of the second aspect, the catalytic nucleic acid enzyme is a DNAzyme comprising or consisting of a sequence defined by SEQ ID NO: 138.

In a third aspect, the present invention provides a method for detecting the ce of at least one target comprising: (a) providing two or more oligonucleotide partzymes, wherein at least a first oligonucleotide me and a second oligonucleotide partzyme self—assemble in the presence of said target to form at least a first catalytically active multi-component nucleic acid enzyme me); (b) providing the isolated polynucleotide substrate of the first or second aspect, wherein said polynucleotide substrate is capable of being modified by said first ”MNAzyme, 'wherein said modification of said polynucleotide substrate by said MNAzyme‘provides a detectable effect; (c) contacting said two or more oligonucleotide partzymes with a sample vely ning said target under conditions permitting: (1) the self-assembly of said at least first MNAzyme, and (2) the catalytic activity of said at least first MNAzyme; and (d) detecting said detectable effect.

In one embodiment of the third aspect, the , is an MNAzyme assembly facilitator.

In one embodiment of the third aspect, the target is a nucleic acid.

In one embodiment of the third aspect, the target is a c acid that hybridizes to one or more sensor arms of said MNAzyme by base pair complementarity.

In one embodiment of the third aspect, the nucleic acid is selected from the group consisting of DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, tRNA, mRNA, snoRNA, stRNA, stNA, pre- and pri— . microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons, or any ation thereof.

In one embodiment of the third aspect, the nucleic acid is amplified.

In one embodiment of the third aspect, the cation comprises one or more of: polymerase chain reaction (PCR), strand displacement amplification (SDA), loop- mediated isothermal cation (LAMP), rolling circle - amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (35R), nucleic acid sequence based amplification (NASBA), or reverse transcription polymerase chain reaction (RT-PCR).

In one embodiment of the third aspect, the cleotide substrate hybridises with substrate arms of said MNAzyme at a temperature of between 50°C and 90°C, between 50°C and 65°C, between 50°C and 60°C, between 52°C and 58°C, between 66°C and 76°C, between d 76°C, between 64°C and 70°C, between 70°C and 76°C, between 70°C and 75°C, between 72°C and 76°C, 52°C, 58°C, 64°C, 66°C, 68°C, 70°C, 72°C, or 76°C.

In one embodiment of the third aspect, the method further comprises providing: (a) two or more additional oligonucleotide partzymes capable of self-assembling in the presence of a ent target to form a second catalytically active MNAzyme; and IS (b) at least one additional polynucleotide substrate; v wherein said additional polynucleotide substrate is capable‘of being modified by said second e in the presence of said different target.

In one embodiment of the third aspect, the additional polynucleotide substrate is not capable of being modified by said first MNAzyme.

‘ 'In one embodiment of the third aspect, the ing in part (d) comprises use of .fluorescence spectrosCopy, surface plasmon resonance, mass spectroscopy, NMR, electron spin resonance, polarization fluorescence spectroscopy, circular dichroism, immunoassay, tography,» etry, ochemical, photometry, graphy, electronic methods, UV, visible light or infra-red spectroscopy, enzymatic, methods, or any ation thereof.

In one embodiment of the third aspect, the ing in part (d) comprises use of fluorescence spectroscopy.

In one embodiment of the third aspect, the detecting in part ((1) comprises detection ,of a FRET detectable effect. ‘ In one embodiment of the third aspect, the catalytic core of said e comprises DNA or an analogue thereof.

In a fourth aspect, the present ion provides use of the isolated polynucleotide substrate of the first or second aspect as a substrate for a catalytic nucleic acid enzyme. ' In one embodiment of the fourth aspect, the catalytic nucleic acid enzyme is a multi-component nucleic acid enzyme (MNAzyme), said MNAzyme comprising at'least two or more oligonucleotide partzymes wherein at least a first oligonucleotide me and a second oligonucleotide partzyme self- assemble in the ce of an MNAzyme assembly facilitator to form a catalytically active component nucleic acid enzyme (MNAzyme), wherein each of said at least first and said second oligonucleotide partzymes 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 partzymes act as sensor'arms of the MNAzyme, the substrate arm n of the first and second oligonucleotide partzymes act as substrate arms of the MNAzyme, and the catalytic core portion of the first and second oligonucleotide partzymes act as a catalytic core of the e; and wherein the sensor arms of the MNAzyme interact with said MNAzyme assembly facilitator so as to maintain the first and second oligonucleotide partzymes in proximity for association of their respective catalytic core portions to form the catalytic core of the e, said catalytic core capable of modifying said polynucleotide substrate, and wherein said substrate arms of said e engage said cleotide substrate so that said catalytic'core of said MNAzyme can modify said polynucleotide substrate.

In one embodiment Of the third or fourth aspect, the catalytic core portion of each said oligonucleotide partzyme comprises DNA or an analogue thereof.

In one embodiment of the fourth aspect, the assembly tator is a target to be identified, detected or tated. * In one embodiment of the third or fourth aspect, the first and second oligonucleotide partzymes comprise respective sequences defined by: SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO:. 16 and SEQ ID NO: 14; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 40 and SEQ ID NO: 41; SEQ ID NO: 42 and SEQ ID NO: 43; SEQ ID NO: 44 and SEQ ID NO: 45; SEQ ID NO; 46 and SEQ ID NO: 45; SEQ ID INC: 47 and SEQ ID NO: 63; SEQ ID NO: 48 and SEQ ID NO: 49; SEQ ID NO: 50 and.

SEQ ID NO: 51; SEQ ID NO: 52 and SEQ ID NO: 51; SEQ ID NO: 38 and SEQ ID NO: 55; SEQ ID NO: 56 and SEQ ID NO: 57; SEQ ID NO: 58 and SEQ ID NO: 59; SEQ ID NO: 60. and SEQ ID NO: 61; SEQ ID NO: 62 and SEQ ID NO: 63; SEQ ID NO: 64 and SEQ ID NO: 65; SEQ ID NO: 66 and SEQ ID NO: 67; SEQ ID NO: 62 and SEQ ID NO: 68; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 46 and SEQ ID NO: 55; SEQ ID NO: 46 and SEQ ID NO: 59; SEQ ID NO: 38 and SEQ ID’ N03 45; SEQ ID NO: 58 and SEQ ID NO: 45; SEQ ID NO: 62 and SEQ ID NO: 45; SEQ ID NO: 46 and SEQ ID NO: 63; SEQ ID NO: 71 and SEQ ID‘NO: 68; SEQ ID NO: 98 and SEQ ID NO: 99; SEQ ID NO: 100 and SEQ ID NO: 103; SEQ ID NO: 104 and SEQ ID NO: 105; SEQ ID NO: 106 and SEQ ENG: 107; SEQ ID NO: 108 and SEQ ID NO: 109; SEQ ID NO: 110 and SEQ ID NO 111; SEQ ID NO: 112 and SEQ ID NO: 113; SEQ ID NO: 116 and SEQ ID NO: 117; SEQ ID NO: 118 and SEQ ID NO: 119; SEQ ID NO: 120 and SEQ ID NO: 121; SEQ ID NO: 122 and SEQ ID NO: 119; SEQ ID NO: 155 and SEQ ID NO: 156; SEQ ID NO: 157 and SEQ ID NO: 158; SEQ ID NO: 159 and SEQ ID NO: 160; SEQ ID NO: 168 and SEQ ID NO: 169; SEQ ID NO: 179 and SEQ ID NO: 180; SEQ ID NO: 181 and SEQ ID NO: 182; SEQ ID NO: 183 and SEQ ID NO: 184 or SEQ ID'NO: 185 and SEQ ID NO: 186.

In. one embodiment of the third or fourth aspect, said oligonucleotide substrate and said first and second oligonucleotide partzymes are defined by a combination of sequences as set forth in Table 6, 8, 10, 13, 16, 20, 22 24.

In one embodiment of the fourth aspect, the catalytic nucleic acid enzyme is a DNAzyme, and the DNAzyme and oligonucleotide substrate are defined by a combination of sequences as set forth in Table 1.5.

In one embodiment of the fourth aspect, the target is a nucleic acid that hybridizes to one or more sensor arms of said MNAzyme by base pair complementarity.

In one embodiment of the fourth aspect, the polynucleotide substrate hybridises with said catalytic c acid enzyme at a temperature of between 50°C and 90°C, between 50°C and 65°C, between 50°C and 60°C, between 52°C and 58°C, between 66°C and 76°C, between 68°C and 76°C, between 64°C and 70°C, between 70°C and 76°C, between 70°C and 75°C, between 72°C and 76°C, 52°C, 58°C, 64°C, 66°C, 68°C, 70°C, 72°C, or 76°C.

In a fifth aspect, the present invention provides a kit sing'the ed polynucleotide substrate of the first or second aspect.

In one embodiment of the fifth aspect, the kit further comprises a catalytic nucleic acid enzyme capable of catalytically ing said cleotide substrate.

In one embodiment of the fifth aspect, the catalytic nucleic acid enzyme is a multi- component nucleic acid enzyme (MNAzyme).

In a sixth aspect, the present invention provides a kit sing the isolated polynucleotide substrate of the first or second aspect and a plurality of oligonucleotide partzymes designed to assemble a multi-component nucleic acid enzyme (MNAzyme) capable of detecting at least one target, wherein said MNAzyme is capable of catalytically modifying the cleotide substrate.

In one embodiment of sixth aspect, said oligonucleotide substrate and Said plurality of oligonucleotide partzymes are defined by a combination of sequences'as set forth in Table 6,8, 10, 13, 16, 20, 22 and/or 24.

In a Seventh aspect, the present invention provides an assembly comprising a solid ' support bound to a polynucleotide substrate of the first‘or second .

In one embodiment of the first or third to seventh aspects, any one or more of N1- are deoxyribonucleotides.

, N15 In one embodiment of the first or third to seventh aspects, any one or more of N1- N15 are ribonucleotides.

In one ment of the first or third to seventh s, all of Nt-le are deoxyribonucleotides.‘ I In one embodiment of the first or third to seventh s, all of Nl—le are ribonucleo'tidesf In one embodiment of the first or third to seventh aspects, .Ng—le comprises a mixture of deoxyribonucleotides and ribonucleotides.

In one embodiment of the seventh aspect, the assembly comprises a ity of different sOlid supports bound to a plurality of different cleotide substrates.

In one embodiment of the first, second, fourth, or fiflh'aspect, the catalytic nucleic acid enzyme is a DNAzyme.

In one embodiment of the first, second, fourth, or fifth aspect, the tic c acid enzyme is a ribozyme.

In one embodiment of the first, second, fourth, or fifth aspect, the catalytic nucleic acid enzyme is a multi-component nucleic acid enzyme (MNAzyme).

In one embodiment of the above aspects, the catalytic nucleic acid enzyme is capable of modifying the cleotide substrate by cleavage.

In one embodiment of the first, second, or fifth aspect, the catalytic nucleic acid enzyme is an MNAzyme comprising first and second oligonucleotide partzymes, and said ucleotide substrate and said first and second oligonucleotide partzymes are defined by a combination of sequences as set forth in Table 6, 8, 10, 13, 16, 20, 22 and/or 24.

In one embodiment [of the first, second, or fifth aspect, the catalytic nucleic acid enzyme is a DNAzyme, and the DNAzyme and oligonucleotide substrate are defined by a combination of sequences as set forth in Table 15.

In one embodiment of the first, second, or fifth aspect, the polynucleotide substrate is capable ridising to said catalytic nucleic acid enzyme by complementary base pairing.

In one embodiment of the fourth aspect, the polynucleotide substrate hybridises to said catalytic nucleic acid enzyme by complementary base pairing.

In one embodiment of the third and fourth aspect, the polynucleotide substrate hybridises to said MNAzyme by mentary base pairing, In one embodiment of the first, second, fifth, and sixth , the polynucleotide substrate the polynucleotide substrate is capable of hybridising to said MNAzyme by complementary base pairing.

In one embodiment of the above aspects, a portion of the isolated polynucleotide substrate binds to at least one substrate arm of said MNAzyme.

In one embodiment of the above aspects, the polynucleotide substrate is a universal substrate e of being bound and tically modified by more than one different type of catalytic nucleic acid enzyme.

In one embodiment of the above aspects, the polynucleotide substrate is a universal substrate capable of being bound and catalytically modified by more than one different type of multi—component nucleic acid enzyme (MNAzyme).

In one embodiment of the above aspects, at least one of said oligonucleotide partzymes, assembly facilitator or substrate comprises DNA or'an ue thereof.

In one embodiment of the third and sixth aspect, the modifying is cleavage of the polynucleotide substrate by the e.

BRIEF DESCRIPTION OF THE FIGURES Preferred embodiments of the t invention will now be described, by way of -_ example only, with reference to the accompanying figures wherein: Figure 1 is a m ing an exemplary design of a Multi-component nucleic acid (MNAzyme). By way of ary disclosure, a MNAzyme is comprised of two oligonucleotide components (partzyme A and partzyrne B), which self assemble in the presence of an assembly facilitator. When the two partzymes assemble in the ce of the assembly facilitator, a catalytically active MNAzyme forms whiCh is capable of modifying (e.g. cleaving or ng) 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. The presence of an assembly tator molecule (e. g. a target nucleic acid sequence) provides the “input” signal which directs the assembly of partzyme ents in a highly specific n which is amenable to tion. In some embodiments, the assembly facilitator may be, for example, a target nucleic acid sequence present in a test sample. In other embodiments, the assembly facilitator may be, for example a synthetic ucleotide 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 ate by the led MNAzyme can provide a “detectable effect” which may be detected and/or quantified. For e, when the substrate is dual labelled with a fluorophore (F) and a quencher (Q), cleavage of this “reporter substrate” by an active MNAzyme separates the fluorophore and the io quencher resulting in a concomitant increase in fluorescence.

Figure 2 provides a flow chart showing exemplary applications of methods for target detection using MNAzymes. es can be used for (1) direct detection; (2) detecting amplicons generated, for example, by PCR, SDA, LAMP, RCA, TMA, 38R or NASBA either during, or following, target amplification; and (3) initiating a signal amplification e.

Figure 3 provides a depiction of exemplary MNAzymes and a method for target detection using MNAzymes that cleave substrates tethered to a t. In this embodiment, the MNAzyme forms only in the presence of an assembly facilitator (target). When the MNAzyme cleaves the tethered substrate between a fluorophore and quencher, a signal is generated. As shown here, upon ge between fluorophore F and quencher Q, there is a ant increase in fluorescence. In general, the method may be designed such that either fluorophore F or quencher Q'may stay attached to the support once ge occurs, Panel (i): The support shown has only one substrate type (Substrate l) tethered to it. Panel (ii): There may be multiple substrate types tethered in different positions. Each substrate can‘ be cleaved only by a MNAzyme formed in the presence of a specific MNAzyme assembly facilitator molecule — here,vTargets I and 2 facilitate the self-assembly of MNAzymes 1 and 2 respectively. Thus, in this e MNAzyme 1 only self-assembles in the presence of Target 1 and only cleaves Substrate 1. Similarly, MNAzyme 2 only self-assembles in the presence of Target 2 and only cleaves Substrate 2. The signal can be localised by positioning of the substrate on the e, thus allowing specific detection of different assembly facilitators. The exemplary assay in Panel (ii) es two distinct substrate sequences.

Figure 4 shows an exemplary assay using catalytically modified substrate products as MNAzyme assembly facilitator components. In this strategy an initiating MNAzyme (Mt) is formed in the presence of a target (T). The initiating MNAzyme (Mt) cleaves a first substrate (SI) to create a first assembly facilitator component (Slf), which directs ion of a-first cascading MNAzyme (cascading MNAzyme Mel). In this example the first cascading MNAzyme (Mel) comprises two (partzymes and three assembly facilitator ents designated F1, F2 and Slf. Mcl can cleave an additional substrate (82) thus liberating an additional assembly facilitator component (S2f), which directs formation of a second cascading MNAzyme ding MNAzyme Mc2); In this example the second cascading MNAzyme (M02) comprises two partzymes and three assembly tator components designated F3, F4 and 82f. M02 can then cleave more of the first substrate (81) thus creating more of the first ly facilitator component (Sit). This leads to the formation of further first cascading MNAzyme (Mcl) thereby forming an amplification cascade. This exemplary assay requires two distinct substrate sequences.

Figure 5 provides a graph rating Ct values generated by e qPCR using a range of different universal substrates for detection of the human TFRC gene. The identity of the universal substrates used in reactions are indicated on the x-axis (where “2” refers to Sub2, “3” refers to Sub3 etc.). The same primer set and genomic DNA were used for all reactions, and all partzymes had the same target—sensor regions and catalytic . domains: The only differences between reactions were thesubstrate-sensor arms of the partzyrnes and the fluorescently labelled universal substrates. Thus the difference in Ct values between reactions is correlated with efficiency of cleavage of the universal ates. Lower Ct values indicate a faster number of cycles to achieve a threshold leVel of fluorescence, and ore indicate more efficiently cleaved universal ates.

Figure 6 es graphs illustrating the signal to noise ratios resulting from MNAzyme mediated cleavage of a range of universal substrates in an isothermal format.

Target was a synthetic oligonucleotide. The signal to noise ratio was calculated from normalized fluorescence data ted during cleavage reactions at a range of reaction temperatures. The identity of the universal substrates used in reactions are indicatedon the X-axis (where “2” refers to Sub2, “3” refers to Sub3 etc.). In Figure 6 (i) the different columns of data refer to s for different reaction temperatures (as indicated in the legend 52°C,l54°C, 56°C and 58°C). Figure 6 (i) illustrates the signal to noise ratio on the" y-axis. Figure 6 (ii) illustrates the standard deviation of the e of the signal to noise ratio from all four temperatures tested and the different series of substrates are ted by different g of columns.

Figure 7 provides graphs illustrating linear amplification plots generated by MNAzyme qPCR using a range of different universal substrates for detection of "a range - of human genes. Each combination of substrates and targets was run at an annealing temperature of a) 52°C or b) 58°C. The universal substrates tested with each gene are indicated by symbols d at the top left of each plot and were (i) Sub3 and Sub6I with CYP2C9, (ii) Sub6, Sub72, Sub74 and Sub79, with TP53, (iii) Sub60, Sub6l and Sub79 with B2M, (iv) Sub49 and Sub75 with HMBS, (v) Sub2, Sub72 and Sub80 with TFRC and (iv) Sub55, Sub80 and Sub88 with RPL13a. The same primer set and genomic DNA were used for all reactions with each different gene, and all partzymes had the same catalytic domains and target-sensor regions matched to the particular gene. The only differences between reactions were the substrate-sensor arms of the partzymes and the IO fluorescently labelled universal substrates. Thus the difference in the shape of the amplification plot and the Ct values n reactions for the same gene is correlated with efficiency of cleavage of the universal substrates. The steeper the curves and the earlier Ct values indicate a faster number of cycles to achieve a threshold fluorescence, and therefore indicate more efficiently cleaved universal ates.

Figure 8 provides graphs illustrating the signal to noise ratio resulting from DNAzyme ed cleavage of a range of universal substrates in an isothermal format.

The signal to noise ratio was calculated from normalized fluorescence data collected during cleavage reactions at a range of reaction temperatures. The identity of the universal substrates used in reactions is ted on the x’axis (where “2” refers to Sub2, “3” refers to Sub3 etc.). In Figure 8 (i) the different columns of data refer to s for different reaction temperatures (as indicated in the legend. — 50°C to 60°C) with the signal to noise ratio on the y-axis. In Figure 8 (ii) the columns of data refer to the standard deviation of the average of the signal to noise ratio from all. six atures tested and the different series of substrates are indicated by different shading of columns.

Figure 9 provides graphs illustrating linear amplification plots from MNAzyme qPCR‘ med with a range of different sal substrates (Sub44, SubSS, Sub61, Sub65, Sub72 and Sub74) to investigate non-specific cleavage activity with a subset of partzymes (designed with substrate sensor arms to be mentary to Sub44, SubSS, Sub72 and Sub74) for ion of the human RPL13a gene. Each universal substrate Was tested individually with partzyme pairs designed to bind with full complementarity to the other substrates to determine if a signal could be detected. The partzymes complementary to (i) Sub72, (ii) Sub74, (iii) SubSS and (iv) Sub44, were tested with all substrates. In Panel (a) the bottom line of the table indicates the ate to which the partzymes in the experiment exhibit full complementarity. The other rows of the table show an alignment of the sequences of the other substrates tested in each experiment with differences between the bottom substrate and other ates sequences indicated by letters that are grey and underlined. In Panel (b) linear amplification plots are illustrated.

The normalised fluorescence (y—axis) is plotted against the cycle number (x-axis). dual amplification curves are labelled on the right of the plot to indicate which cation curve relates to each substrate. The threshold fluorescence is ted by a solid horizontal line above the x-axis. An increasing signal above the threshold fluorescence indicates ge of the sal substrate.

Figure 10 provides graphs illustrating the signal to noise ratio resulting from DNAzyme mediated cleavage of each universal substrate individually with DNAzymes designed to bind with full complementarity to the other substrates, in an isothermal format. The normalized signal to noise ratio was calculated from normalized fluorescence data collected at reaCtion temperatures of (i) 52°C or (ii) 58°C: The identity of the sal substrates used in the reactions is indicated on the x-axis. The different columns of data refer to the cleavage s for each DNAzyme as indicated in the legend, with the signal to noise ratio on the y-axis.

Figure 11 illustrates ntial amplification plots generated with amplification at either 52°C (Panel (i)) or 58°C (Panel (ii)) for two multiplex MNAzyme qPCR reactions Multiplex 1 using series 1 substrates (Sub2, Sub3, Sub4, Sub6 and Sub7) and Multiplex 2 using series 2 and 3 substrates , Sub6l, Sub74, Sub79 and Sub80). Both multiplexes measured the human genes TFRC, HPRT, TP53, RPL13a and CYP2C9 in a single reaction vessel. For each different gene measured in the two multiplex formats the same primer sets and genomic DNA were used at both temperatures and for all universal substrates, and all partzymes had the same catalytic domains and target-sensor regions matched to the particular gene. The only difference between reactions was the substrate- sensor arms of the partzymes and the fluorescently labelled universal substrates. The universal substrates tested with each gene are indicated at the top left of each plot. The ication plots for Multiplex 1 (crosses) and Multiplex 2 (circles) were overlayed to allow for a direct comparison n the two multiplexes at two different DNA trations, 100 ng (cross and circle plot on the left) and 391 pg (cross and circle plot on the right). The difference in the shape of the amplification plots correlated with efficiency of cleavage of the universal substrates. The steeper the curves and the earlier Ct values indicate a faster number of cycles to achieve a threshold fluorescence, and therefore indicate more efficiently cleaved universal substrates.

TIONS Certain terms are used herein which shall have the meanings set forth as follows.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polynucleotide substrate” also-includes a plurality of polynucleotide substrates.

The term “comprising” means ding principally, but not necessarily ”.

Furthermore, variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings.

Use of the term “about” herein in reference to a recited numerical value includes the recited cal value and numerical values within plus or minus ten percent of the d value.

Use of the term “between” herein when referring to a range of cal values encompasses the numerical values at each endpoint of the range. For example, a polynucleotide of between 10 nucleotides and 20 nucleotides in length is inclusive of a IS polynucleotide of 10 nucleotides in length and a polynucleotide of 20 nucleotides in length.

The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a single- or double-stranded polymer of deoxyribonucleotide and/or ribonucleotide bases, and/or analogues, derivatives, variants, fragments or combinations thereof, including but not limited to DNA, ated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, stNA, pre- and pri-microRNA, other ding RNAs, mal RNA, derivatives thereof, amplicons thereof or any combination'thereof. By way of non-limiting example, the source of a nucleic acid may be selected fiom the group consisting of tic, mammalian, human, , plant, fungal, bacterial, viral, archael sources or any combination thereof.

The term “oligonucleotide” typically denotes a segment of DNA or a DNA- containing nucleic acid molecule, Or RNA or RNA-containing molecule, or a combination thereof. An oligonucleotide may thus comprise or consist of deoxyribonucleotide and/or ‘30 ribonucleotide bases, and/or analogues, derivatives, variants, fragments or combinations thereof, ing but not limited to DNA, methylated DNA, alkylated DNA, RNA, , methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, stNA, pre- and pri—microRNA, other non-coding RNAs, ribosomal RNA, tives thereof, amplicons thereof or any combination thereof. Examples of 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 MNAzymes including, but not limited to partzymes,‘ and assembly facilitators.

The term “pyrimidine nucleotide” encompasses any tide comprising a pyrimidine base including, but not d to, cytosine, thymine and uracil. A pyrimidine nucleotide may se a ribose sugar molecule (ie. a “pyrimidine ribonucleotide”) or a deoxyribose sugar molecule (ie. a “pyrimidine deoxyribonucleotide”).

The term “purine nucleotide” encompasses any nucleotide comprising a purine base including, but not limited to, adenine and guanine. A purine nucleotide may comprise a ribose sugar molecule (ie. a e ribonucleotide”) or a deoxyribose sugar molecule (ie. a e deoxyribonucleotide”).

The terms “nucleic acid enzyme”, “catalytic nucleic acid”, “nucleic acid with catalytic activity”, and “catalytic nucleic acid enzyme” are used herein interchangeably and shall mean a DNA or DNA-containing molecule or x or an RNA or RNA- containing le or complex, or a combination thereof being a DNA-RNA hybrid molecule or complex, which may bind 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. The terms above include uni-molecular c acid enzymes which may comprise a single DNA or DNA-containing le (also knoWn in the art as a “DNA enzyme”, “deoxyribozyme” or “DNAzyme”) or an RNA or RNA—containing molecule (also known in the art as a “RNA enzyme” or “ribozyme”) or a combination thereof, being a DNA-RNA hybrid molecule which may ize at least one substrate and catalyse a ation (such as ligation or cleavage) of the at least one substrate. The terms above e 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. The terms “nucleic acid enzyme”, “catalytic nucleic “nucleic acid with catalytic activity”, and “catalytic nucleic acid enzyme” include within their meaning MNAzymes.

The terms “MNAzyme” and “multi-component nucleic acid enzyme” as used herein have the same g and refer to two or more ucleotide sequences (cg. 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 ons including cleavage of a ate, on of ates and other enzymatic modifications of a ate 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”. With reference to Figure l, partzymes A and B each bind to an assembly facilitator (e.g. a target DNA or RNA ce) 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 e, formed by the interaction of the catalytic domains of partzymes A and B. Cleavage of a DNA/RNA chimeric reporter ate is exemplified in the drawing. The MNAzyme cleaves the substrate between a fluorophore and a quencher dye pair, thus generating signal. The terms “multi— component nucleic acid ” and “MNAzyme” 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.

It will be understood that the terms “MNAzyme” and “multi-component nucleic acid enzyme” as used herein ass all known MNAzymes and modified MNAzymes including those disclOsed in any one or more of BCT patent publication numbers WO/2007/041774, WO/2008/040095, W02008/122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011-0143338 (the contents of each of these documents are orated herein by reference in their entirety). Non— limiting examples of MNAzymes and modified MNAzymes encompassed by the terms “MNAzyme” and “multi-component nucleic acid enzyme” include MNAzymes with cleavage tic activity (as exemplified herein), disassembled or lly assembled MNAzymes comprising one or more assembly inhibitors, es comprising one or more aptamers (“apta-MNAzymes”), MNAzymes comprising one or more truncated sensor arms and optionally one or more stabilizing ucleotides, MNAzymes comprising one or more activity inhibitors, multi-component nucleic acid inactive proenzymes (MNAi), and MNAzymes with ligasev catalytic activity (“MNAzyme ligases”), each of which is described in detail in one or more of 7/041774, WO/2008/040095, W02008/122084, US 2007-0231810, US 2010—0136536, and/or US 2011-0143338.

As used , the terms “partzyme”, “component partzyme”, “partzyme component”, “component oligonucleotide”, “oligonucleotide component” and nucleotide partzyme” refer to a DNA—containing or RNA-containing or DNA- RNA—containing oligonucleotide, two or more of which, only in the presence of an MNAzyme ly facilitator as herein defined, can er form an ”MNAzyme.” In certain preferred embodiments, one or more ent mes, and preferably at least two, may comprise three regions or domains: a ytic” domain, Which forms part of the catalytic core that catalyzes a modification; a “sensor arm” , 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 l. Partzymes may comprise at least one additional component including but not limited to an aptamer, referred to herein as an “apta- partzyme.” 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 or a substrate.

The terms “assembly facilitator molecule”, “assembly facilitator”, “MNAzyme assembly facilitator molecule”, and “MNAzyme assembly facilitator” as used herein refer to entities that can facilitate the self-assembly of component partzymes to form a catalytically active e by interaction with the sensor arms of the MNAzyme. As used herein, assembly facilitators may facilitate the assembly of MNAzymes which have cleavage, ligase or other enzymatic activities. In preferred embodiments an assembly facilitator is required for the self-assembly of an MNAzyme. An assembly facilitator may be comprised of one molecule, or may be sed of two or more “assembly facilitator components” that may pair with, or bind to, the sensor arms of one or more oligonucleotide “partzymes”. 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, stNA, pre— and pericroRNA, other non-coding RNAs, ribosomal RNA, tives thereof, ons, or any combination f. The nucleic acid may be amplified. The amplification may comprise one or more of: polymerase chain reaction (PCR), strand displacement amplification, loop-mediated rmal cation, rolling circle cation, transcription-mediated amplification, self-sustained sequence replication, ligase chain reaction, nucleic acid sequence based amplification, or reverse transcription polymerase chain reaction (RT-PCR).

An “assembly facilitator component” is a molecule which can be used to control the assembly of active MNAzymes or facilitate the transition from inactive MNAzyme components to active MNAzymes.

The term “target” as used herein includes any natural or synthetic entity, constituent or analer which is sought to be detected, identified or quantitated bya method which uses a particular nucleic acid enzyme, such as an MNAzyme(s), with or without an additional amplification step and/or cascade. s therefore encompass the broadest range of detectable es, constituents or analytes for which methods of ive detection, identification and/or quantification are desirable. Some exemplary targets include, but are not limited to, nucleic acid, protein, polypeptide, peptide, roteins, lipids, lipoproteins, entire organisms, cells, viruses, bacteria, archaea, yeast, fungi, dies, 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 l5 target may also be an assembly facilitator or assembly facilitator component.

A “detectable effect” is an effect that can be detected or quantified as an indication that ation of substrate/s has occurred. The magnitude of the effect may be indicative of the quantity of an input such as an assembly facilitator (eg. a target). The able effect may be detected by a variety of methods, including cence 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 f.

The terms “polynucleotide substrate” and “substrate” as used herein include any single- or double-stranded polymer ‘of deoxyribonucleotide or ribonucleotide bases, or - analogues, derivatives, variants, nts or ations thereof, including but not limited to DNA, methylated DNA, alkylated DNA, RNA, ated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, siRNA, stNA, pre- and croRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons thereof or any combination thereOf (including mixed polymers of deoxyribonucleotide and cleotide bases), which is e of being recognized, acted upon or modified by an enzyme including a catalytic nucleic acid . A ucleotide substrate” or “substrate" may be modified by various enzymatic activities ing but not limited to cleavage or ligation. Modification of a “polynucleotide substrate” or “substrate” may provide a “detectable effect” for monitoring the catalytic activity of a enzyme.

A “reporter substrate” as used herein is a substrate 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 e, or to another molecule. A er 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 .

As used herein, a ic substrate” or a “universal substrate” is a substrate, for example, a reporter substrate, that is 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 tates 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 substrate. These universal ates can each be independently labelled with one or more labels. In red IS embodiments, independently detectable labels are used to label one or more universal substrates to allow the creation of a convenient system for independently or simultaneously ing a variety of assembly facilitators using MNAzymes. In some embodiments, substrates cleaved by es could be reconstituted, and hence recycled, using an e or DNAzyme ligase. In some embodiments, substrate(s) cleaved or d by MNAzymes can be further used as components or modulators of additional MNAzyme(s) or DNAzyme(s).

In some embodiments, “universal ates” may be tethered to a solid support in different positions to provide a substrate array. In such embodiments, the tethered universal substrates may all be labelled with the same fluorophore. In certain cases, each universal substrate can be cleaved only by an MNAzyme formed in the presence of a specific MNAzyme assembly facilitator molecule and signal can be localised by positioning of the substrate on the surface, thus allowing specific detection of different assembly facilitators.

The term “product” refers to the new molecule or molecules that are produced as a result of enzymatic modification of a substrate. As used herein the term “cleavage product” refers to a new molecule produced as a result of cleavage or endonuclease activity by an . The term “ligation t” refers to a new molecule produced as a result of the ligation of substrates by an enzyme.

As used herein, use of the terms “melting temperature” and “Tm” in the context of a polynucleotide substrate of the present invention will be understood to be a reference to ' the g temperature (Tm) as calculated using the Wallace rule, whereby Tm = . 2°C(A+T) + 4°C(G+C) (See Wallace et al., (1979) Nucl. Acids Res. 6(11):3543-3558), unless specifically indicated otherwise.

As used herein, the term “base” will be understood to ass the entire ribonucleotide or deoxyribonucleotide to which the base is attached, ABBREVIATIONS The following abbreviations are used herein and throughout the specification: IO MNAzyme: multi-component nucleic acid enzyme, er multipartite nucleic acid enzyme; DNAzyme: deoxyribonucleic acid enzyme; Ribozyme: ribonucleic acid enzyme; me: Partial enzyme containing oligonucleotide PCR; polymerase chain reaction; qPCR: Real-time quantitative PCR; NF-HzO.‘ nuclease-free water; LNA: locked nucleic acid; . fluorophore; Q: quencher; N= A, C, T/U, G, or any analogue thereof; N’= any nucleotide mentary to N, or able to base pair with N; (N); any number ofN; - (Nyx: any number ofN’; W: A or T; R: A, G, or AA; rN: any ribonucleotide; (rN)x2 any number of rN; rR: A or G ribonucleotide; rY: C or U; ribonucleotide M: A or C; 'H: A, C, or T/U; D: G, A, or T/U; JOE or 6—JOE: 6-carboxy-4’,5'-dichloro-2',7'-dimethoxyfluorescein; .

FAMor 6-FAM: 6-Carboxyfluorescein. , BHQ] : Black Hole Quencher® l BHQZ: Black Hole Quencher® 2 113: Iowa Black® FQ 1BR: Iowa Black® RQ shRNA: short hairpin RNA siRNA.‘ short ering RNA mRNA: messenger RNA tRNA.‘ ‘transfer RNA snoRNA: small nucleolar RNA stRNA: small temporal RNA stNA: small modulatory RNA pre-microRNA: precursor microRNA pri-microRNA: primary microRNA UV ultra violet IS DETAILED DESCRIPTION OF [LLUSTRATIVE EMBODIMENTS It is to be understood at the outset, that the figures and examples provided herein are to exemplify rather than limit the present invention and its various embodiments.

A need exists for tic "nucleic acid substrates With ties that facilitate improved catalytic nucleic acid function. In particular, many applications involving es and DNAzymes will benefit significantly from the provision of new universal substrate families having sed capacity for catalytic modification by different MNAiymes with, the same or ct target specificities. For example, these substrate families would be advantageous in increasing the efficiency and/or cy of lex assays involving MNAzymes. Additional universal substrates are, particularly useful in applications where substrate arrays are d by tethering substrates to solid supports.

The present invention provides a set of guidelines for producing universal oligonucleotide substrates with a higher probability of being catalytically modified (e.g. cleaved) efficiently over a broad‘ ature range with improved performance at ed temperatures. These guidelines include, but are not limited to, any one or more of: (i) seven or more cytosine nucleotides in the ten bases surrounding the two central ribonucleotides; (ii) bases immediately adjacent to the two central ribonucieotides are cytosines (N3 and N9); (iii) total pyrimidine content of the oligonucleotide substrate is greater than 64%; (iv) total Tm of the oligonucleotide substrate is 66°C or' greater, applicable if the reaction temperature for catalytic ation (cg. cleavage) of the oligonucleotide substrate by the nucleic acid enzyme is above 50°C; and/or (v) a low number of e nucleotides (e.g. three, two, one or none) in the 10 bases surrounding the two central cleotides.

The development of these guidelines has facilitated the development of catalytic nucleic acid enzyme substrates with features that augment catalytic nucleic acid function.

It has been fied that the catalytic modification of nucleotide/s within a substrate targeted by a given nucleic acid enzyme can be enhanced by the presence of certain specific nucleotides proximate to those which are catalytically modified.

Accordingly, certain aspects of the present invention relate to polynucleotide lO substrates for catalytic nucleic acid enzymes. The polynucleotide substrates may comprise a series of pyrimidine nucleotides 5' (ie. upstream) and/or 3' (ie. downstream) of nucleotide/s that are catalytically modified by a nucleic acid enzyme that targets the substrate. The dine nucleotides may be cytosine nucleotides.

Other aspects of the t invention relate to the use of polynucleotide substrates l5 described herein as substrates for c acid enzymesteg. a DNAzyme, ribozyme or an MNAzyme). In certain embodiments, the substrates are used as substrates for MNAzymes. In certain embodiments, the substrates are used as substrates for DNAzymes.

Additional aspects of the present invention relate to methods for detecting a target molecule. The methods comprise modifying a polynucleotide substrate described herein to e a detectable effect. In n embodiments, the methods comprise modifying the polynucleotide substrate using an MNAzyme that is capable of detecting a . r aspects of the t invention relate to kits comprising one or more polynucleotide substrate/s described herein. The kits may comprise a nucleic acid enzyme capable of catalytically modifying the substrate/s. in certain embodiments, the nucleic acid enzyme may be an MNAzyme. 1. tic nucleic acid enzyme substrate/s The present invention es polynucleotide substrates for catalytic nucleic acid enzymes. The present ion also provides substrate families the members of which have increased capacity for catalytic modification by different nucleic acids (e.g.

MNAzymes) with the same or ct target specificities.

The polynucleotide substrates se at least one sequence motif that can be modified by a catalytic nucleic acid enzyme. No limitation exists regarding the particular type of catalytic nucleic acidenzyme that may modify a polynucleotide substrate of the present invention. The sequence motif may comprise any one or more of at least one DNA nucleotide, at least one RNA nucleotide, at least one analogue of a ‘DNA nucleotide, and at least one analogue of a RNA nucleotide. A Non-limiting examples of le sequence motifs include those recognised and modified by DNAzymes (cg. 10—23 DNAzymes; 8-17 es; “7281”, “7248” and “7Q10” DNAzyme ligases; “UVIC” thymine dimer photoreversion es, “DAB22” carbon-carbon bond forming DNAzymes; and derivations thereof), ribozymes (e.g. hammerhead mes; homodimeric ribozymes, heterodimeric ribozymes; and derivations thereof), and MNAzymes (see, for e, MNAzymes describedin PCT ' patent publication numbers WO/2007/04l774, WO/2008/040095 and W02008/122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011? 0143338; each of which is incorporated herein by reference in its entirety).

Non-limiting examples of suitable sequence motifs include those set out in Table 1 below.

Table l: Exemplary catalytic motifs Substrate Catal tic Motif 3-17 DNAzyme , (N')xmx 9 _(N‘)x -23 DNAzyme (N')x LBJ! (N')x I N = A, C, 7', G or any analogue; N’ =.anynucleotide mentary to N;(N)x or (N’)x = any number of nucleotides;_ W = A or T; R = A, G or AA; rN = any ribonucleotide base;(rN)x é any number of cleotides; rR = A or G ribonucleotia’es; rY = C or U ribonucleotides; M = A or C; H = A, C ‘or T; D =G,AorT Catalytic nucleic acids have been shown to tolerate only certain modifications in the area that forms the catalytic core (Perreault et £11., 1990 Nature 344(6266): 565-7.; Perreault et al., 1991 Biochemistry 30(16): ; Zaborowska et al.,_2002 J Biol Chem. 277(43):?40617-22; Cruz et aI., 2004 Chem Biol. Jan;ll(I): 57-6; Silverman, 2004 Chem Biol. Jan;1 1(1): 7-8). Examples of sequences sible forcatalytic activity of DNAzymes are listed in Table 2. ' Table 2: Exemplary sequences for some active DNAzymes and their ates DNAz mete Substratese'uence , (N)XTNNNAGCNNNWCGR(N)X - . .

- (NW M”N N )X SEIDN0:189 (N)XGGMTMGHNDNNNMGD(N)X 23 , .

- (NMR Yr (MK SEOIDNO:19Q - For the DNAzyme sequence N is generally A, C, T or G or any analogue, in some instances N can be U; for the substrate sequence N = A, C, 171], G or any analogue; ; N' = any nucleotide complementary to N; (N)x or (N’)X = any number of nucleotides; 'W = A or T, in some ces W can be U; R = A, G or AA; rN = any ribonucleotide base; (rN)x = any number of ribonucleotides; rR = A or G ribonucleotide; rY =‘C or U cleotide; M = A or C; H = A, C or T, in some ces H can be U; D = G, A or T, in some instances D can be U.

The polynucleotide substrates may comprise multiple sequence motifs: The motifs may be recognised and modified by one type of catalytic nucleic acid enzyme.

Alternatively, different sequence motifs within the substrate may be reCOgnised and modified by ent types of catalytic nucleic acid enzymes.

As noted above, polynucleotide substrates of the present invention comprise at least one sequence motif capable of modification by a tic nucleic acid enzyme. In'some embodiments, nucleotides in the proximity of the sequence motif' are pyrimidine nucleotides. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides preceding and/or succeeding (i.e. following) the sequence motif may be pyrimidine nucleotides. Any one or more of the pyrimidine nucleotides maybe cytosine nucleotides.

In other embodiments, the sequence motif is preceded and/or succeeded (ie. followed) directly (ie. in continuous sequence) by one or more pyrimidine nucleotides.

For e, the sequence motif may be directly preceded and/or directly succeeded by a 615 sequence of l, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine tides. Any one or more of the pyrimidine nucleotides may be cytosine nucleotides.

In further embodiments, l, 2, 3, 4, 5, 6, 7, 8, 9, or l0 or more nucleotides within ten nucleotides 5' (ie. upstream) and/or within ten nucleotides 3' (ie. downstream) of the sequence motif are pyrimidine nucleotides. Any one or more of the pyrimidine nucleotides may be cytosine nucleotides.

In still further embodiments, more than 9, more than 10, or more than 1 1 nucleotides of the polynucleotide ate may be ne nucleotides. For example, the substrate may comprise or consist of 10, 11, 12, 13, 14, 15, or more than 15 nucleotides, and 9, '10, 11 or more than 11 of these nucleotides may be cytosine nucleotides.

Additionally or alternatively, less than 5, less than 4, less than 3, or less than 2 nucleotides of the polynucleotide ate may be guanine nucleotides. For example, the substrate may comprise or t of 10, 11, 12, 13, 14, 15, or more than 15 tides, and 4, 3, 2, 1 or none of these nucleotides may be guanine nucleotides.

No ular limitation exists regarding the length of a polynucleotide substrate of the t invention. For example, the substrate may be less than 100, 75, 50, 40, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. For example, the substrate may be between 5 and 30, 10 and 15, 10 and 20, 10 and 25,10 and 30,16 and 23,16 and 21,16 and 18, 18 and 21,18 and 23, or 21 and 23 ' nucleotides in length.

A polynucleotide substrate 0f the present invention may be designed to possess a specific melting temperature (Tm) as calculated using the Wallace rule, whereby Tm = 2°C(A+T) + 4°C(G+C) (see Wallace 2‘: al., (1979) Nucl. Acids Res. 6(11):3543-3558). In certain embodiments, the substrate may be recognised and catalytically modified by an e, and the Tm of the bases that are bound by. the e substrate arm/s of the MNAzyme may be between about 52°C and about 76°C, between about55°C and about 75°C,- between about 60°C and about 70°C, between about 65°C and about 70°C, n about 64°C and 68°C, or between 64°C and 70°C (as calculated using the Wallace rule).

In other embodiments, the ate may be recognised and catalytically modified by an MNAzyme or a DNAzyme, and the Tm of the bases that are bound by the partzyme substrate arm/s of the MNAzyme may be between 68°C and 90°C, between 66°C and 76°C, between 68°C and 76°C, between 64°C and 70°C, between 70°C and 76°C, between 70°C and 75°C, between 72°C'and 76°C, 52°C, 58°C, 64°C, 66°C, 68°C, 70°C, 72°C, or . 76°C.

By way of non-limiting example only, a polynucleotide substrate of the present invention may comprise a sequence defined in any one or more of SEQ ID NOS: 25—27, 29-30, 72-90, or 172-175. In n embodiments, the polynucleotide substrate may consist of a sequence defined in any one or more of SEQ ID NOS: 25-27, 29-30, 72-90, or 172-175.

In some ments, a polynucleotide substrate of the present invention may comprise a sequence defined by SEQ ID NO: 28. In other embodiments, the polynucleotide substrate may consist of a sequence defined by SEQ ID NO: 28.

In some embodiments, polynucleotide substrates of the t invention are capable of catalytic modification by an MNAzyme comprising two oligonucleotide ,partzymes. The ces of the polynucleotide substrate and the oligonucleotide partZymes may be any specific combination of three sequences (as ed by SEQ ID NOS) that is shown in Table 6, 8, 10, I3, 16, 20, 22 and/or 24.

In other ments, polynucleotide substrates of the present invention are capable of catalytic modification by a DNAzyme. The ces of the polynucleotide substrate and DNAzyme may be any specific pair of sequences (as depicted by SEQ ID NOS) that is shown in Table 15.

Polynucleotide substrates of the present invention 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.

Non-limiting examples of additions or substitutions include LNA phosphoramidite, 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2‘-O-methylcytidine, 5- carboxymethylaminomethyl thiouridine, dihydrouridine, 2'-O-methylpseudouridine, beta D4galactosquueosine, 2'-O-methylguanosine, e, N6-isopentenyladenosine, 1- methyladenosine, 1-methylpseudouridine, l-rnethylguanosine, l-methylinosine, 2,2— dimethylguanosine, 2-methyladenosine, 2-methylguanosine, ylcytidine, 5— cytidine, N6-methyladenosine, ylguanosine, 5-methylaminomethyluridine, -methoxyaminomethyl—2-thiouridine, beta osylmethyluridine, 5- methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6—' isopentenyladenosine, N-((9-beta-ribofuranosylmethylthiopurine~6— yl)carbamoyl)threonine, N-((9—beta—ribofuranosylpurineyl)N-methy]— oyl)threonine, uridine-S-oxyacetic acid methylester, uridine-S-oxyacetic acid (v), wybutoxosine, pseudouridine, queosine, 2-thiocytidine, ‘S-methyl-Z-thiouridine, 2- thiouridine, 4‘thiouridine, S-methyluridine, N-((9-beta-D—ribofuranosylpurine-6— bamoyl)threonine, 2'-O—methyl-5—methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-aminocarboxypropyl)uridine, beta D-arabinosyl uridine, and beta D-arabinosyl thymidine.

Non-limiting examples of derivatives e fimctionally equivalent nucleic acids or nucleotides, including any fusion les produced integrally (e. g. by recombinant means) or added post-synthesis (eg. by chemical means). Such fusions may comprise oligonucleotides of the invention with RNA or DNA added thereto or conjugated to a polypeptide (e.g. puromycin or other polypeptide), a small molecule (e.g. psoralen), a microcarrier or nanocarrier, or an antibody.

Non-limiting examples of analogues include compounds having a physical structure that is related to a DNA or RNA le or residue, and may be capable of forming a hydrogen bond with a DNA or RNA residue or an ue 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 assed 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 es of analogues.

Active DNAzymes have been described which contain tide ues, including deoxyinosine, C-S-immidazole deoxyuridine, 3-(aminopropynyl)-7—deaza-dATP, 2’-O— methyl RNA, 2’ O-methyl cap. Other analogues could also be compatible with catalytic activity of es and MNAzymes. Alteration of a nucleic acid with catalytic activity, for example by substitution of one base for another, by substitution of an analogue for a base, or alteration of the sugar component or phosphodiester backbone, can be straight forward for the skilled artisan. For example, alterations can be made during synthesis, or by modification of specific bases after synthesis. Empirical testing of catalytic nucleic acids incorporating alterations such as base changes or base analogues allows for assessment of the impact of the d sequences, or specific analogues, on tic activity. Analogues of the bases A, C, G, T and U are known in the art, and a subset is listed in Table 3.

IS , Table 3: Exemplary nucleotide analogues 2-meth ladenosine N6 meth ladenosme -.methox ridine Name 2-meth lthio—N6-isoenten ladenosine N~((9-beta-ribofi1ranosylmethylthiopurine l carbamo l threonine N-((9-beta-ribofiiranosylpuriney1)N-methyl- carbamo l threonine , —Uridine-S-oxMv acetic acid meth lester .

S-meth lthiouridine 2'-O-meth l—S-meth luridine 2'-O-meth luridine 3- 3-aminocarbox ouro luridine, ac 3 11 beta Dvarabinos luridine beta D-arabinos 1th idine » cleotide substrates of the present invention may incorporate additional entities such as ed nucleic acids, rticles, microparticles, proteins, antibodies, RNA, DNA, nucleic acid analogues, proteins, glycoproteins, lipoproteins, peptide nucleic acids, locked nucleic acids, peptide-nucleic acid chimeras, or any combination thereof.

The nanoparticles may be gold nanoparticles.

Polynucleotide substrates of the present invention may be catalytically d by a catalytic c acid enzyme. Non-limiting examples of potential catalytic - modifications e ge of c acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, aminoiacid 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 rticle formation, biphenyl isomeriZation, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion and orarhidate cleavage.

In certain applications, it may be desirable to detect product/s arising from catalytic modification of polynucleotide substrates of the present ion. This can be achieved using any number of standard techniques known in the art.

For example, the substrate may comprise a detectable portion and a quencher portion, wherein upon modification» of said substrate by a catalytic nucleic acid, a detectable effect provided by said detectable portion is increased or decreased. The detectable effect may be detected by fluorescence spectroscopy, e plasmon resonance, mass spectroscopy, NMR, electron spin resonance, zation fluorescence spectroscopy, circular dichroism, immunoassay, chromatography, radiometry, electrochemical, photometry, scintigraphy, electronic methods, UV, visible light or infra red oscopy, enzymatic methods or any ation thereof.

Additionally or. alternatively, product’s g from catalytic modification of polynucleotide substrates of the present invention may be detected on the basis of size (cg. by standard ophoresis), nucleic acid sequencing, fluorescence reSonance energy transfer, chemiluminescence, potentiometry, mass spectrometry, plasmon nce, colorimetry, polarimetry, flow try, scanometry, and DNA sequencing or any combination thereof.

Nucleic acid product/s arising from catalytic modification of polynucleotide ates of the t invention may be ed-in order to assist detection using . techniques such as, for example the polymerase chain reaction (PCR).

Polynucleotide substrates of the present invention may be recognized and modified by catalytic nucleic acid enzymes (e.g. MNAzymes) designed to detect a target that differs from the substrate to be modified by the enzyme. Accordingly, polynucleotide substrates of the present invention may be “generic” or “universal” substrates that are recognized by and acted on catalytically by a plurality of catalytic nucleic acid enzymes (e.g. a plurality of MNAzymes), each of which can recognize a different target. The use of such substrates may facilitate the pment ‘of separate assays for detection, identification or fication of a wide variety of targets using catalytic nucleic enzymes which recognize a universal substrate. The universal substrates may each be independently ed with one or more labels. In certain embodiments,independently detectable labels may be used to label one or more universal substrates to allow for the ndent or simultaneous detection of a variety of targets using es. For. example, a series of universal substrates may be used in a multiplex reaction allowing simultaneous detection of multiple targets.

Polynucleotide substrates of the present invention may be provided bound, attached or tethered to an insoluble or solid support for use in various applications (eig. enzymatic cascades or any other signal transduction cascades). The support may be an insoluble material, or 'a matrix which retains the ate and excludes it from freely moving in the bulk of the reaction mixture. Such supports are known in the art for immobilizing or localizing substrates, including nucleic acid targets. The skilled addressee will appreciate that the support can be selected from a wide variety of matrices, rs, and the like in a variety of forms including beads convenient for use in microassays, as well as 'other materials compatible with the reaction conditions. In certain preferred embodiments, the [0 support can be a plastic material, such as plastic beads or wafers, or that of the well or tube in which a particular assay is conducted. In n embodiments, the support may be a microcarrier or a nanocarrier. The attachment of the substrate to the support may be designed so that‘upon modification (e.g. cleavage) of the substrate by the catalytic nucleic acid (e.g. e), a portion of the modified substrate remains ed to the support, while the other is freed to move into the bulk of the reaction mixture, away from the portion ing attached. 2. Exemplary s cleotid'e substrates of the present invention may be used in any number of potential applications utilising catalytic nucleic acids which recognise/modify the substrates.

For e, the substrates may be used in applications involving DNAzymes (e.g. -23 DNAzymes; 8-17 DNAzymes; “7281”, “7248” and “7Q10” DNAzyme ligases; “UVIC” thymine dimer photoreversion DNAzymes, “DABZZ” carbon-carbon bond forming DNAzymes; and tions thereof), ribozymes (e.g. hammerhead mes; homodimeric ribozymes, heterodimeric ribozymes; and derivations thereof), and/or MNAzymes.

In certain embodiments of the invention, the substrates may be used as substrates for MNAzymes. The features of es and various applications using MNAzymes are described in detail in PCT patent publication numbers 7/041774, 8/040095 and W02008/122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011-0143338 (the contents of each of‘ these documents are incorporated herein by reference in their entirety).

MNAzymes are capable of self-assembling from two or more oligonucleotide ents, also referred to herein as partzymes. The partzyme oligonucleotides self- le in the presence of an MNAzyme self assembly facilitator to form an MNAzyme. MNAzymes are ore catalytically active nucleic acid s. In some embodiments, the presence of an MNAzyme can be detected, and is indicative of the presence of a target, because the MNAzyme forms only in the presence of the target, - wherein the target comprises the assembly facilitator.

In preferred embodiments, the MNAzyrhe structures are based on one or more DNAzymes and/or ribozymes. More preferred are those e ures which are based on a particular DNAzyme structure. Presently preferred structures are based on DNAzymes including the 10-23 and 8-17 DNAzymes. In various embodiments the MNAzymes comprise either or both ribonucleotide bases and deoxyribonucleotide bases.

In more preferred embodiments, an MNAzyme structure is based at least in part on the structure of a DNAzyme. In other preferred embodiments, MNAzymes comprise at least some deoxyribonucleotide bases or ues thereof. In more preferred embodiments, the catalytic core of an MNAzyme comprises one or more deoxyribonucleotide bases or ues thereof. In still more preferred embodiments, one or more deoxwibonucleotide bases or analogues thereof are involved in the catalysis of a subStrate. In other embodiments, at least one deoxyribonucleotide base, or its analogue, in the catalytic. core improves catalytic activity. In yet other embodiments, there is a strict requirement for at least one deoxyribonucleotide base, or its analogue, in the catalytic core of the MNAzyme for catalysis to occur at a measurable rate, relative to that of a able MNAzyme without the deoxyribonucleotide base present.

The MNAzymes may contain one or more substitutions such as analogues, derivatives, .modified or altered bases, ribonucleotides, tions 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 ate arms and/or in the catalytic core portions, such that the molecule retains tic activity. Substitutions and modifications to arms that bind the substrate or assembly facilitator may be well ted and in fact are the basis of allowing ing of the molecules to different substrates/assembly facilitators. For example, modification of the sensor arms will allow tailoring to ent assembly facilitators, while modification of the substrate arms will allow tailoring to different substrates.

The e may comprise either deoxyribonucleotides or ribonucleotides, or even both. es comprising at least one and more preferably, all, deoxyribonucleotide component oligonucleotides are preferred. Also preferred are es comprising at least one deoxyribonucleotide base, or its analogue, within the catalytic core of the MNAzyme. Even more preferred are those embodiments where such a base is required for catalytic activity.

MNAzyme assembly and disassembly may also 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. Accordingly, disassembled or partially led MNAzymes may be prevented from assembling into a catalytically active MNAzyme in the presence of an assembly facilitator by modulating the microenvironment, thus providing a “molecular switch”.

A basic example of a MNAzyme structure icted in Figure l. The ure shown comprises partzyme A and partzyme B, the sensor arms (i) of which have base- ' paired with an MNAzyme assembly tator molecule, for example a target DNA or IS RNA. mes A and B by interacting with the ly facilitator have allowed the partial catalytic cores (iii) to come into close proximity and thereby form a single catalytic core. The substrate arms (ii) of the MNAzyme have interacted with and base— paired with a substrate, depicted here as a Reporter Substrate. Thus the MNAzyme has self-assembled and this process is tated through the presence of the MNAzyme ly tator molecule. In the absence of assembly facilitator, no MNAzyme will form. Modification (in this case, cleavage) of a polynucleotide substrate of the present invention is catalyzed, by the catalytic core of the MNAzyme at the MNAzyme Modification Site (e.g. Cleavage Site within the substrate denoted by a cross (X)). The polynucleotide substrate in this particular embodiment comprises a detectable portion having a detectable signal, for example fluorophore F, and a quencher portion Q having a quenching effect on the detectable signal F through the action of quencher Q. Upon cleavage at the e Cleavage Site, there is a substantial se in detectable signal, here fluorescence, which can readily detected and quantified if so desired.

Figure 1 can further be understood to depict an example of a basic method of using es to detect a target, which in some ments comprises an assembly tator. More specifically, partzyme A and partzyme B are shown in Figure 1, each comprising a substrate arm portion (ii), catalytic core portion (iii), and a sensor arm portion (i). In the presence of a target, the sensor arm portions of partzyme A and partzyme B can begin to hybridize to, and base pair with complementary portions, of the target, for example a DNA or RNA sequence. Upon contacting the target in this fashion, the MNAzyme self-assembles forming a catalytic core which can modify a substrate which is bound by the substrate arms. Preferably the presence of the MNAzyme is detected through'the detection or measurement of its catalytic ty. The substrate arms of the thus assembled MNAzyme can engage a polynucleotide substrate of the present invention through the interaction of the complementary sequences on the substrate arms and the substrate. Once the substrate is soengaged with the substrate arms, the core can promote the modification (e.g. cleavage) of the substrate, which can in . catalytic turn be measured or detected, directly or indirectly.

The skilled artisan will readily appreciate that the methods described herein may e amplification of a target , during or after MNAzyme catalytic activity. Such target amplification finds particular application in. embodiments of the t invention where the amOunt of target being, sought tovbe detected, identified or quantified is of such quantum so as to provide a signal that may ise not be detectable. Such. amplification may se one or more of: polymerase chain reaction (PCR), strand '15 displacement amplification (SDA), Sloop-mediated rmal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self— sustained sequence replication (3SR),- nucleic acid sequence based amplification (NASBA), or e transcription polymerase chain reaction (RT-PCR).

Figure 2 provides exemplary applications of methods for target detection using MNAzymes. Strategy 1 exemplifies MNAzymes adapted for detection of s including DNA, RNA and proteins. As described above (see description of Figure 1) an MNAzyme composed of tWo te oligonucleotides with recognition sequences for both a target and a substrate forms when the ‘oligonucleotides recognize and bind a target.

The substrate, e.g. reporter substrate, is modified by the catalytic action of the MNAzyme and causes generation of a detectable signal, either directly egy 1), during or after target amplification (Strategy 2) or via a signal e (Strategy 3). In some embodiments, both target and signal amplification occur simultaneously or sequentially.

Strategy 2 of Figure 2 exemplifies the use of an MNAzyme adapted to monitor the accumulation of amplicons during, or ing, in vitro amplification of nucleic acid targets. Techniques for in vitro amplification‘of nucleic acid sequences are known in the art. These include ques mediated by a DNA rase, such as‘the polymerase chain reaction (“PCR”) (see, for example, U.S. Patent No. 4,683,202; U.S. Patent No. 4,683,195; U.S. Patent No. 4,800,159; U.S.i Patent No. 4,965,188; U.S. Patent 'No. ,176,995), strand displacement cation (“SDA”), rolling circle amplification (“RCA”), reverse transcription'polymerase chain reaction R) and loop-mediated isothermal amplification (“LAMP”). Other target amplification techniques are mediated by an RNA polymerase, for example, transcription-mediated amplification ), ustained sequence replication (“33R”) and nucleic acid sequence replication based amplification (“NASBA”). The amplification products (“amplicons”) produced by PCR, RT—PCR, SDA, RCA and LAMP are composed of DNA, whereas RNA amplicons are produced by TMA, 38R and NASBA.

With further reference to gy 2 of Figure 2 an MNAzyme that recognises and s a polynucleotide substrate of the present invention can be used in conjunction with target cation methods which e, for example, the aforementioned PCR, l0 RT-PCR, SDA, RCA, LAMP, TMA, 38R and NASBA. The accumulation of amplicons produced by PCR using either asymmetric or symmetric primer ratios can be monitored using es. Examples 1, 2, 4, 6, 8 and 9 of the present specification demonstrate the detection of PCR ons in real time utilising MNAzymes that catalytically modify various polynucleotide substrates of thepresent invention. l5 Again referring to strategy 2 of Figure 2, a target nucleic acid may be amplified in ance with a procedure for amplifying that nucleic acid (ie. DNA or RNA).

Preferably, standard methods of in vitro amplification are used. The amplicons generated during the cation may serve as target assembly facilitators for an e. The MNAzyme activity, which is made detectable by modification Of a polynucleotide substrate of the present invention by the MNAzyme, is indicative of the presence of the target. The skilled artisan will appreciate that assays of this nature can be conducted in a single vessel under conditions that permit both the target nucleic acid amplification and the MNAzyme assembly and catalytic activity. Additionally or atively, they can be conducted subsequent to, or at time points throughout, the target nucleic acid amplification, by removing samples at the end or during the course of the amplification reactions.

Strategy 3 of Figure 2 shows an overview of a method of using a MNAzyme to initiate amplification of a signal through the use of a signal cascade. Example 3 of the present specification demonstrates the isothermal direct detection of a target utilising es that catalytically modify various polynucleotide substrates of the present invention, which could be used to te a signal cascade.

The skilled see will appreciate that methods or protocols that combine target amplification with catalytic nucleic acid activity may require specific reaction conditions (e.g. those described in es 1, 2, 4, 6, 8 and 9 of the present specification).

Preferably, reaction conditions are compatible with both polymerase activity (for amplification), and catalytic nucleic acid modification of a substrate (for detection).

Protocols for determining conditions for concurrent catalytic activity and polymerase activity at high ature, such as during PCR, have been described for DNAzymes.

The influence of factors including DNAzyme arm length, buffer, temperature, nt ion concentration and effects of additives are known in the art. DNA enzymes are suited for use in combination with in vitro amplification strategies. For example, they are not irreversibly denatured by exposure to high temperatures during amplification.

In certain embodiments, a polynucleotide substrate of the present invention capable of recognition and modification by an MNAzyme may be bound, attached or tethered to an insoluble or solid support. For example, with reference to Figure 3, Panel (i), an exemplary method for detecting targets using an e and a polynucleotide substrate of the present invention anchored to a support is depicted. In this embodiment, the substrate is preferably a substrate with a detectable portion comprising a detectable signal, for example a fluorophore, and a quencher portion which diminishes or eliminates the able signal while the detectable portion and the quencher portion of the substrate remain in cloSe ity, for example, until the substrate is modified (e.g. by cleavage).

The substrate is attached to a support. Preferably the support is an insoluble material, or a matrix which retains the substrate and excludes it from freely moving in the bulk of the reaction mixture. The ment of the substrate to the support may be designed such that upon modification (e.g. by cleavage) of the substrate by the MNAzyme, either the detectable portion or the quencher portion, but not both, remains attached to the support, while the other is freed to move into the bulk of the on mixture, away from the portion remaining attached. Thus, in a cleavage example, the detectable signal vastly increases as the quencher portion and the detectable n are separated upon cleavage.

In the embodiment shown in Figure 3, Panel (i), the fluorophore-containing detectable. portion remains ed after cleavage. This has the benefit of allowing localization of the signal on the support but in certain instances, the hore/s may be released into solution. In a further embodiment where, for example, on occurs, the quencher may be d to a fluorophore thus decreasing the detectable signal.

In certain embodiments, multiple universal substrates may be tethered to a solid support in different positions to provide a substrate array. With reference to Figure 3 Panel (ii), two substrates may be ed at defined positions on a solid surface. Each universal substrate can be cleaved only by an MNAzyme formed in the presence of a c e assembly facilitator molecule (e.g. target 1 or target 2) and signal can be sed by oning of the ate on the surface (position 1 or position 2), thus allowing specific ion of different assembly facilitators. In such embodiments tethered universal substrates may all be labelled with the same fluorophore. In other embodiments tethered universal substrates may be labelled with different fluorophores.

The strategy depicted in Figure 3 (ii) can be extended to create universal substrate arrays with many different universal substrates attached in defined positions on a solid surface.

In such embodiments increasing the number of universal substrates that are available for use in arrays may provide an advantage by allowmg the on of more x arrays.

Such universal of use in universal substrates may have for . arrays utility highly . multiplexed analysis of target analytes. The present invention provides onal universal substrates with features that may t tic activity function which may be useful in improving the capacity to perform increasingly more complex analysis using MNAzymes.

In certain ments, a polynucleotide substrate of the present invention may be recognised and modified by a e to provide an assembly facilitator, ly l5 facilitator component, or partzyme for a second ent MNAzyme.

With reference to Figure 4, an initiating MNA'zyme (Mt) may be formed in the presence of a target (T). The initiating MNAzyme (Mt) cleaves a (first) cleotide substrate of the present invention (SI) to create a first assembly tator component ($11), which directs formation of a first ing MNAzyme (cascading MNAzyme Mel). In this example the first cascading MNAzyme (Mel) comprises two partzymes and three assembly facilitator components designated Fl F2 and Slf. Mcl can cleave an additional substrate (82) thus liberating an additional assembly facilitator ent (82f), which directs formation of a second cascading MNAzyme (cascading MNAzyme Mc2). In this example the second cascading MNAzyme (Mc2) comprises two partzymes and three assembly facilitator components designated F3, F4 and 82f. Mc2 can then cleave more of the first substrate (Sl) thus creating more of the first assembly facilitator component (Slf). This leads to the formation of further first cascading MNAzyme (Mcl) thereby forming an amplification cascade. The skilled addressee will recognise that Figure 4 shows three assembly facilitator components are required to facilitate active MNAzyme assembly, More or less assembly tator components could be ed in a similar schema.

The skilled artisan will readin understand that the methods described herein may be optimized using a variety of experimental parameters in order to optimize the ion, identification and/or quantification of a target, and/or the ition and catalytic modification of a polynucleotide substrate of the present invention by a catalytic nucleic aCid (e.g. an MNAzyme or a DNAzyme). The particular experimental parameters that are zed, and the level of such optimization, will depend upon the particular method being employed and the particular target and/or substrate involved. Such parameters include, but are not limited to, time, ature, concentration of salts, detergents, cations and other reagents including but not limited to dimethylsulfoxide (DMSO), and length, complementarity, GC content and melting point (Tm) of nucleic acids.

In some embodiments, for example those methods involving detection of sequence variation and/or detection of methylated DNA, the experimental parameters, and preferably ing the temperature at which the method is performed, may be zed so as to discriminate between binding of an MNAzyme component nucleic acid to a target nucleic acid that does or does not comprise a ce variation or a ated nucleotide, respectively. The temperature at which such methods may be performed may be in the range of about 20°C to about 96°C, about 20°C to about 75°C, 20°C to about 60°C or about 20 to about 55°C.

In some ments, optimized reactions for practicing the methods of using MNAzymes and DNAzymes are provided herein. In such optimized reactions, catalytic ty is increased by up to 10, 20, or 30% above unoptimized reactions. More preferred reaction conditions improve catalytic activity by at least 35%, or 40%, and preferably up to 50% or more. In still more preferred embodiments, optimized reactions have an increase of catalytic activity of more than 50%, and up to 66%, 75% or even 100%. In yet more preferred embodiments, a fully optimized reaction method will offer 100, 200 or even 300% or more increase in catalytic activity. Other red reaction conditions can improve the catalytic activity by up to 1,000% or more over methods practiced with unoptimized reaction conditions. A highly preferred reaction condition for optimizing the methods provided herein is the inclusion of certain divalent cations. The catalytic ty of most nucleic acid enzymes may be ced in a concentration- dependent fashion by the concentration of divalent cations. Preferred optimized reactions are optimized for one or more of Bali Sr”, Mg2+, Ca2+, Ni”, C02”: Mn”, Zn“, and Pb“.

In some embodiments, the use of polynucleotide substrates of the present invention 3o in assays with nucleic acid enzymes (cg. MNAzymes or DNAzymes) may increase a detectable effect (c.g. an increase or decrease in cent signal) arising from tic modification of the substrate by the enzyme above the able effect gained using a known substrate in the same assay under the same conditions. For example, the detectable effect may be increased by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, or more than 50% compared to the known substrate. In certain embodiments, the detectable effect is a fluorescent signal.

In some ments, the methods of the invention involve using a polynucleotide substrate for an MNAzyme in combination with an MNAzyme comprising two oligonucleotide partzymes. The sequences of the polynucleotide substrate and the oligonucleotide mes may be any specific combination of three sequences (as depicted by SEQ ID NOs) that is shown in Table 6, 8, 10, 13, 16, 20, 22 and/or 24.

In some'embodiments, the methods of the invention involve using a ucleotide substrate in combination with a DNAzyme. The sequences of the polynucleotide substrate and DNAzyme may be any specific pair of ces (as depicted by SEQ ID NOS) that is shown in Table 15. 3. Kits Also provided herein are kits comprising one or more polynucleotide substrates of IS the present invention.

The kits may comprise additional reagents for practising the methods sed herein. For example, the kits may comprise one or more catalytic nucleic acids capable of recognising and modifying the substrate. Non—limiting examples of suitable catalytic nucleic acids include DNAzymes (e.g. 10-23 DNAzymes; 8-17 DNAzymes; “7Z81”, “7248” and “7Q10” DNAzyme ligases; “UVlC” thymine dimer photoreversion DNAzymes, “-DAB22” carbon-carbon bond forming DNAzymes; and tions thereof), ribozymes (e.g. hammerhead mes; homodimeric ribozymes, heterodimeric ribozymes; and derivations thereof), and MNAzymes.

Kits of the present invention may be “compartmentalised” kits. A compartmentalised kit encompasses any kit in which reagents are provided in separate containers such as, for example, 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 contamination of samples and reagents, and/or allow the on of agents or ons of each container from One compartment to another in a quantitative n. Such kits may also e a container which will accept a sample to be , a container which contains reagents to be used in the assay, containers which contain wash reagents, and containers which contain a detection reagent.

In certain embodiments, the kits comprise one or more polynucleotide substrates of the present invention and a plurality Vof oligonucleotide partzymes designed to assemble an MNAzyme capable of recognising and catalytically modifying the polynucleotide substrate in the presence of target. The target may act as an assembly facilitator g assembly of the oligonucleotide partzymes into a tically active MNAzyme capable of recognising and modifying the cleotide ate.

In some embodiments, the kits comprise a polynucleotide substrate of the present . invention and an MNAzyme comprising two ucle'otide partzymes. The ces of the polynucleotide substrate and the oligonucleotide partzymes may be any specific combination of three ces (as depicted by SEQ ID NOS) that is shown in Table 6, 8, , 13, 16, 20, 22 and/or 24.

IO In other embodiments, the kits se a polynucleotide substrate of the present invention and a DNAzyme. The sequences of the polynucleotide substrate and DNAzyme may be any specific pair of sequences (as depicted by SEQ ID NOS) that is shown in Table 15. dual oligonucleotide partzymes _may be t in the same container.

Alternatively, individual oligonucleotide partzyme/s may be present in separate containers. It will be understood that not all components-for all MNAzymes intended to be used in a given method need necessarily to be provided in a kit as such component/s may be generated as part of a cascade reaction.

In other embodiments, components for additionalcatalytic nucleic acids with, for example, either ge or ligase activityimay also form part of the kits of the present invention. In yet other embodiments the kits of the present invention may include DNAzymes or components thereof.

Kits of the present invention may include instructions for using the kit components in order to conduct desired methods.

Kits and methods of the invention may be used in conjunction with automated analysis equipment and systems including, but not limited to, real time PCR es.

Kits of the present invention may include additional reagents for ting target amplification reactions (eg. PCR) including, for example, oligonucleotide primers, buffers, magnesium ions, polymerase enzymes and the like.

Kits of the present invention may comprise one or more assemblies comprising one or more solid supports and one or more polynucleotide substrates of the present invention. one or more of the solid ts may be bound to one or more of the polynucleotide substrates. In certain embodiments, the kits may comprise one or more assemblies comprising a plurality of different solid supports. The plurality of different solid supports may be bound to a ity of different cleotide substrates of the present invention.

EXAMPLES In the following examples, the ability of es based on the 10-23 DNAzyme, and 10-23 DNAzymes, to efficiently cleave universal substrates was tested.

The universal substrates tested included some of those previously known in the art (Table 4) and the novel substrates designed according to either all or a subset of the design guidelines of the present invention (Table 5). - These examples demonstrate the IO robustness, as indicated by efficient cleavage in a range 20f conditions, of universal substrates ed according to all or a subset of the design guidelines of the t invention.

Table 4: Previously known universal substrates used in Examples » Sub? (SEQ ID NO 24) TTAACATGGCACguTGGCTGTGATA Sub4 (SEQ ID NO. 171) CATGGCGCACguTGGGAGAAGTA *Uppercase letters indicate DNA, lower case letters indicate RNA.

Table 5: Improved universal substrates from the current invention used in es Name Sequence" Sub44 ID NO: . (SEQ 25) CAGGTCTCCTCguCCCTATAGTGA Sub45 (SEQ ID NO- 26) ACGGGTCCCguCTCCTTTGGAA Sub46 (SEQ ID NO: 27) ACCGCACCTguCCCCAGCTC ‘Sub49 (SEQ ID NO: 28) TAAACTTGGCTCguTGGCTGTGA’I‘A mm m 29> - mm In 74> - mm m 75> «em m 76> <sm m m me In 78.) we m 79> «am In em me In 81> we m 82) mm m 83> «SEQ ID 84> <sm In 85, - me In es) «sEQ as Sub89 (SEQ ID NO: 89) ACCGCACCTCguCCCTCCTCCT Sub90 (SEQ ID NO: 90) CTCGACCCTCguCCCTCGTCCA 16) (SEQ ID NO: 172) GCACCTCguCCCCAGC » Sub55 (18) (SEQ ID NO: 173) CGCACCTCguCCCCAGCT Sub55 (23A) (SEQ ID NO: 174) AACCGCACCTCguCCCCAGCTCA Sub55 (23C) (SEQ ID NO: 175) CACCGCACCTCguCCCCAGCTCC ' *Uppercase letters indicate DNA, lower case letters indicate RNA.

Example 1: Use of sal substrates with MNAzymes'in real-time quantitative PCR (qPCR), at an annealing temperature of 52°C.

MNAzymes can be’ used to monitor amplification of target nucleic acids in real-time using in vitro target amplification methods such as PCR, referred to as MNAzyme qPCR.

Further, reaLtime monitoring during qPCR using MNAzyme substrates labelled with fluorophore and quencher pairs tes a curve on which a threshold line, of an arbitrary level of fluorescence, can be placed over the exponential phase of the reactions, ‘10 producing a value which can be known as a Ct (cycle threshold). ons that produce a lower Ct value are indicative of more efficient cleavage of a specific ate since such reactions reach the threshold cycle faster. In this example amplification and detection are performed in a one-step process, wherein PCR amplification and ' MNAzyme-mediated detection occur simultaneously in a single tube. The amount of time taken to reach the threshold fluorescence, ed by the Ct value ted, can be 1 V influenced by the sequence of the universal substrate.

In this example, previously known universal substrates from series 1 (Sub2, Sub3, Sub6 and Sub7, see Table 4) are compared to new improved universal substrates, series 2 (Sub44, Sub45, Sub46, Sub49, SubSS and Sub60, see Table 5) that are the Subject of the t invention to ine if the series 2 substrates have the same, higher or lower level of activity in real-time PCR as series ‘1 substrates. The level of activity was determined by the Ct obtained for each reaction containing individual substrates during real-time PCR. 1.1." Partzyme Oligonucleotides In the experiments conducted to measure the efficiency of cleavage of universal substrates previously known. (Table 4) and novel universal substrates (Table 5) in real- time, all the me oligonucleotides VA and B were designed with sensor arms complementary to the same sequence of the human RPLPO gene. The sequences of the A and B partzymes are listed below from 5’ to 3’, where the bases underlined hybridizeto their matched substrate. The “-P” indicates 3’ phosphorylation of the oligonucleotide.

SEQ ID NO: 1 partzyme A RPLPOA/Z-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGAGGAAACCTTi SEQ ID NO: 2 partzyme B RPLPOB/Z-P: TGCCCAGGGAGGCTAGCTGTGGAGACGGATTACACCTTCi SEQ ID No: zyme A RPLPOA/3—P: CAAACGAGTCCTGGCCTTGTCTACAACGAGGTTGTGCTG SEQ ID NC: 4 partzyme B /BeP: GTGAGGCTAGCTGTGGAGACGGATTACACCTTC ,SEQ ID NO: 5 partzyme A RPLPOA/6—P: CAAACGAGTCCTGGCCTTGTCTACAACGAGAGGCGTGAT SEQ ID NO: 6 me B RPLPOB/G—P: CTGGGAGGAAGGCTAGCTGTGGAGACGGATTACACCTTC 'SEQ ID NO: 7 partzyme A RPLPOA/7-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGTGCCATGTTAA SEQ ID NO: 8 partzyme B RRLPOB/7-P: TATCACAGCCAAGGCTAGCTGTGGAGACGGATTACACCTTC SEQ ID NO: 9 partzyme A RPLPOA/44eP: CAAACGAGTCCTGGCCTTGTCTACAACGAGAGGAGACCTG SEQ ID NO: 10 partzyme B RPLPOB/44—Pt TCACTATAGGGAGGCTAGCTGTGGAGACGGATTACACCTTC SEQ ID NO: 11 partzyme A RPLPOA/45-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGGGACCCGT SEQ ID NO: 12 partzyme B RPLPOB/45—P: TTCCAAAGGAGAGGCTAGCTGTGGAGACGGATTACACCTTC SEQ ID NO: 13 partzyme A RPLPOA/46—P: CAAACGAGTCCTGGCCTTGTCTACAACGAAGGTGCGGT SEQ ID NO: 14 partzyme B RPLPOB/46~P: GAGCIGGGGAGGCTAGCTGTGGAGACGGATTACACCTTC SEQ ID NO: 15 partzyme A RPLPOA/49-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGAGCCAAGTTTA SEQ ID NO: 16 partzyme A RPLPOA/SS-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGAGGTGCGGT SEQ ID NO: 17 partzyme A RPLPOA/60-P: CAAACGAGTCCTGGCCTTGTCTACAACGAGTGGTTGGC SEQ ID NO: 18 partzyme B RPLPOB/60—P: GTCGTGTTGGAGGCTAGCTGTGGAGACGGATTACACCTTC 1.2. Reporter Substrates The reporter ates tested in this example are. shown below with the sequence, ’ to 3’. The lower case bases represent RNA and the upper case bases represent DNA. In the current example the substrates, other than Sub60, were end labelled with a 6-FAM moiety at the 5’ end and a quencher moiety at the 3’ end. The quencher le was either Black Hole Quencher l (indicated by a “B” in the name of the substrate below) or Iowa Black® FQ (indicated by an “IB” in the name of the substrates below). Sub60 was end labelled with a quencher moiety at the 5’ end and a FAM moiety at the 3 ’ end (due to the 5’ terminal base being a “G” which is known to quench FAM fluorescence). Cleavage of the substrates was monitored between 510-530 nm (FAM on ngth range on CFX96 d)) with excitation between 450—490 nm (FAM tion wavelength range on CFX96 (BioRad)).

SEQ ID NO: 21 SubZ—FIB: AAGGTTTCCTCguCCCTGGGCA SEQ ID NO: 22 Sub3-FB: ‘ CAGCACAACCguCACCAACCG SEQ ID NO: 23 SubG—FIB: ATCACGCCTCguTCCTCCCAG SEQ ID NO: 24 Sub7-FB: TTAACATGGCACguTGGCTGTGATA 3'5 SEQ ID NO: 25 Sub44—FIB: CAGGTCTCCTCguCCCTATAGTGA SEQ ID NO: 26 Sub45-FIB: ACGGGTCCCguCTCCTTTGGAA SEQ ID NO: 27 ‘Sub46—FIB: ACCGCACCTguCCCCAGCTC SEQ ID No: 28 FB: TAAACTTGGCTCguTGGCTGTGATA SEQ ID NO: 29 SubSS—FIB: ACCGCACCTCguCCCCAGCTC IO SEQ ID NO: 30 Subso-IBF: GCCAACCACguCCAACACGAC 1.3. PCR primersfor amplification ofRPLPO The target PCR amplicon for this example was ted by in vitro PCR amplification of human genomic DNA using the oligonucleotide PCR primers listed below. Primer sequences are written 5’ to 3’.

SEQ ID NO: 31 Forward primer 5RPLPO: CTATCATCAACGGGTA SEQ ID NO: 32 Reverse primer 3RPLPO: GCCCACTGTGGTCCTGGTG 1.4. Target sequence .

The target sequence for this example was a PCR amplicon of the RPLPO gene generated by in vitro PCR amplification of human c DNA extracted from K562 cells. 1.5. Reaction Components: Amplification and detection ofa target sequence Real-time PCR amplification and detection of the target ce was performed in ' a total reaction volume of 25 uL. All reactions were conducted in a CFX96 Real-Time PCR Detection System (Bio-Rad). The cycling parameterswere 95°Cfor 10 minutes, 10 cycles of 95°C for 15 seconds and 60°C for 30 seconds (—1°C per cycle for the latter temperature), 40 cycles of 95°C for 15 s and 52°C for 60 seconds (data collected at the 52°C step). ons were set up withsubstrates and their-associated partzymes as, in Table 6. Each set of reaction conditions were run in duplicate and contained 80 nM 5RPLPO and 400 nM of 3RPLPO, 200 nM each of partzyme A and partzyme B, 200 nM of substrate, 8 mM Mng, 200 pM of each dNTP, 10 units RiboSafe RNase inhibitor (Bioline), 1 x Immobuffer (Bioline), 2 units of se (Bioline) and either genomic DNA template (50 ng) or no-DNA target (nuclease free H20 (NF~H20)). Separate reactions were set up to test each substrate with its matched partzymes. The same PCR primers were used for all reactions and all partzymes had the same target-sensing portions. Any ences in efficiency of reactions will ore be attributable to differences in the ncy of cleavage of the substrates.

Table 6: Partzyme combinations used for each universal substrate Sub2 RPLPOA/Z-P RPLPOB/Z-P - SEO ID NO: 21 SEO ID N021 SEO IDNO: 2 ,RPLPOA/3-P RPLPOB/3-P SEQ ID NO; 22 SE 11) NO: 3 SEQ ID NO: 4 RPLPOA/6-P RPLPOB/6-P SEO ID NO: 23 SEO ID NO: 5 . SEO IDNO: 6 Sub7 RPLPOA/7-P RPLPOB/7-P - SEO ID NO: 25 SEO ID NO; 9 ' - SEO ID NO: 10 Sub45 ‘ RPLPOA/45-P RPLPOB/45-P SE ID NO: 26 SEO 11) NO: 11 SEQ ID NO: 12 Sub46 RPLPOA/46-P RPLPOB/46-P SEQ ID NO: 27 SEQ ID NO: 13 SEQ ID NO: 14 Sub49 /49-P - RPLPOB/7-P SE ID NO: 28 SE IDNO: 15 SE lDNO: 8 SEQ ID NO: 29 SE ID NO: 16 SEQ ID NO: 14 RPLPOA/60-P ‘ RPLPOB/éO-P SEQ ID NO: 30 SEQ 11) NO: 17 SEQ ID NO: 18 1.6. Results: Amplification oftarget and cleavage ofreporter substrate Each MNAzyme qPCR reaction containing human genbmic DNA, with each ent substrate, showed [an increase in fluorescence over time for the real-time detection of RPLPO from human genomic DNA. For all substrates, the fluorescence of the no—DNA target control was lower than that in the DNA target-containing reactions.

This demonstrates that the increase in fluorescence ed in target—containing reactions is due to target dependent assembly of catalytically active MNAzymes that then cleaved one of the universal substrates.

The series 1 and 2 substrates all crossed the threshold producing a Ct value, as seen in Table 7.-The series 1 substrates had Ct values in the range from 16.9 (Sub6) to 18.4 (Sub3 and Sub7) and the series 2 substrates had Ct values in the range of 17.1 (Sub55)vto 19.2 ). This indicates that the series 2 substrates are highly active and very able to series 1 substrates under the reaction conditions tested.‘ These s trate that, on average, the substrates that were cleaved with the st efficiency (i.e. lowest Ct) were those with a higher number of pyrimidines in the eight bases surrounding the ribonucleotides in the substrate (underlined in Table 7).

Table 7: Efficiency of cleavage of universal substrates (listed in order of cleavage efficiency based on Ct) # pyrimidines in 8 Name SequenceA bases surrounding ribonucleotides ATCACGCCTCguTCCTCCCAG SEQ ID NO: 23 AAGGTTTCCTCguCCCTGGGCA - SEQ ID NO: 21 ACCGCACCTCguCCCCAGCTC Sub55 SEQ ID NO: 29 ' CAGGTCTCCTCguCCCTATAGTGA SW44 SEQ ID NO: 25 ACCGCACCTguCCCCAGCTC S“b46 SEQ ID NO: 27.

GCCAACCACguCCAACACGAC SW60 SEQ ID NO: 30 .

Sub3 CAGCACAACCguCACCAACCG SEQ ID NO: 22 TTAACATGGCACguTGGCTGTGATA Sub7 SEQ ID NO: 24 Sub49 TAAACTTGGC'TCguTGGCTGTGATA SEQ ID NO: 28 ACGGGTCCCguCTCCTTTGGAA Sub45 SEQ ID NO: 26 " ase represent DNA and lowercase represent RNA * Tm given here equates to the g temperature of the bases bound to the two partzymes calculated using the Wallace rule. When the substrate is bound to the MNAzyme based on the 10-23 DNAzyme'the “g” ribonucleotide remains unbound therefore does not contribute to rall bound Tm.

~ Only one replicate due to experimental error.

Example 2: Use of universal substrates with MNAzymes qPCR at an annealing temperature of 58°C.

MNAzymes can be used to monitor amplification of target nucleic acids in real-time using in vitro target amplification methods such as PCR. Furthermore, real-time monitoring during qPCR using MNAzyme substrates labelled with fluorophore and quencher pairs generates a curve on which a threshold line, of .an ary level of fluorescence, can be placed over the exponential phase of the reactions,‘producing a value which can be known as a Ct (cycle old). Reactions that produce a lower Ct value are indicative of more efficient cleavage of.a specific substrate since such reactions reach the threshold cycle faster. In this example, amplification and detection are performed in a one-step process, wherein PCR amplification and MNAzyme-mediated detection occur simultaneously in a single tube. Where all other reaction conditions are the same the Ct value can be ced by the sequence of the universal substrate. The annealing/detection temperature for MNAzyme qPCR used in the art is between 50 and 54°C. This temperature was dictated by the fact that the universal substrates known in the art had a limitation on the temperature at which they were efficiently cleaved with 54°C being the upper limit for the series 1 universal substrates; There is a need for universal substrates that cleave at higher temperatures to allow greater flexibility in design of primers and partzymes that anneal at higher temperatures. This design flexibility for primers and partzymes could be of great benefit for many ations such as genetic targets of interest that have high percentages of G and C bases in their ce, requiring higher reaction atures and hence partzymes and primers with higher Tms for specific detection. 1 Investigation into efficiency of cleavage of substrates based on the mance of the series 1 and 2 substrates, lead to the development of ines to aid in a third round of substrate designs, resulting in the series 3 subStrates. These guidelines included but were not limited to (i) seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (Nit-N13), (ii) bases immediately adjacent to the cleotides are cytosines (N3 and N9) (iii) total content of substrate has >64% pyrimidines and (iv) total Tm of the oligonucleotide is 66°C or greater (where this latter guideline is only applicable if the reaction temperature for substrate cleavage is above 50°C).

In this example, the series 1 universal substrates (Sub2, Sub3 and Sub6) are ed to the series 2 universal substrates (Sub44, Sub 45, Sub46, Sub60T and , and the series 3 ates (Sub61, Sub65, Sub72, Sub73, Sub74, Sub75, Sub77, Sub79, Sub80, Sub82, Sub83, Sub84, Sub85, Sub86, Sub87, Sub88, sub89 and Sub90) to e the cleavage efficiency of all substrates in real-time PCR at 58°C to ensure that the design guidelines produce universal substrates with a high ility of applicability to MNAzyme qPCR at an elevated temperature. The level of cleavage efficiency was determined by measuring the Ct value for reactions containing different universal substrates. 2.1. me Oligonucleotides . In the experiments conducted to measure the efficiency of ge iof the series I, 2 and 3 universal substrates in real—time PCR, all the partzyme oligonucleotides A and B were ed with sensor arms mentary to the same sequence of the human TFRC gene. The sequences of the A and B partzymes are listed below from 5’ to 3’, where the bases underlined hybridize to their matched universal substrate. The “-1” indicates 3’ phosphorylation of the ucleotide.

SEQ ID NO: 34 partzyme A TFRCA/z-P: . _GGAATATGGAAGGAGACTGTCACAACGAGAGGAAACCTT o SEQ ID NO: 35 partzyme B TFRCB/Z—P: TGCCCAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID No; 36 partzyme A TFRCA/35P: GGAATATGGAAGGAGACTGTCACAACGAGGTTGTGCTG IS SEQ ID No: 37 partzyme B TFRCB/3~P: CGGTTGGTGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 38 partzyme A TFRCA/6-P: GGAATATGGAAGCAGACTGTCACAACGAGAGGCGTGAT SEQ ID NO: 39' partzyme B TFRCB/S-P: CTGGGAGGAAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 40’ partzyme A TFRCA/44—p: GGAATATGGAAGGAGACTGTCACAACGAGAGGAGACCTG SEQ ID' NO: 41 partzyme B TFRCB/44—P: TCACTATAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 42 partzyme A TFRCA/45—P: GGAATATGGAAGGAGACTGTCACAACGAGGGACCCGT SEQ ID NO: 43 partzyme B TFRCB/45—P: iTTCCAAAGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 44 par-tzyme A ’IjFRCA/46-P: ' GGAATATGCAAGGAGACTGTCACAACGAAGGTGCGGTI SEQ ID NO: 45 partzyme B TFRCB/46—P: GGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 46 partzyme A TFRCA/SS-P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCGGT SEQ ID NO: 48 partzyme A TFRCA/60~P; GGAATATGGAAGGAGACTGTCACAACGAGTGGTTGGC SEQ ID NO: 49 partzyme B TFRCB/60—P: TTGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 50 partzyme A TFRCA/6th: GGAATATGGAAGGAGACTGTCACAACGAGGGGTCGAG 'SEQ ID NO: 51 partzyme B TFRCB/6l—P: TGGCGTGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 52‘ partzyme A TFRCA/65—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTCGAGA SEQ ID NO: 55 partzyme B TFRCB/72-P: CTGGGAGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 56 me A TFRCA/73-P: GGAATATGGAAGGAGACTGTCACAACGAGGGGACGCCA> SEQ ID NO: 57‘ partzyme B TFRCB/73—P: IS CACGAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 58 partzyme A TFRCA/74—P: TGGAAGGAGACTGTCACAACGAGGGGAGTGAT SEQ ID NO: 59 partzyme B TFRCB/74-P: CTGGGAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 60 partzyme A TFRCA/754P: GGAATATGGAAGGAGACTGTCACAACGAGAGGAGGGTCA "SEQ ID NO: 61 partzyme B TFRCB/75—P: GGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID No: 62 me A TFRCAX77~Pz GGAATATGGAAGGAGACTGTCACAACGAGAGGGAGGAG SEQ ID NO: 63 partzyme B TFRCB/77-P: AGGAGGAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 64 partzyme A TFRCA/79-P: IGGAATATGGAAGGAGACTGTCACAACGAGGGGAGAGGA SEQ ID N04 65 partzyme B TFRCB/79—P: GGTTGAAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 66 partzyme A TFRCA/804pz GGAATATGGAAGGAGACTGTCACAACGAGAGGGCGGTT SEQ ID NO: 67 partzyme B TFRCB/80~P: GGTTCACGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 68 partzyme B TFRCB/SZ— P: TGGACGAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID.NO; 69 .partzyme A TFRCA/83- P: GGAATATGGAAGGAGACTGTCACAACGAGGGGAGCGGA SEQ ID NO: 70 partzyme B TFRCB/83—P: GTTGCAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 71 partzyme A TFRCA/90— P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGTCGAG IO 2.2. Reporter Substrates The reporter substrates for this example are shown below with the sequence, 5’ to 3’. The lower case bases represent RNA and the upper case bases represent DNA. In the t e, the substrates were end ed with a 6-FAM moiety at the 5’ end (indicated by a “F” in the name of the substrates below) and an Iowa Black® FQ quencher - moiety 'at the. 3’ end (indicated by a “1B5 in the name of the substrates below). The sequence of Sub60 was been modified to include a “T” at the 5’ end, this enabled it to be ’ end-labelled with 64FAM. The partzyme A substrate binding sequence has not changed and therefore cleavage efficiency is comparable to the Sub60 ce in Example 1 which lacks the extra “T” at the 5’ end. Cleavage of the substrates was monitored between, 510-530 nm (FAM emission'wavelength range on CFX96 (BioRad)) with excitation between'450-49O nm (PAM excitation ngth range on CFX96 (BioRad)).

SEQ ID NO: 21 SubZ-FIB: AAGGTTTCCTCguCCCTGGGCA SEQ ID NO; 22 Sub3—FIB: CAGCACAACCguCACCAACCG SEQ ID NO: 23 Sub6-FIB: ATCACGCCTCguTCCTCCCAG SEQ ID NO: 25 FIB: CAGGTCTCCTCguCCCTATAGTGA SEQ ID NO: 26 Sub45—FIB: ACGGGTCCCguCTCCTTTGGAA SEQ ID NO: 27 Sub46rFIB: ACCGCACCTguCCCCAGCTC SEQ ID NO: 29 FIB: ACCGCACCTCguCCCCAGCTC SEQ ID NO: 72 Sub50T*FIB: TGCCAACCACguCCAACACGAC SEQ ID-NO: 73‘ FIB: CTCGACCCCguCTCCACGCCA SEQ ID NO: 74 Sub65*FIB: TCTCGACCTCguCTCCACGCCA SEQ ID NO: 75 Sub72-FIB: ' CCTCguCTCCTCCCAG SEQ ID NO: 76 Sub73-FIB: TGGCGTCCCCguCCCCTCGTG SEQ ID NO: 77 Sub74-FIB: ATCACTCCCCguCCCCTCCCAG SEQ ID NO: 78 Sub75-FIB: TGACCCTCCTCguCTCCCCACTA SEQ ID NO: 79' FIB: CTCCTCCCTCguCCCTCCTCCT SEQ ID NO: 80 Sub79—FIB: TCCTCTCCCCQuCCCCTTCAACC SEQ ID NO: 81 Sub80—FIB: AACCGCCCTCguCCCGTGAACC SEQ ID NO: 82 Sub82-FIB: CTCCTCCCTCguCCCTCGTCCA SEQ ID No} 83 Sub83—FIB: TCCGCTCCCCguCCCCTGCAAC SEQ ID NO: 84 Sub84-FIB: ACCGCACCTCgfiCTCCTCCCAG SEQ ID NO: 85 "Sub85—FIB: ACCGCACCTCguCCCCTCCCAG SEQ ID NO: 86 Sub86—FIB: ATCACGCCTCguCCCCAGCTC SEQ ID NO: 87 Sub87-FIB: VATCACTCCCCguCCCCAGCTC SEQ ID NO: 88 Sub889FIB: CTCCTCCCTCguCCCCAGCTC SEQ ID NO: 89 Sub89—FIB: ACCGCACCTCguCCCTCCTCCT SEQ ID NO: 90 SubQO-FIB: CTCGACCCTCguCCCTCGTCCA 2.3. Target sequence and PCRprimersfor amplification of TFRC The target sequence for this e was a PCR amplicon from the TFRC gene generated by in vitro amplification of human genomic DNA, extracted from the IM9 cell line (Promega), using the oligonucleotide PCR primers listed below. The sequence in bold in the primer sequences corresponds to a sal tag (Ul or U2) that ses the Tm of the primer t affecting the specificity of the primer to the gene target. This tag improves amplification efficiency in PCR reactions. Primer sequences are listed 5’ to SEQ ID NO: 91 Forward primer 5TFRC_U1: GCTAAAACAATAACTCAGAACTTACG ’ SEQ ID NO: 92 Reverse primer 3TFRC__U2 : CAGCTTTCTGAGGTTACCATCCTA 2.4. Reaction Components: Amplification and quantification et sequence Real-time PCR amplification and detection of the target sequence was performed in a total reaction volume of 25 uL. All reactions were conducted in a CFX96 Real-Time PCR Detection System ad). Reactions were set up with substrates and their associated partzymes as in Table 8. The cycling parameters were, 95°C for 2 minutes, 50 cycles of 295°C for 15 seconds and 58°C for 60 seconds (data collected at the 58°C step).

Each set of reaction conditions were run in duplicate and contained 40 nM STFRC‘UI, _25 200 nM of 3TFRC_U2, 200 nM each of partzyme A and partzyme B, 200 nM substrate, 8 mM MgC12, 200 HM of each dNTP, 10 units RiboSafe RNase tor (Bioline), l x Immobuffer (Bioline), 2 units of MyTaqHSTM DNA polymerase (Bioline) and either genomic DNA template (50 ng) or no target (NF-H20).

Table 8: Partzyme combinations used for each universal substrate lISflflflflflHllllllIlIIflfli5HflHEIIIIIIHIIHiEflfiiiEIIIIIII Sub2 Z-P TFRCB/Z P SEQ in NO: 21 SEQ ID NO: 34 SEQ ID NO: 35 Sub3 ' TFRCA/3-P TFRCB/3—P SEQ ID NO: 22 SEQ ID NO: 36 SEQ ID NO 37 IIIIIIIIIIIII ' , TFRCA/G-P 4 TFRCB/6—P SE 0, ID NO: 23 SEQ ID NO: 39 Sub44 TFRCA/44 TFRCB/44-P . Sub45 TFRCA/45-P TFRCB/45-P SEO ID NO: 26 SEO ID NO: 42 SE ID NO: 43 . SEQ ID NO: 27 SEO ID NO: 44 SEQ ID NO: 45 Sub55 SS-P 1 TFRCB/46-P SEQ ID NO: 72 SEQ ID NO: 48 ' . SEQ ID NO: 49 SEQ ID NO: 73 SEO ID NO: 50 SEQ ID NO: 51 TFRCA/65-P TFRCB/6LP SEQ ID NO: 74 SEQ ID NO: 52 SE ID NO: 51 » 6-P TFRCB/72-P SEQ ID NO: 75 SEQ ID NO: 38 SEQ ID NO: 55 Sub73 TFRCA/73-P TFRCB/73-P SEO ID NO: 76 SEO ID NO: 56 SEO IDNO: 57 ' SEO ID NO: 77 SEQ ID NO: 58 SEO ID NO: 59 TFRCA/75-P TFRCB/75-P SEO ID NO: 78 SEO ID NO: 60 . SEO ID NO: 61 - TFRCA/77-P TFRCB/77-P SEQ ID NO: 79' SEQ ID NO: 62 ‘ SEO ID NO: 63 TFRCA/79-P TFRCB/79-P SEO ID NO: 80 SEQ ID NO: 64 - SEQ ID NO: 65 TFRCA/BO—P TFRCB/80-P SEO ID NO: 81 SEO ID NO: 66 SEO ID NO: 67 Sub82 TFRCA/77-P TFRCB/82-P SEO ID NO: 82 SEQ ID NO: 62 SEO ID NO: 68 ,- Sub83 TFRCA/83-P TFRCB/83-P SEO ID NO: 83 SEO ID NO: 69 SEO ID NO: 70 Sub84 TFRCA/SS-P TFRCB/72-P SEO ID NO: 84 ‘ SEO ID NO: 46 SEO ID NO: 55 TFRCA/SS-P A 74—P.

. SEO ID NO: 85 . SEO ID NO: 46 SEO ID NO: 59 TFRCA/6-P TFRCB/46—P SEQ ID NO: 86 SEO ID NO: 38 SEQ ID NO: 45 ' Sub87 TFRCA/74-P ' A TFRCB/46-P SEO ID NO: 87 SEO ID NO: 58 SEO ID NO: 45 TFRCA/77-P TFRCB/46-P SE ID NO: 83 SEQ ID NO: 62 SEO ID NO: 45 TFRCA/SS-P TFRCB/77-P SEQ ID NO: 89 SE. ID NO: 46 SEQ ID NO: 63 Sub90 TFRCA/90-PV TFRCB/82-P 'SE ID NO: 90 SEO ID NO: 71 SEO ID NO: 68 2.5. Results: Amplification et and cleavage ofreporter substrate Each MNAzyme qPCR reaction containing human genomic DNA showed an. increase in fluorescence over time for the realtime detection of TFRC from human genomic DNA. For all reactions the fluorescence of the no-DNA target l was lower than that in the DNA target-containing ons. This demonstrates that the increase in fluorescence produced in target-containing reactions is due to target dependent assembly of catalytically. active MNAzymes that then d one of the universal reporter substrates.

Comparison of the Ct values for each sal substrate (Figure 5 and Table 9) IO show the series 1 substrates (Sub2, Sub3'and Sub6) and series 2 substrates (Sub44, Sub45, Sub46 and Sub60T) all have Ct values > 27, whereas the other series 2 and all series 3 substrates tested (Sub55, Sub6l, Sub65, Sub72, Sub73, Sub74, Sub75, Sub77, Sub79, Sub80, Sub82, Sub83, Sub84, Sub85, Sub86, Sub87, Sub88, Sub89 and Sub90) had Ct values less than 27. This indicates that the latter series 2 universal substrate and all series 3. universal substrates tested showed increased ncy of the MNAzyme cleavage reaction at a higher annealing/detection temperature than that previously possible for MNAzyme qPCR. This ed efficiency of cleavage now permits efficient and robust detection of target using MNAzyme qPCR at a higher temperature than previously possible. This may also prove beneficial when using DNA polymerase formulations that require a higher temperature for amplification.

Of note is the importance of the nature. of the nucleotide sequence of these ntly cleaved substrates and the proximity of specific nucleotides to the ribonucleotides of the substrates. These features form the basis of a set of guidelines that result in universal substrates with a higher probability of being cleaved efficiently at elevated temperatures. These design guidelines include but are not limited to (not all may be necessary): (i) seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (N4-N13); (ii) the bases immediately adjacent to the cleotides are cytosines (Ng‘ and N9); (iii) total content of ate has >64% ’pyrimidine’s; (iv) total Tm of the oligonucleotide is 66°C or r (where this latter guideline is only applicable if the reaction temperature for substrate cleavage is above 50°C) (Table 9). In addition, it was ed that a low number of guanine nucleotides (e.g. three, two, one or none)" in the 10 bases surrounding the ribonucleotides is also beneficial.

All universal substrates in Figure 5 that had at Ct < 27 at an annealing temperature of 58°C obeyed three or more of these design guidelines (Table 9).

Table 9: Efficiency of cleavage of universal substrates (listed in order of cleavage efficiency based on Ct) Sub80 AACCGCCCTCguCCCGTGAACC SEQ ID NO: 81 ACCGCACCTCucccc__g_____AGCTc SEQ ID NQ 29 ACCGCACC__T_______CguCCCCTCCCAG SEQ ID NO. 85 TCCTCTC__C_________CCguCCCCTCCTACC SEQ ID NO. 80 CTCGA__C___g____cccuCTCCACGCCA SEQ ID NO. 73 CCTCuCTCC‘ACGCCA SEQ ID NO 74 --fl-— , Sub89 ACCGCACCTCguCCCTCCTCCT SEQ ID NO: 89 --- CCTCguCCCTCGTCCA Sub90 N kll \l SEQ ID NO: 90 sub87 ATCACTCCCCguCCCCAGCTC NVq SEQ ID NO: 87 Sub83 TCCGCTCCCCguCCCCTGCAAC \1 N Myr 00 SEQ ID NO: 83 Sub86 ATCACGCCTCgECCCCAGCTC. N9v SEQ ID NO: 86 _—SEQID NO: N?\N \l o m9N SEQ ID NO: 82 Sub74 ATCACTCCCCQECCCCTCCCAG \I o N0 ha SEQ .

ID NO: 77 S“b84 ACCGCACCTCguCTCCTCCCAG \l o N9m SEQ ID NO: 84 EHIIHAVSub75 TGACCCTCCTCuCTCCCCACTA \l o N9u) SEQ ID NO: 78 Sub73 TGGCGTCCCCuCCCCTCGTG \J o_ N37‘w SEQ ID NO- 76 CTCCTCCCTC CCCTCCTCCT \1 o N_O\ DJ ATCACGCCTC CTCCTCCCAG Sub2 AAGGTTTCCTCguCCCTGGGCA N\l N SEQ ID NQ 21 S“b6°T§ TGCCAACCACCCAACACGAC N \l b) SEQ ID NO: 72 ACCGCACCTguCCCCAGCTC S“b46 N\l (I! SEQ ID NO 27 ACGGGTCCCguCTCCTTTGGAA Sub45 NQ G SEQ ID NQ 26 AACCguCACCAACCG lN0° CAGGTCTCCTCguCCCTATAGTGA N 00 A ATCACGCCTCguTCCTCCCAG . l|H||||||IIEI||||||||||||||||llllllllllllillllillIlilIlillIlHIlllHIlII:||||H|||:||IIH|1O ase bases represent DNA and lowercase bases represent RNA and position of base in a substrate is represented by (NJ-N l-N1-N3'N4'N5'Ng'NrNg‘l'R'fY‘N9’N ,o-N, l-N ,2-N 13-N .4-N . 5-(Nx) + % C/T (pyrimidines) of sequence length shown above for each substrate, does not include ribonucleotides * Tm given here equates to the melting temperature of the bound bases calculated using the Wallace rule only calculated for bases that hybridize to their complement. When the substrate is bound to the MNAzyme based on the 10-23 DNAzyme the “g” cleotide remains unbound therefore does not contribute to the overall bound Tm.

~ The number of the design guidelines (i), (ii), (iii) and/or (iv) that have been met by the substrate sequence.

§ The additional “T” in Sub60T is not bound by a partzyme arm and is therefore not included in the IO calculations of% OT and Tm.

Example 3: Use of universal substrates with MNAzymes in a format for direct detection of a nucleic acid target. igation into efficienéy of ge of substrates based on the performance of the series 1 and 2 ates, lead to the development of guidelines to aid in designing a third round of substrates, series 3. These guidelines included but were not d to (i) seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (N4- N13), (ii) bases immediately adjacent to the ribonucleotides are cytosines (N8 and N9) (iii) total content of substrate has >64% pyrimidines and (iv) total Tm of the oligonucleotide is 66°C or greater (where this latter ine is only able if the reaction temperature for substrate cleavage is above 50°C).

MNAzymes can be used to directly detect target nucleic acids in an isothermal reaction without any target amplification. This method of direct target detection can be used to assess the efficiency of cleavage of substrates. Partzymes were designed to test the efficiency of cleavage of a range of Sal substrates when coupled with direct detection of the gene TFRC at a range of temperatures. In this example, the previously known series 1 universal ates (SubZ, Sub3, and Sub6) were compared to the series 2 universal substrates (Sub44, Sub45, Sub46, Sub49, Sub55 and Sub60T) and series 3 substrates (Sub61‘, Sub65, Sub72, Sub73, Sub74, Sub75, Sub77, Sub79, Sub80, Sub82, 3O Sub83, Sub84, Sub85, Sub86, Sub87, Sub88, Sub89 and Sub90) to determine if the design guidelines d from analyses of series 1 and 2 substrates would be useful in the development of series 3 substrates that are cleaved with the same or a higher level of activity as substrates from series 1 and 2. The level of cleavage efficiency was determined by calculating the signal to noise ratio (from s of a “test” reaction containing template and a no template control reaction) afler 10 s over a temperature range of 52, 54, 56-and 58°C. The standard deviatiOn of the signal to noise ratios over this ature range was also calculated as a measure of robustness of the substrates with regards to ature. 3.1. Partzyme Oligonucleotides In the experiments conducted to measure the cleavage efficiency of series 1, 2 and 3 universal substrates described in Tables 4 and 5 using direct target detection, all the partzyme oligonucleotides A and B were designed with sensor arms mentary to the same sequence of the human TFRC gene. The sequences of the A and B partzymes . are listed below from 5’ to 3’, where the bases underlined hybridize to the substrate; The “-P” indicates 3’ phosphorylation of the oligonucleotide.

SEQ ID NO: 34 partzyme A TFRCA/Z—P: ' GGAATATGGAAGGAGACTGTCACAACGAGAGGAAACCTT SEQ ID No; 35 partzyme B Z—P: TGCCCAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT IS SEQ ID NO: 36 .partzyme A TFRCA/3—P: GGAATATGGAAGGAGACTGTCACAACGAGGTTGTGCTG SEQ ID NO: 37 partzyme B ’I‘FRCB/3eP: CGGTTGGTGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 38 partzyme A TFRCA/6—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGCGTGAT SEQ ID NO: 39 partzyme B TFRCB/S—P: CTGGGAGGAAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 40 partzyme A TFRCA/44~P:l GGAATATGGAAGGAGACTGTCACAACGAGAGGAGACCTG SEQ ID NO: 41 ‘partzyme B TFRCB/44—P: TCACTATAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 42 partzyme A TFRCA/45—P: GGAATATGGAAGGAGACTGTCACAACGAGGGACCCGT SEQ ID NO: 43 partzyme B TFRCB/45-P: TTCCAAAGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT, SEQ ID NO: 44 partzyme A TFRCA/46—P: GGAATATGGAAGGAGACTGTCACAACGAAGGTGCGGT SEQ ID NO: 45 partzyme B TFRCB/46—P: IGAGCTGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 93 partzyme A TFRCA/49—P: GGAATATGGAAGGAGACTGTCACAACGAGAGCCAAGTTTA SEQ ID NO: 94 partzyme B TFRCB/49—P: TATCACAGCCAAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 46 partzyme A TFRCA/SS—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCGGT SEQ ID NO: 48 partzyme A TFRCA/60—P: GGAATATGGAAGGAGACTGTCACAACGAGTGGTTGGC SEQ ID NO: 49 partzyme B TFRCB/60—P: GTCGTGTTGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 50 partzyme A TFRCA/6l-P: TGGAAGGAGACTGTCACAACGAGGGGTCGAG SEQ ID NO '51 me B sl—Pi TGGCGTGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 52 partzyme A TFRCA/65—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTCGAGA SEQ ID NO: 55 partzyme B TFRCB/72—P: CTGGGAGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 56 partzyme A TFRCA/73—P: GGAATATGGAAGGAGACTGTCACAACGAGGGGACGCCA SEQ ID NO: 57 _partzyme B TFRCB/73~P: CACGAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 58 me A TFRcA/74—P: GGAATATGGAAGGAGACTGTCACAACGAGGGGAGTGAT SEQ ID NO: 59 partzyme B TFRCB/74—P: . CTGGGAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 60 partzyme A TFRCA/755P: GGAATATGGAAGGAGACTGTCACAACGAGAGGAGGGTCA SEQ ID NO: 61 me B TFRCB/75-P: TAGTGGGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 62 partzyme A TFRCA/77—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGAGGAG SEQ ID NO: 63‘ partzyme B TFRCB/77—P: AGGAGGAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO; 64 partzyme A TFRCA/79-P: GGAATATGGAAGGAGACTGTCACAACGAGGGGAGAGGA SEQ ID NO: 65 partzyme B TFRCB/79-P: GGTTGAAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 66 partzyme A TFRCA/BO—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGCGGTT SEQ ID NO: 67 partzyme B BO-P: GGTTCACGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 68 partzyme-B 82—P: TGGACGAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 69 partzyme A TFRCA/BB-Pzi . GGAATATGGAAGGAGACTGTCACAACGAGGGGAGCGGA SEQ ID NO: 70 partzyme B TFRCB/83-P: GTTGCAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 71 partiyme A TFRcA/QO-P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGTCGAG 3.2. Reporter Substrates The‘reporter ates for this example are shown below with the sequence, 5’ to 3’. The lower case bases represent RNA and the. upper case bases represent’DNA. In the current example, the substrates were. end labelled with a 6-FAM moiety at the 5’ end (indicated by a “F” in the name of the'substrates below) and an Iowa Black® FQ quencher moiety at the 3’ end (indicated by an “1B”. in the name of the substrates . Cleavage of the substrates was monitored betWeen 510-530 nm (FAM on wavelength range on CFX96 (BioRad)) with excitation between 450-490 nm (FAM excitatiOn wavelength . range on CFX96 (BioRad)), SEQ ID NO: 21 SubZ—FIB: AAGGTTTCCTCguCCCTGGGCA SEQ ID NO: 22, Sub3-FIB: CAGCACAACCguCACCAACCG SEQ ID NO: 23 'Subs-FIB: ATCACGCCTCguTCCTCCCAG SEQ ID NO: 25 Sub44~FIB: CAGGTCTCCTCgUCCCTATAGTGA SEQ‘ID NO: 26 Sub45-FIB:- ACGGGTCCCguCTCCTTTGGAA SEQ ID NO: 27 Sub46-FIB: ACCGCACCTguCCCCAGCTC SEQ ID NO: 28 Sub495FB: TAAACTTGGCTCguTGGCTGTGATA SEQ ID NO: 29 SubSS-FIB: CCTCguCCCCAGCTC SEQ ID NO: 72 Sub60T—FIB: TGCCAACCACguCCAACACGAC SEQ ID NO: 73 SubGl-FIB: CTCGACCCCguCTCCACGCCA SEQ ID NO: 74 Sub65~FIB$ TCTCGACCTCguCTCCACGCCA SEQ ID NO: 75 —FIB: ATCACGCCTCguCTCCTCCCAG SEQ ID No: 76 Sub73—FIB: IS TGGCGTCCCCguCCCCTCGTG SEQ ID NO: 77 Sub74—FIB: ATCACTCCCCguCCCCTCCCAG SEQ ID NO: 78 Sub75-FIB: TGACCCTCCTCguCTCCCCACTA SEQ ID NO: 79. FIB: CTCCTCCCTCguCCCTCCTCCT SEQ ID NO: 80 Sub79-FIB: TCCTCTCCCCguCCCCTTCAACC SEQ ID NO: 81 ‘SubSO-FIB: AACCGCCCTCguCCCGTGAACC SEQ ID NO: 82 Sub82—FIB: CTCCTCCCTCguCCCTCGTCCA SEQ ID NO: 83 Sub83—FIB: TCCGCTCCCCguCCCCTGCAAC SEQ ID NO: 84 Sub84—FIB: ACCGQACCTCguCTCCTCCCAG SEQ ID NO: 85 Sub85—FIB: ACCGCACCTCguCCCCTCCCAG .SEQ ID NO: 86' VSub86—FIB: ATCACGCCTCguCCCCAGCTC SEQ ID NO: 87 Sub87—FIB: ATCACTCCCCguCCCCAGCTC SEQ ID NO: 88 Sub88—FIB: CTCCTCCCTCguCCCCAGCTC SEQ ID NO: 89 Sub89-FIB: ACCGCACCTCguCCCTCCTCCT SEQ ID NO: 90 Sub90-FIB: CTCGACCCTCguCCCTCGTCCA 3.3. Target sequence The target sequence for this example was a synthetic oligonucleotide AF-TFRC with the sequence, 5" to 3’ below. This target ce has the same Sequence as a n of the TFRC gene, SEQ ID NO: 95 Assembly tator AF-TFRC:C IS AGTCTGTTTTCCAGTCAGAGGGACAGTCTCCTTCCATATTCC 3. 4. Reaction Components: Direct isothermal detection oftarget sequence Detection of the target sequence was measured by an increase in fluorescent signal caused by\ cleavage of the er substrate by the catalytically active MNAzyme. The total volume of all ons was ~25 uL and all reactions were conducted on the C-FX96TM Real-Time PCR Detection Systems (BioRad), with each combination of partzymes and substrates (Table 10) being tested at 52°C, 54°C, 56°C, 58°C and 60°C. Fluorescence for each reaction was programmed to be read after 1 second for the first 50 cycles and then programmed to be read after 25 seconds for the next 50 cycles. All reactions contained 1 x PCR Buffer II (Applied Biosystems), 10 mM MgC12, and 0.2 uM of Partzymes A and B and 0.2 uM ate (tested in combinations as in Table 10). Each reaction was performed in duplicate as either a “test” with 10 nM target sequence (AF-TFRC) or no- template control (NF- H20).

Table 10: Partzyme combinations used for each substrate Substrate &m2 TFRCAQJ’ TFRCBQJ" .

Sub3 3—P TFRCB/3-P SEQ ID NO: 22 SEQ ID NO: 36 SEQ ID NO: 37 - IISMIIIIIIIIIIIIIIIEERIHIEHIIIIIIIIIIEERIEHEHIIIIIIII . SEQ ID NO: 23 SEQ IDNO: 38 SEO ID NO: 3.9 TFRCA/44 TFRCB/44-P SEQ ID NO. 25 SE ID NO: 40 SEO ID NO: 41 Sub45 TFRCA/45-P TFRCB/45-P SEO ID NO: 26 SEO IDNO: 42 SEO IDNO: 43 SEO ID NO: 27 SEO IDNO: 44 SEO ID NO: 45 - TFRCA/49-P 49-P SEO ID NO: 28 SEO ID NO: 94 . SEQ ID NO: 93 SEQ ID NO: 29 SEQ ID NO: 46 SEQ ID NO: 45 Sub60T ' . TFRCA/60-P TFRCB/60-P Sub61 TFRCA/61 -P TFRCB/6 1 -P SEO ID NO‘: 73 SEO IDNO: 50 4 SEO IDNO: 51' Sub65 TFRCA/6S-P TFRCB/61-P SEQ ID NO: 74 SEO ID NO: 52 Sub72 , 6-P TFRCB/72-P SEO IDNO: 75 SEO ID NO: 38 SEO ID NO: 55 ' Sub73 TFRCA/73-P TFRCB/73-P SEO ID NO: 76 SEO IDN0156 SEO ID NO: 57 TFRCA/74-P TFRCB/74-P SE ID NO: 77 SE IDNO: 58 SE ID NO: 59 SEQ ID NO: 78 SEQ ID NO: 60 SEQ ID NO: 61 SEO ID NO: 79 SEO ID NO: 62 SEQ ID NO: 63 TFRCA/79-P TFRCB/79-P SEO ID NO: 80 SEO ID NO: 64 SEO ID NO: 65 TFRCA/80-P TFRCB/80-P SEO IDNO: 81 SEO ID NO: 66 SEO ID NO: 67 TFRCA/77-P TFRCB/82-P ‘ SEO ID NO: 82 SE ID N05 62 SE ID NO: 68 TFRCA/83-P TFRCB/83-P SEQ ID NO: 83 SEQ ID NO: 69 ' SEO ID NO: 70 SE ID NO: 34 . SE ID NO: 46 SE ID NO: 55 Sub85 TFRCA/SS-P SEQ ID NO: 85 SE ID NO: 46 SEQ ID NO: 59 Sub86 TFRCA/6-P TFRCB/46-P SEO IDNO: 86 SE ID NO: 33 SE ID 140545 TFRCAX74-P TFRCB/46-P SEO ID NO: 87 SEO ID NO: 58 .SEO ID NO: 45 TFRCA/77-P TFRCB/46-P SEQ ID NO: 88 SEO ID NO: 62 SEO ID NO: 45 SS-P TFRCB/77-P SEO ID NO: 89 SEO ID NO: 46 SEO ID NO: 63 TFRCA/90-P TFRCB/82-P ' SEO ID NO: 90 SEQ ID NO: 71 SEQ ID NO: 68 3.5. Results: Direct isothermal detection oftarget sequence Each reaction with each universal ate showed an increase in fluorescence over time for reactions containing the tic template AF-TFRC (target sequence ponding to a portion of the TFRC gene). For all substrates, the fluorescence of the no-template control was lower than that in the target ce—containing reactions. This demonstrates that the increase in fluorescence ed in target-containing reactions is due to target dependent assembly of catalytically active MNAzymes that then cleaved the universal reporter substrate.

For each reaction, the raw fluorescent data points obtained from the CFX96 were IO ised by dividing each data point by the value obtained for the paired no-template reaction at the first reading. This normalised data was then used to calculate the signal to noise value at approximately the 10 minute mark by dividing test data points by the no template data points. This calculation was performed for each substrate at each reaction ature. The signal to noise value provides a measurement of the efficiency of the cleavage of substrates (Figure 6, (i)), with a high signal to noise indicating efficient ge. The standard deviation of the signal to noise ratio for each substrate over the temperature range was also calculated and plotted to determine substrates with consistently high activity over the tested range of temperatures (Figure 6, (ii)). A low value for this standard deviation indicates l change of signal to noise levels between temperatures. This suggests that these substrates are robust with respect to temperature.

Analysis of the signal to noise ratio for each substrate at the range of temperatures (Figure 6, (i)) shows that the series I substrates (Sub2, Sub3 and Sub6) had a higher signal to noise at the lower temperatures measured. However the cleavage efficiency of these substrates dropped dramatically as the reaction temperature was increased to 58°C.

A similar pattern was seen for a subset of the series 2 substrates (Sub44, Sub45, Sub46, and Sub6OT). The series 2 ate, Sub49, performed poorly at the lower temperatures and ly better at the higher but overall had lower signal to noise than other substrates.

The series 2 substrate Sub60T showed a decrease in signal to noise with increasing ' temperature, and overall lower signal to noise at the lower temperatures than the majority of other series 1 and 2 substrates. The other series 2 substrate (SubSS), and a subset of the series 3 substrates (Sub6l, Sub65, Sub74, Sub79, Sub80, Sub82, Sub83, Sub84, Sub85, Sub86, Sub87, Sub88, Sub89 and Sub90) displayed high fluorescence levels and hence were efficiency cleaved across all the temperatures tested. The series 3 substrates Sub73, Sub75 and Sub77 yed roughly the same signal to noise value across the temperatures tested, however the overall signal to noise level was low. These three substrates all gave good results with relatively low Ct values when tested with MNAzyme qPCR (see Example 2). Comparison of the data for these substrates for Examples 2 and 3 may indicate that when a constant temperature in the range of 52 to 58°C is used the turnover of these ates is lower thus affecting the cleavage efficiency. This decreased substrate turnover could be related to the “oft” rate of cleaved products. In Example 2, the cleaved products would'dissociate from the partzyme ate arms at least once a cycle when the ature was increased to above 90°C as part of the thermocycling profile of PCR.

Overall, the substrates that conform to the design guidelines (Table 9) showed a greater signal to noise ratio across the tested temperature range than the substrates that fell outside these guidelines (Figure 6, (i)). More specifically reactions with SubSS, Sub61, Sub65, Sub72, Sub74, Sub79, Sub80, Sub82, Sub83, Sub84, Sub85, Sub86, Sub87, Sub88, Sub89 and Sub90 displayed high signal .to noise values at every is temperature tested demonstrating that these are robust substrates over a range of temperatures. This ement was further evident when the rd deviation was calculated from the signal to noise ratio across the temperatures for each substrate (Figure 6, (ii)). This measure of variability paired with the absolute values of signal to noise indicated that, over the temperature range tested, the series 2 substrate SubSS, and the series 3 ates Sub61, Sub65, Sub74, Sub79, Sub80, Sub82, Sub83, Sub85, Sub86, Sub87 Sub88, Sub89 and Sub90, had a high signal to noise ratio with little variability across a broad temperature range. There were three series 3 substrates, Sub65, Sub72 and Sub84, that only match three of the four design ines and two of these, Sub72 and Sub84, had a ly greater standard deviation of the signal to noise than the series 3 substrates that match all four of the design guidelines as specified in Table 9.

Substrates that had signal to noise values less than 1.6 at 3 or more temperatures (Sub60, Sub73, Sub75 and Sub77) were'not considered robust with respect to the range of temperatures tested.

These data suggest that compliance with all four of these design guidelines (Table 9) will, in general, produce'substrates that are cleaved efficiently and robustly over a range of temperatures.

A study of the sequence of the most successful substrates from series 2 and 3 shows that these substrates share common features. Substrates that have little variation between signal to noise ratios over the temperatures tested (Figure 6, (i) and (ii)) generally contained seven or more ne nucleotides within the bases N4 to N13. This indicates that the core region, i.e. the 10 bases surrounding the ribonucleotides, is highly influential on substrate activity. In addition, it was ed that a low number of guanine nucleotides (e.g. three, two, one or none) in the 10 bases nding the ribonucleotides is also beneficial.

Example 4: Use of universal substrates with MNAzyme qPCR, at annealing temperatures of 52°C and 58°C.

MNAzymes can be used to monitor cation of target nucleic acids in ime using in vitro target amplification methods such as PCR. Further, real-time monitoring during qPCR using MNAzyme substrates labelled with fluorophores and quencher pairs generates a curve that can indicate the efficiency of a reaction by its Ct value and steepness (reaction rate). In this example amplification and detection are performed in a ep process, wherein PCR amplification and MNAzyme-mediated detection occur simultaneously in a single tube. The rate of production of signal red by Ct and steepness of reaction enrves) at different annealing temperatures such as 52°C and 58°C (the temperature which data was collected), can be influenced by the sequence of the universal substrate.

The ing/detection temperature for MNAzyme qPCR, used in the art is between 50 and 54°C. This temperature was dictated by the fact that the universal substrates known in the art had a limitation on the temperature at which they were efficiently cleaved with 54°C being the upperlimit for the series 1 universal substrates.

There is a need for universal substrates that clea‘ve at higher temperatures to allow greater flexibility in design of primers and pa'rtzymes that anneal at higher temperatures. This design flexibility for s and partzymes would be of great benefit for many applications such as genetic targets of interest that have high percentages of G and C bases in their sequence, requiring partzymes and primers with higher Tms for specific detection. Utility of universal substrates would be greatly sed if Substrates d that were efficiently cleaved at a range of temperatures between 52 and 58°C.

In this example, partzymes corresponding to series 1, 2 and 3 universal substrates, 3O were designed to target a range of genes, as outlined in Table 11. One skilled in the art would appreciate that any gene sequence or gene transcript or any other nucleic acid amplification product could be used as a target as bed here. Each combination of partzymes and their associated universal substrates were tested at annealing temperatures of 52°C and 58°C in qPCR. The s from this comparison will determine if series 1, 2 and 3 substrates ed to different genes, and at different ing temperatures allow the same, higher or lower level of cleavage efficiency in real-time PCR. The level of cleavage efficiency was determined by measuring the Ct value and looking at the ess of reaction curves for reactions containing different universal substrates.

Table 11. Substrates used to detect different genes by MNAzyme qPCR . ate ’ Sub6l Sub72, Sub74, and Sub79 Sub79 Illliflflflllll Sub80 ’ Sub80 ‘and Sub88 4.1. Partzyme ucleoiides In the experiments conducted to measure the efficiency of cleavage of the universal substrates in real-time PCR, the partzyme oligonucleotides A and B were designed with sensor arms complementary to the human CYP2C9, TP53, 82M, HMBS, RPLi3a or r TFRC genes. The ces of the A and B partzymes are listed below from 5’ to 3’, where the bases underlined hybridize to the substrate. The “-P” indicates 3’ phospho‘rylation of the ucleotide.

SEQ ID NO: 96 partzyme A CYP2C9A/3—P: GGGAAGAGGAGCATTGAGGAACAACGAGGTTGTGCTG SEQ ID NO: 97 partzyme B CYP2C9B/3-sz CGGTTGGTGAGGCTAGCTCCGTGTTCAAGAGGAAGC SEQ ID NO: 98 me’A CYP2C9A/6l—P: GGGAAGAGGAGCATTGAGGAACAACGAGGGGTCGAG SEQ ID No} 99 . partzyme B CYP2C9B/6l-P: TGGCGTGGAGAGGCTAGCTCCGTGTTCAAGAGGAAGC SEQ ID NO: 100 partzyme' A TP53A/6—P: GACGGAACAGCTTTGAGGTGACAACGAGAGGCGTGAT SEQ ID NO: 1011 partzyne B TP53B/6-P: CTGGGAGGAAGGCTAGCTCGTGTTTGTGCCTGTCCTGG SEQ ID NO: 103 partzyme B TPS3B/72—P: CTGGGAGGAGAGGCTAGCTCGTGTTTGTGCCTGTCCTGG ’SEQ ID NO: 104 partzyme A TP53A/74-P: GACGGAACAGCTTTGAGGTGACAACGAGGGGAGTGAT SEQ ID NO: 105 partzyme B TP53B/74~P: CTGGGAGGGGAGGCTAGCTCGTGTTTGTGCCTGTCCTGG SEQ ID NO: 106 partzyme A TP53A/79—P: ACAGCTTTGAGGTGACAACGAGGGGAGAGGA SEQ ID NO: 107 partzyme B TP53B/79—P: GGTTGAAGGGGAGGCTAGCTCGTGTTTGTGCCTGTCCTGG’ w- SEQ ID NO: 108 partzyme A B2MA/6OFP: ‘ATTCAGGTTTACTCACGTCATCACAACGAGTGGTTGGC SEQ ID NO: 109 partzyme B 0-P: GTCGTGTTGGAGGCTAGCTCAGCAGAGAATGGAAAGTCAAAI SEQ ID NO: 110 partzyme A 1—P ATTCAGGTTTACTCACGTCATCACAACGAGGGGTCGAG SEQ ID NO: 111 partzyme B B2MB/61—P TGGCGTGGAGAGGCTAGCTCAGCAGAGAATGGAAAGTCAAA ID NO: 112 . SEQ partzyme A 9—P GTTTACTCACGTCATCACAACGAGGGGAGAGGA 2O SEQ ID N0= 113 partzyme B B2MB/79—P GGTTGAAGGGGAGGCTAGCTCAGCAGAGAATGGAAAGTCAAA SEQ ID NO: 114 * partzyme A HMBSA/49—P: GCCATGTCTGGTAACGGCAAACAACGAGAGCCAAGTTTA SEQ ID.NO: 115 partzyme B HMBSB/495P: TATCACAGCCAAGGCTAGCTTGCGGCTGCAACGGCGGTG SEQ ID NO: 116 partzyme A HMBSA/75-P: GCCATGTCTGGTAACGGCAAACAACGAGAGGAGGGTCA SEQ ID NO: 117 partzyme B HMBSB/75—P: TAGTGGGGAGAGGCTAGCTTGCGGCTGCAACGGCGGTG SEQ ID NO: 34 partzyme A TFRCA/Z—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGAAACCTT SEQ ID NO: 35 partzyme B TFRCB/Z-P: TGCCCAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 38 partzyme A TFRCA/72—P: ' GGAATATGGAAGGAGACTGTCACAACGAGAGGCGTGAT SEQ ID NO: 55 partzyme B TFRCB/72—P: 'CTGGGAGGAGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 66 - partzyme A TFRCA/BO—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGCGGTT SEQ ID NO: 67 _ partzyme B TFRCB/BO—P: GGTTCACGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 118 partzyme A A/55—P TTGACAAATACACAGAGGTCACAACGAGAGGTGCGGT SEQ ID NO: 119 partzyme B RPLl3aB/55-P GAGCTGGGGAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 120 partzyme A A/80wP AATTGACAAATACACAGAGGTCACAACGAGAGGGCGGTT SEQ ID NO: 121 partzyme B RPLlBaB/80—P GGTTCACGGGAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 122 partzyme A RPDlBaA/88—P AATTGACAAATACACAGAGGTCACAACGAGAGGGAGGAG 4.2. . Reporter Substrates In the current e, the substrates were 5’ end labelled with a hore and 3’ end labelled with a quencher moiety. Table 12» depicts. the Substrate — hore/quencher combinations. Some substrates were tested with more than one particular fluorophore/quencher ation. Cleavage of the substrates was red at various emission and excitation wavelengths (Table 12).

Table 12. Substrates and their fluorescent labelling SubstrateW Sub49 ' Sub49-FB .

Sub3-FIB 6-FAM ' “0‘4” “0'5” Sub6l Sub61-FIB Sub75 Sub75-FIB SubSS SubSS-l-IIB ' Sub80 Sub80«HIB . HEX 515-535 560-580 Sub88 Sub88-HIB Sub6-TRBZ‘ sum subn'TRIBR Texas Red n 560-590 610-650 Sub74—TRIBR Sub79 Sub79 TRIBR Sub2-O67OB2 'Quasar670 BHQZ 620-650 675-690 Sub72 - Sub72 067082 -Sub80-67OBZ_——— Sub60 Sub60-Q7OSB2 Sub61 Sub61-O7OSBZ <2uasar705 BPflQZ 672-684 705—730 Sub79 Sub794270532 ' A BHQI; black hole quencher l, BHQ2; black hole quencher 2, IB; Iowa black® FQ, IBR; Iowa black® RQ *CFX96 Real-Time PCR ion System (Biorad) excites, and measures emission of, each fluorophore over a range of wavelengths for each channel.

The reporter substrates tested in this example are shown below with the sequence, ‘5" to 3’. The lower casebases represent RNA and the upper case bases ent DNA; SEQ ID No; 21 SubZ: AAGGTTTCCTCguCCCTGGGCA SEQ ID NO: 22 Sub3: IO CAGCACAACCguCACCAACCG .SEQ ID NO: 23 SUb6: ATCACGCCTCguTCCTCCCAG SEQ ID NO: 28 Sub49: TAAACTTGGCTCguTGGCTGTGATA SEQ ID NO: 29 Sub55: CCTCguCCCCAGCTC SEQ ID NO: 30 Sub60: GCCAACCACguCCAACACGAC‘ SEQ ID NO: 73 Sub6l: CTCGACCCCguCTCCACGCCA 'SEQ ID NO: 75 Sub72: ATCACGCCTCguCTCCTCCCAG SEQ ID NO: 77 Sub74: ATCACTCCCCguCCCCTCCCAG SEQ ID NO: 78 Sub75: TGACCCTCCTCguCTCCCCACTA SEQ ID NO: 80 VSub79: TCCTCTCCCCguCCCCTTCAACC SEQ ID NO: 81 Sub80: AACCGCCCTCguCCCGTGAACC SEQ ID NO: 88 Sub88: CTCCTCCCTCguCCCCAGCTC 4.3. Target ce and PCR primers for amplification of the CYP2C9, TP53, 321W, HMBS, TFRC and RPL13a genes ‘ Human genomic DNA extracted from IM9 cell line (Promega) was used as template for in vitro amplification of the target genes. The amplicons were generated by qPCR using the oligonucleotide PCR primers listed below. Primer sequences are listed from 5’ to 3’. The sequence in bold in the primer sequences corresponds to a universal tag (U1, U2 or U3) that increases'the Tm of the primer without affecting the specificity of the primer to the gene . This tag improves amplification efficiency in PCR reactions.

SEQ ID NO: 91 Forward primer 5TFRC_U1 GCTAAAACAATAACTCAGAACTTACG SEQ ID NO: 92 Reverse primer U2' CAGCTTTCTGAGGTTACCATCCTA SEQ ID NO: 12.3 Forward primer 5B2M,U1 GCTAATCTTTTCCCGATATTCCTCAG SEQ ID NO: 124 Reverse primer 3BZM’_,U2 CAGCCCAGACACATAGCAATTCAG.

SEQ ID NO: 125 Forward primer 5TE53_U3 TACTGCCTCTTGCTTCTC SEQ ID NO: .126 Reverse primer 3TP53_U2 CAGCTCTGTGCGCCGGTCTCTC SEQ ID NO: 127 Forward primer 5RPL13a_U3I CTAAACCGGAAGAAGAAACAGCTCA SEQ ID NO: 128 e primer 3RPL13a__U2 CAGGAGGAATTAACAGTCTTTATTGG SEQ ID NO: 129' Forward primer 5CYP2C9_U3 CTAACCTCATGACGCTGCGGAA SEQ ID NO: 130 Reverse primer 3CYP2C9_U2 _CAGATATGGAGTAGGGTCACCCA ‘30 SEQ ID NO: 131 Forward primer U3 CTAAACCCACACACAGCCTACTTTC SEQ ID‘ NO: 132 Reverse primer 3HMBS_U2 CAGAGCCCAAAGTGTGCTGGTCA 4.4. Reaction Components: ication and quantificotion oftarget sequence Real-time PCR amplification and detection of the target sequence was performed in a total reaction volume of 25 uL. All reactions were conducted in a CFX96 Real-Time PCR Detection System (Bio-Rad). Reactions were set up with substrates and their associated panzymes as in Table 13. The cycling parameters were either; I) 95°C for 2 minutes, 50 cycles of 95°C for 15 seconds and 52°C for 60 seconds (data collected at the 52°C step) or ‘2) 95°C for 2 minutes, 50 cycles of 95°C for 15 s and 58°C for 60 seconds (data collected at the 58°C step).

IO Each set of on conditions was run in duplicate and contained 40 nM forward primer and 200 nM of reverse primer, 200 nM each of panzyme A and partzyme B, 200 nM substrate, 8 mM MgC12, 200 MM of each dNTP, 10 units RiboSafe RNase inhibitor (Bioline), 1 x Immobuffer (Bioline), 2 units of MyTaqHSTM DNA polymerase (Bioline) - and either c DNA template (100 ng) or no target (NF-H20).

Table 13: Oligonucleotide combinations used for each universal substrate Sub3 CYP2C9A/3—P CYP2C9B/3—P 5CYP2C9__U1 and SEQ ID 'NO: 22 SEO ID NO: 96 SEQ ID NO: 97 3CYP2C9__U2 Sub61 CYP2C9A/61—P SEQ ID NO: 129 and SEQ SEQ 11) NO: 73 - SEQ ID NO: 98 SEQ ID NO: 99 ID NO: 130 respectively Sub6 TP53A/6—P TP53B/6-P SE0 ID NO: 23 SE0 11) NO: 100 SE ID NO: 101 Sub72 TP53A/6-P 5TP53__U1 and SE0 11) NO: 75 SE0 ID NO: 100 SEQ 11) No: 103 U2 Sub74 TP53A/74-P SEQ ID NO: 125 and SEQ . SEQ 11) NO: 77 SEQ 11) NO: 104 SEQ ID NO: 105 ID NO: 126 respectively SE ID NO: so SE0 ID NO: 106 SE01D NO: 107 SE0 ID NO: 30 SEQ 11) NO: 108 SE0 ID NO: 109 Sub61 B2MA/61-P ’1-P 5B2M_Ul and 3BZM_U2 SEQ ID NO: 123 and SEQ SE0 11) NO: 73 SE0 113110: 110 SE0 ID NO: 111 ID NO: 124 respectively SEQ ID NO: 80 SEQ ID NO: 112 SEO ID NO: 113 5HMBS_U1 and SEO ID NO: 28 SEO ID NO: 114 SEQ ID NO: 115 U2 Sub75 HMBSB/75-P . SEQ ID NO: 131 and SEQ SEQ ID NO: 72 SEQ ID NO: 116 SEQ ID NO: 117 ID NO: 132 respectively TFRCA/Z-P TFRCB/Z-P 5TFRC_U1 and SEO ID NO: 21 SEC IDNO: 34 SEC IDNO: 35 3TFRC_U2 SEQ ID NO: 91 and SEQ ID SEQ ID NO: 75 SEQ ID NO: 38 SEQ ID NO: 55 NO: 92 respectively RPL13aA/55-P RPL13aB/55-P - SE IDNO: 29 SE IDNO: 11s SEQ IDNO: 119 5RPL13a_U1 and Sub80 RPL13aA/80-P 3RPLl3a_U2 SEO ID NO: 81 SEO IDNO: 120 SEO lD NO: 121 SEQ 1D NO: 127 and SEQ ID NO: 128 tively SEO IDNO: 88 SEO IDNO: 122 SEQ IDNO: 119 4. 5. Results: Amplification oftarget and cleavage ofreporter substrate Each MNAzyme qPCR reaction containing human genomic DNA showed an se in fluorescence over time for the real-time detection of the genes CYP2C9, TP53, B2M, HMBS, RPLl3a and TFRC, at annealing temperatures of both 52°C and 58°C (Figure 7). For all universal substrates, the fluorescence. of the no—DNA target control was lower than that in the DNA target-containing reactions. This trates that the increase in fluorescence produced in target-containing reactions is due to target dependent assembly of catalytically active MNAzymes that then d one of the universal reporter substrates.

Results from MNAzyme qPCR detection of the CYP2C9 and TP53 genes showed that all sal substrates tested performed equivalently at 52°C with less than 0.5 Ct difference between the substrates and similar slopes of the amplification curves (Table 14 and Figure 7, (i)a and (ii)a respectively). The series 1 substrates tested (Sub3 and Sub6) performed worse at the higher temperature of 58°C than the series 3 substrates tested (Sub61, Sub72, Sub74 and 9) with a difference of more than 1 Ct between the substrates and a much shallower slope for the amplification curves for Sub3 and Sub6_ indicating a less efficient reaction (Table 14 and Figure 7, (i)b and (ii)b respectively).

These data show that the improved design of these series 3 substrates , Sub72, Sub74 and Sub79) leads to more efficient cleavage at 58°C, and that this improved performance at elevated temperatures does not prevent these substrates from being efficiently cleaved at a lower temperature.

Results from MNAzyme qPCR ion of the B2M and HMBS genes showed that all the universal ates tested performed equivalently at 52°C with only approximately 0.5 Ct difference between the substrates and similar slopes of the amplification curves (Table 14 and Figure 7, (iii)a and (iv)a respectively). The series 2 substrates tested (Sub60 and Sub49) performed worse at the higher temperature of 58°C than the series 3 substrates tested (Sub61 and Sub75, and Sub79) with a difference of more than 1 Ct between the ates and a shallower slope for the amplification curves for Sub60 and Sub49 indicating a less efficient reaction (Table 14 and Figure 7, (iii)b and (iv)b respectively). These data show that the improved design of these series 3 substrates (Sub61, Sub75 and Sub79) leads to more efficient cleavage at 58°C, but that this improved performance at elevated temperatures does not prevent the substrates from being efficiently cleaved at a lower temperature. The better performance of the series 3 substrates versus the series 2 substrates at 58°C is attributable to the fact that the series 3 substrates follow all the design guidelines for highly active ates and the series 2 substrates do not (see Table 9).

Results from MNAz‘yme qPCR detection of the TFRC gene showed that all l0 universal substrates tested performed equivalently at 52°C with only approximately 0.5 Ct difference between the substrates and similar slopes of the cation curves (Table 14 and Figure 7, (v)a). The series 3 substrates tested (Sub72 and Sub80) both performed better at 58°C than the series 1 substrate tested (Sub2) with a difference of more than 1 Ct between the substrates and a shallower slope for the amplification curve for Sub2 IS indicating a less efficient reaction (Table 14 and Figure 7, (v)b). The series 3 substrate, Sub80, performed better at 58°C than the series 3 substrate, Sub72, with a greater than 1 Ct difference between the ates. Sub80 follows all of the design guidelines for highly active substrates and Sub72 does not (see Table 9).

Results from MNAzyme qPCR detection of the RPL13a gene showed that at 52°C the series 2 substrate Sub55 and the. series 3 substrate Sub88 were better than the series 3 substrate Sub80 (Table 14 and Figure 7, . At 58°C the series 3 substrate Sub80 and the series 2 Sub55 med the same, and both these substrates performed better than the series 3 substrate Sub88 (Table 14 and Figure 7, (vi)b). The series 2 substrate Sub55 meets all the all of the design ines for highly active substrates (see Table 9) and would therefore be expected to perform well. The series 3 Sub88 also meets all of the design guidelines for highly active substrates (Table 9) but has not performed as well as Sub55. Overall, the design guidelines show a high probability of producing substrates that are efficiently cleaved under MNAzyme qPCR conditions.

Overall, with a range of ent target ces, the series 1, 2 and 3 substrates performed comparably in MNAzyme qPCR performed at 52°C.. At 58°C, the series 3 substrates rformed the series 1 and 2 substrates, with the exception of the series 2 ate Sub55 which, as explained above, falls within the all the design guidelines for highly active substrates. These data show that the design guidelines do, in general, produce substrates that are robust and efficiently cleaved at a range of atures in the context of thermocyeling protocols used for qPCR.

Of note is that some of the series 3 substrates, indicated by (A) in Table 14, have very high Tm’s, and therefore at lower temperatures they may have poorer turnover over of the d substrate and hence even though the activity is comparable to the other substrates, the final fluorescence value is lower.

Table 14. Substrate cleaved by MNAzymes targeted to different genes.

U!00 0 _ Amplifi- Diff in. - , Gene Substrate cation Ct cation ‘ Ct Diff in Ct c'urve" curve* I“ -Sub3 22.9 NO“ \] .

CYP2C9 <0.5 ‘ > 1.0 ‘ 932361= S‘gbig) u Sub61 ' ' u . NN.m NM N. ix.) L» o -Sub72 22 7‘ (Sub79 & < 0.5 Sub6) > Sub74 (1:181 caps;_“ 22.7 ‘ 1.5 Sub79 22.6 Sub60 N:5o Sub60== ‘~ (Sub60 & Sub61 3201 Sub61 NPo _ >Sub79 Sub61) Sub79 (Asub79) N5‘0x Sub49 Sub49 = N5" ~ HMBS Sub75 1 Sub75 ("Sub75) 25.0 27.0- - NS" eo 27.1 32722: (Sub80 & TFRC Sub72 N9’N 25.7 Sub80 ("Sub80) Sub80 N5”4: Sub55 N,0w Sub55 = Sub88 > RPLl3a Sub80 N H O Sub88) < SubSS > Sub80 0.5 ' Sub88 ("Sub80) Sub88 N9w *The shape of the amplification curve (steepness and time taken to reach plateau) demonstrates the reaction efficiency. ' " Tm of these Substrates is too high for lower reaction atures and this can result in poor turnover over ofthe d portions of the substrate and thus lower final fluorescence values. e 5: Use of sal substrates with DNAzymes The 10-23 DNAzyme is a unimolecular structure that can directly bind to, and modify, a substrate ce. The 10-23 DNAzyme has utility in in vitro diagnostic applications. Due to similarity in the catalytic region, the 10-23 DNAzyme can bind and cleave substrates that are ble by the MNAzyrne based on the 10-23 DNAzyme.

Unlike the MNAzyme, the DNAzyme does not need a target sequence to form the active IO core, and therefore the binding and uent cleavage of the substrate by the 10-23 DNAzyme is not influenced by the target sequence and does not utliize a split catalytic . core. The ability of matched 10-23 DNAzymes tocleave the Series 1 sal substrates (Sub2, Sub3, and Sub6), the series 2 universal substrates ‘(Sub44, Sub45, Sub49, SubSS and Sub60T) and the series 3 substrates (Sub6l, Sub72, Sub73, Sub74, Sub75, Sub’l7, Sub79, Sub80, Sub84, Sub85, Sub86, Sub87, Sub88, and Sub89) was measured to determine if the design guidelines of the present invention lead to the development of substrates that can be d by' the 10—23' DNAzyme with high activity and robustly over a range of temperatures. 5.1. 10-23 e Oligonucleotides A series of 10-23 DNAzymes were designed with sensor arms complementary to the substrates described above and listed in Tables 4 and 5. The sequences of the DNAzymes are listed below from 5’ to 3’, where the bases underlined hybridize to the substrate and the bases in italics form the catalytic core. Some DNAzyme sequences below contain an extra G at the very 5‘ and 3’ ends (e.g. D255). These added bases do not hybridize with the substrates and do not impact on the efficiency at which the DNAzyme cleaved the substrate.

SEQ ID NO: 133 DNAzyme D22 TGCCCAGGGAGGCTAGCTACAACGAGAGGAAACCT’I‘ '30 SEQ ID NO: 134 DNAzyme Dz3‘ CGGTTGGTGAGGCTAGCTACAACGAGGTTGTGCTG SEQ ID NO: 135 DNAzyme Dz'6 CTGGGAGGAAGGCTAGCTACAACGAGAGGCGTGA'I‘ SEQ ID NO': 136 DNAzyme 0244 TCACTATAGGGAGGCTAGCTACAACGAGAGGAGACCTG SEQ ID NO: 137 DNAzyme D245 TTCCAAAGGAGAGGCTAGCTACAACGAGGGACCCGT SEQ ID NO: 138 DNAzyme D249 TATCACAGCCAAGGCTAGCTACAACGAGAGCCAAGTTTA SEQ ID NO: 139 DNAzyme D255 GGAGCTGGGGAGGCTAGCTACAACGAGAGGTGCGGTG SEQ ID NO: 140 ’DNAzyme D260 GTCGTGTTGGAGGCTAGCTACAACGAGTGGTTGGC SEQ ID NO: 141 DNAzyme D261 w_ GTGGCGTGGAGAGGCTAGCTACAACGAGGGGTCGAGG' SEQ ID NO: 142 'DNAzyme D272 GCTGGGAGGAGAGGCTAGCTACAACGAGAGGCGTGATG SEQ ID NO: 143 DNAzyme 02731 GCACGAGGGGAGGCTAGCTACAACGAGGGGACGCCAG SEQ ID NO: 144 e D274 AGGGGAGGCTAGCTACAACGAGGGGAGTGATG SEQ ID NO: 145 DNAzyme D275 GGGAGAGGCTAGCTACAACGAGAGGAGGGTCAG SEQ ID NO: 146 .DNAzyme D277 GAGGAGGAGGGAGGCTAGCTACAACGAGAGGGAGGAGG SEQ ID NO: 147 DNAzyme D279 GGGTTGAAGGGGAGGCTAGCTACAACGAGGGGAGAGGAG SEQ ID NO: 148 ‘DNAzyme D280 GGGTTCACGGGAGGCTAGCTACAACGAGAGGGCGGTTGG SEQ ID NO: 149 DNAzyme D284 GCTGGGAGGAGAGGCTAGCTACAACGAGAGGTGCGGTG SEQ ID NO: 150 DNAzyme D285 GCTGGGAGGGGAGGCTAGCTACAACGAGAGGTGCGGTG SEQ ID NO: 151 DNAzyme D286 GGAGCTGGGGAGGCTAGCTACAACGAGAGGCGTGATG SEQ ID NO: 152 DNAzyme D287 QGAGCTGGGGAGGCTAGCTACAACGAGGGGAGTGATG SEQ ID NO: 153 DNAzyme D288 GGAGCTGGGGAGGCTAGCTACAACGAGAGGGAGGAGG SEQ ID NO: 154 DNAzyme D289 GAGGAGGAGGGAGGCTAGCTACAACGAGAGGTGCGGTG .2. Reporter Substrates In the current example, the substrates were end labelled with a 6-FAM moiety at the ’ end (indicated by a “F”lin the name of the substrates below),and an .Iowa Black® FQ quencher moiety at the 3’ end (indicated by a “1B” in the name. of the substrates below).

Cleavage of the substrates was red between 510-530 nm‘ (FAM emission wavelength n CFX96 (BioRad)) with excitation between 450-490 nm ~(FAM excitation ngth range on CFX96 (BioRad)). The reporter substrates for this IO e are shown below with the seqUence, 5’ to 3’. The lower case bases represent RNA and the upper case bases represent DNA.

SEQ'ID NO: 21 Subz-FIB: -AAGGTTTCCTCguCCCTGGGCA SEQ ID No; 22 Sub3-FIB: CAGCACAACCguCACCAACCG SEQ ID NO: 23” SubG—FIB: ATCACGCCTCguTCCTCCCAG SEQ ID NO: 25' Sub44-FIB: CAGGTCTCCTCguCCCTATAGTGA SEQ ID NO: 26 Sub45—FIB: ACGGGTCCCguCTCCTITGGAA SEQ ID NO: 28 ’Sub49-FB: TAAACTTGGCTCguTGGCTGTGATA SEQ ID NO: 29 SufiSS-FIB: ‘ACCGCACCTcguCCCCAGCTC SEQ ID NO: 72 -FIB: 'TGCCAACCACguCCAACACGAC SEQ ID NO: 73 Sub6l—FIB: CTCGACCCCguCTCCACGCCA ' SEQ ID NO: 75 Sub72—FIB: ATCACGCCTCguCTCCTCCCAG SEQ ID NO: 76 Sub73-FB: TGGCGTCCCCguCCCCTCGTG ”SEQ ID NO: 77 . SubZ4—FIB:' ATCACTCCCCguCCCCTCCCAG SEQ ID NO: 78 Sub75—FIB: TGACCCTCCTCguCTCCCCACTA SEQ ID NO: 79 Sub77-FB: CTCCTCCCTCguCCCTCCTCCT SEQ ID NO: 80' Sub79—EIB: TCCTCTCCCCguCCCCTTCAACC SEQ ID NO: 81 FIB: CCTCguCCCGTGAACC SEQ ID NO: 84 Sub84—FIB: ACCGCACCTCguCTCCTCCCAG SEQ ID NO: 85 SubSS—FIB: ACCGCACCTCguCCCCTCCCAG SEQ ID NO: 86 Sub86—FIB: ATCACGCCTCguCCCCAGCTC '15 SEQ ID NO: 87 Sub87—FIB:- ATCACTCCCCguCCCCAGCTC SEQ ID No: 88 1 Sub88¥FIB:- CTCCTCCCTCguCCCCAGCTC SEQ ID NO: 89 Sub89-FIB: ACCGCACCTCguCCCTCCTCCT .3. Reaction ents: Cleavage of a substrate by a DNAzyme at temperatures n 50°C and 60°C Cleavage of a substrate was ed by an increase in fluorescent signal caused by ' the binding and subsequent cleavage by a matched DNAzyme. Separate reactions were set up to measure the ge of each Substrate with its matched DNAzyme (oligonucleotides as in Table 15). Reactions contained 1 x PCR Buffer II (Applied Biosystems), 10 mM MgC12, 200 nM Substrate andNF-HZO in total volume of 25 uL.

Each reaction was run in duplicate as either a “test” (addition of 1 nM DNAzyme) or “control” (addition of NF-H20) reaction. ons were performed on a CFX96TM Real- Time PCR Detection System (BioRad) at 50, 52, S4, 56, 58 and 60°C. Fluorescence for each reaction was programmed to be read after 1 second for the first 50 cycles and then programmed to be read after 25 seconds for the next 50 cycles.

Table 15: DNAzymes tested with matching Substrates Sub2 SE ID NO: 21 ' Sub84 SEQ ID NO: 84 D284 SEO ID NO: 149 .5. Results: Cleavage ofa substrate by a DNAzyme at various temperatures Each test reaction containing DNAzymes with matched substrates showed an increase in fluorescence over time. There was no increase in fluorescence of water only control reactions (no DNAzyme added). This trates that the increase in cence produced in the DNAzyme-containing ons was due to the binding and ‘ subsequent catalytic cleavage of the reporter substrate by the DNAzyme.

For each ate data set (test and control reactions), the raw fluorescence data points were exported into Excel (Microsoft), duplicate values were averaged and then normalised. isation was performed by dividing each averaged data point by the ed value of the no-DNAzyme reaction at the first reading of reactions containing the same substrate (e.g. the averaged data for the test ons for Sub6l was divided by the averaged fluorescence at cycle 1 for the Sub61 no-DNAzyme control reaction; the [5 averaged data for the no-DNAzyme control reactions for Sub6l was divided by the averaged fluorescence at cycle 1 for the Sub61 no—DNAzyme control reaction.) These normalised data were then used to calculate the signal to noise ratio at approximately 10 minutes after the start of the on, by dividing the normalized fluorescence of the test reaction at 10 s by the normalized fluorescence of the no-DNAzyme reaction at 10 s. This ation of signal to noise was performed for each combination of DNAzyme and substrate and at each temperature tested. The signal to noise value was then plotted on a bar graph to compare the efficiency of cleavage of each substrate by its 'matched DNAzyme at the various temperatures (Figure 8, (i)). The rd deviation of the signal to noise ratio for each substrate over the ature range was also calculated and plotted to determine substrates with consistent signal to noise over the tested range of temperatures (Figure 8, (ii)). This suggests that these substrates are robust with t to temperature.

Due to experimental error there are no data for Sub2 at 54°C or for Sub79 at 58°C, however this has minimal impact on the overall interpretation of the data.

Analysis of the signal to noise ratio for each substrate e 8, (i)) shows that the series 1 substrates (Sub2, Sub3 and Sub6) had high signal to noise at the lower temperatures measured. r the cleavage efficiency of these substrates dropped dramatically as the reaction temperature was increased. A similar pattern was seen for a subset of the series 2 substrates (Sub44, Sub45, Sub49 and Sub6OT). The other series 2 substrate (Sub55), and the majority of the series 3 substrates tested , Sub72, Sub74, Sub75, Sub79, Sub80, Sub84, Sub85, Sub86, Sub87, Sub88 and Sub89) displayed high signal to noise across all the temperatures tested. The series 3 substrates Sub73 and Sub77 had uniformly low signal to noise at all temperatures tested indicating that although the new design guidelines provide a good probability of design of robust ates (83% success rate for this application), there will still be some sequences that meet these guidelines but that are unsuitable for a subset of MNAzyme and/or DNAzyme diagnostic applications. The results for Sub73 and Sub77 also prove that methods utilizing DNAzymes for mass screening of potential substrate sequences to find those that are suitable for MNAzyme diagnostic ations may be missing out on detection of substrates that would be robust and efficiently cleaved by MNAzymes in qPCR applications and vice versa (see Example 2 for the successful utilization of Sub73 and Sub77 in MNAzyme qPCR, Figure 5).

Overall, the majority of the substrates that conform to all of the design guidelines (Table 9) showed a greater signal to noise ratio across the tested temperature range than the substrates that fell outside one or more of these guidelines (Figure 8, (i)). More specifically reactions with Sub55, Sub6l, Sub72, Sub74, Sub75, Sub79, Sub80, Sub84, Sub85, Sub86, Sub87, Sub88 and Sub89 displayed high signal to noise values at every ature tested, demonstrating that these are robust substrates over a range of' temperatures. This improvement was further evident when the standard deviation was calculated from the signal to noise ratio across the temperatures for each substrate (Figure 8, (ii)). This measure of variability ted that, over the temperature range , the series 2 substrate, Sub55, and the series 3 ates Sub61, Sub72 Sub74, Sub75, Sub79, Sub80, Sub82, Sub83, Sub85, Sub86, Sub87, Sub88 and Sub89, had similar signal to noise across the temperatures tested demonstrating that they are robust substrates across a broad temperature range. Note that although Sub60T, Sub73 and Sub77 do demonstrate a low standard deviation in signal to noise over the temperature range, the absolute signal to noise is very low at all temperatures ing these substrates from being a robust ate in this application.

Example 6: Testing for non-specific ge of universal substrates with MNAzyme qPCR.

MNAzymes can be used to monitor amplification of target nucleic acids in real»time using in vitro target amplification methods such as PCR. Amplification and detection are performed in a one-step process, wherein PCR amplification and MNAzyme-mediated detection occur simultaneously in a single tube. Multiple targets can be amplified and detected in a single reaction vessel using partzymes with target sensor arms specific to the individual targets. Partzymes for the detection of a first target will bind to and cleave a first ate, partzymes for the detection of a second target will bind to and cleave a second substrate and so on. For the detection of targets to be specific, there can be no non-specific cleavage of a substrate by partzymes designed to cleave any 'other substrate in the on mix.

The degree of complementarity of the substrate sensor arms of MNAzyme mes with other substrates present in the reaction impacts on the specificity of binding. Full mentarity of bases closest to the ribonucleotides are more‘crucial to specific cleavage. The design guidelines for creation of efficiently cleaved universal substrates include aints around the sequence composition of universal substrates i.e. seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (N4- 3O N13); the bases immediately adjacent to the ribonucleotides are cytosines (N8 and N9); total content of ate has >64% pyrimidines. These constraints may lead to similarities in the sequence of the substrate close to the ribonucleotides, and possibly result in non-specific cleavage of a universal substrate by partially matched partzymes, ally in a multiplex format where a range of universal substrates are present with their associated partzymes in a single reaction mix. If a substrate is cleaved in a non- specific manner by partially matched partzymes designed to specifically cleave a second substrate, then that particular combination of substrates may not be le for use in multiplex format.

In this example, the sal substrates Sub44, SubSS, Sub61, Sub65, Sub72 and Sub74 were tested for non-specific cleavage activity by the partzymes associated with Sub44, Sub55, Sub72 and Sub74. This involved testing each universal substrate individually with partzyme pairs designed to bind with full complementarity to the other substrates to see if a signal was detected in a MNAzyme qPCR format. The partzymes of Sub72 and Sub74 were chosen for this test as their respective substrates differ by only 3 bases (Figure 9, (i)a and (ii)a). The mes of Sub55 were chosen to be tested with Sub6l and Sub65 as they are very similar in the l region around the ribonucleotides The partzymes of Sub44 were chosen as a control as they are . 3) (Figure 9, (iii)a). less similar to the other substrates (Figure 9, (iv)a). 1.5 6.1. Partzyme Oligonucleotides In the experiments conducted to test for non—specific cleavage by non- complementary partzymes, partzyme oligonucleotides A and B were designed with target ' sensor arms complementary to the human RPL13a gene and ate sensOr arms complementary to each of the universal ates as discussed above. The sequences of the A and B mes are listed below from 5’ to 3", where the bases underlined hybridize to the substrate. The “-P” indicates 3’ phosphorylation of the oligonucleotide.

SEQ ID NO: 155 partzyme A RPLl3aA/44—P: AATTGACAAATACACAGAGGTCACAACGAGAGGAGACCTG SEQ ID NO: 156 me B‘RPL13aB/44-P: TCACTATAGGGAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 118 partzyme A RPLlBaA/SS—P TTGACAAATACACAGAGGTCACAACGAGAGGTGCGGTV SEQ ID NO: 119 partzyme B RPL13aB/55-P GAGCTGGGGAGGCTAGCTCTCAAGACCCACGGACTCCT ' SEQ ID NO: 157 partzyme A RPLl3aA/72-P: AATTGACAAATACACAGAGGTCACAACGAGAGGCGTGAT SEQ ID NO: 158 partzyme B RPLl3aB/72»P: CTGGGAGGAGAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 159 partzyme A RPL13aA/74—P: AATTGACAAATACACAGAGGTCACAACGAGGGGAGTGAT SEQ ID NO: 160. meB RPLl3aB/74—P: CTGGGAGGGGAGGCTAGCTCTCAAGACCCACGGACTCCT 6.2. Reporter Substrates In the current e, the substrates were end labelled with a 6-FAM moiety at the ’ end (indicated by a “F” in the nameof the substrates below) and an Iowa Black® FQ quencher moiety'at the 3’ end (indicated by a g‘IB” in the name of the substrates below).

Cleavage of the substrates was monitored at 530 nm (FAM emission ngth) with excitation at 485 nm (FAM excitation wavelength). The reporter ates for this example are shown below With the sequence, 5" to 3’. The lower case bases represent RNA and the upper case bases represent DNA.

SEQ ID NO: 25 Sub44-FB: CAGGTCTCCTCguCCCTATAGTGA SEQ ID NO: 29 SubSS—FB: IACCGCACCTCguCCCCAGCTC ,SEQ ID NO: 73 Sub615FB;” CTCGACCCCguCTCCACGCCA SEQ ID NO: 74 Sub65-FB: CCTCguCTCCACGCCA SEQ ID NO; 75 Sub72—FB: ATCACGCCTCguCTCCTCCCAG SEQ ID NO: 77 .'Sub744FB: ATCACTCCCCQuCCCCTCCCAG 6.3. Target sequence and PCR primersfor amplification ofRPLI'3a The target sequence for this example was a PCR amplicon from the RPL13a gene ' generated by in yitro PCR cation of human genomic DNA extracted from the 1M9 cell line (Promega) using the oligonucleotide PCR primers listed. below. The reporter substrates for this example are shown below with the sequence, 5’ to 3’. .30 SEQ ID NO: .' 161 Forward Vpriimer SRPLlBa: ACCGGAAGAAGAAACAGCTCA SEQ ID NO: 162 Reverse primer 3RPLl3ai GAGGAATTAACAGTCTTTATTGG 16.4. on ents: cation and measurement ofspecific and non- specific cleavage of universal substrates Real-time PCR amplification and detection of the target sequence wasperformed in a total reaction volume of 2S uL. All reactions were conducted in an Mx3005P QPCR system (Stratagene). The cycling parameters were, 95°C for 2 minutes, 40 cycles of 95°C for 15 seconds and 52°C for 60 seconds (data collected at 52°C). Reactions were. setup with substrates and partzymes as in Table 16. Each set of reaction conditions was tested in duplicate and contained 40 nM 5RPL13a and 200 nM of 3RPL13a, 200 nM of partzyme A, 200 anpartzyme B, 200 nM substrate, 8 mM MgC12, 200 uM of each dNTP, 10 units RNasin (Promega), l x Immobuffer (Bioline), 2 units of MyTaqHSTM DNA polymerase (Bioline) and either genomic DNA template (50 mg). or no target (NF- H20).

Table 16: Partzyme combinations used for each universal ate Substrate 4 Sub44 . Specific cleavage . SEO ID NO: 25 SEQ ID No RPLl3aA/44—P SEQ ID NO: 155 ‘ ' SEQ ID NO: 73 RPLl 3aB/44-P SEQ ID NO: 156 SEQ ID NO: 74 SEO ID ‘NO: 75 Sub74‘ Non-specific cleavage SE 0 ID NO: 77 RPL13aA/55-P Sub44 Non-spec1fic cleavage SEQ ID NO: 118 SE 0 ID NO: 25 SubSS' ' Specific cleavage RPLl3aB/55-P SEQ ID NO: 119 SEQ ID NO: 29 Sub61 Non-specific cleavage SEQ ID NO: 73 ' Sub6‘5 ecific cleavage » SEQ ID NO: 74 Sub72 A Non-specific cleavage SE 0 ID NO: 75 Sub74_ Non-specific cleavage SEO ID NO: 77 Sub44 Non-specific cleavage SEQ ID NO: 25 Sub55 Non-specific cleavage SEQ ID NO: 29 A/72-P SEQ ID‘NO: 157 ' ‘ SEQ ID NO: 73 RPLl3aB/72-P SEQ ID NO: 158 : 74 Sub72 Specific cleavage SEQ ID NO: 75 Sub74 ecific cleavage SE 0 ID NO: 77 Sub44 Non-specific cleavage ' SEO ID NO: 25 SEOIDNO:29 - RPLl3aA/74-P SEQ ID NO: 159 V and ' SEO ID NO: 73 RPL13aB/74-P SEQ ID NO: 160 ' SEC IDNO: 74 ' SEQ ID NO: 75 .

SEO ID NO: 77 6.5. ' Results: Measurement of specific and potential non-specific cleavage by a MNAzyme There was an increase in fluorescence in all reactions that contained genomic DNA and a universal substrate with partzymes With substrate sensor arms that were fully complementary to the universal substrate (i.e. reactions testing for specific ge). The fluorescence of the no-DNA target controls was lower than the fluorescence in the reactions testing for specific cleavage, demonstrating that the increase in fluorescence in reactions g for specific cleavage was due to target dependent assembly of catalytically active MNAzymes that then d the fully complementary universal reporter substrates.

There was no increase in fluorescence in any reaction testing cross-reactivity (Figure 9, (i)b - (iv)b). This demonstrates that partzymes designed to cleave these closely related universal ates only cleaved substrates with which they had filll complementarity. These data show that these universal substrates are compatible in multiplex MNAzyme qPCR assays.

Example 7: Testing for non-specific cleavage of universal ates with DNAzymes The 10—23 DNAzyme is a unimolecular structure that can directly bind to and modify a substrate sequence. Unlike the MNAzyme, the 10-23 DNAzyme does not need a target ce to form the active core therefore the binding and subsequent ge of IS the substrate by the 10-23 DNAzyme is not influenced by the target sequence or having a split catalytic core.

The degree of complementarity of the sensor arms of DNAzymes with the substrate impacts on the specificity of binding. Full complementarity of bases closest to the cleotides are more crucial to specific cleavage. The design guidelines for creation of efficiently cleaved universal substrates include constraints around the ce composition of universal substrates i.e. seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (N4-Ni3); the bases ately adjacent to the ribonucleotides are cytosines (N3 and N9); total t of substrate has >64% pyrimidines. These constraints may lead to similarities in the ce of the substrate close to the ribonucleotides, and ly result in ecific cleavage of a universal substrate by partially matched es, especially in a multiplex format where a range of universal'substrates are present with their associated es in a single reaction mix. If a substrate is cleaved in a non—specific manner by partially matched DNAzymes designed to specifically cleave a second substrate, then that particular combination of substrates may not be suitable for use in multiplex format.

In this example, the universal substrates Sub55, Sub6l, Sub72, Sub74, Sub75, Sub79, Sub80 and Sub85 and their associated 10-23 DNAzymes were tested for non- specific cleavage activity. This involved testing every uniVersal substrate individually with the DNAzymes, designed to bind with full complementarity to all the other substrates to see if a signal could bedetect in an isothermal detection format. These substrates were chosen to be tested as they have similar sequences in ent areas of the substrate. 7.1. 10-23 DNAzyme Oligonucleotides The 10-23 DNAzymes used in the experiments conducted to test for non-specific cleavage by non-complementary DNAzymes are listed below from 5’ to 3’, where the bases underlined hybridize to the, substrate and the bases in italicsform the catalytic core.

Some DNAzyme ces below contain an extra G at the very 5’ and 3’ ends. These added bases do not hybridize with the substrate sequence and do not impact on the IO efficiency at which the DNAzyme cleaved the substrate. ‘ SEQ ID. NO: 139 DNAzyme D255 GGAGCTGGGGAGGCTAGCTACAACGAGAGGTGCGGTG SEQ ID NO: 141 DNAzyme D261 GTGGCGTGGAGAGGCTAGCTACAACGAGGGGTCGAGG ,' SEQ ID NO: 142 DNAzyme 13272 GCTGGGAGGAGAGGCTAGCTACAACGAGAGGCGTGATG SEQ ID NO: 144 ‘ e D274 GCTGGGAGGGGAGGCTAGCTACAACGAGGGGAGTGATG SEQ ID NO: 145 DNAzyme D275 GTAGTGGGGAGAGCCTAGCTACAACGAGAGGAGGGTCAG SEQ ID NO: 147 DNAzyme D279 GGGTTGAAGGGGAGGCTAGCTACAACGAGGGGAGAGGAG SEQ ID NO: 148’ DNAzyme D280 GGGTTCACGGGAGGCTAGCTACAACGAGAGGGCGGTTGG SEQ ID‘ NO: 150 DNAzyme D285 GCTGGGAGGGGAGGCTAGCTACAACGAGAGGTGCGGTG 7.2.. Reporter ates In the current example, the substrates were end labelled with a 6-FAM moiety at the .5’ end (indicated by a “F” in the name of the substrates below) and an Iowa Black® FQ quencher moiety at the 3’ end (indicated by a “IB” in the name of the substrates below).

Cleavage of the substrates was monitored n 510-530 nm (FAM emission wavelength range on CFX96 d)) with excitation between 450-490 nm (FAM excitation wavelength range on CFX96 (BioRad)). The reporter substrates for this example are shown below with the sequence, 5’ to 3’. The lower case bases represent RNA and the upper case bases represent DNA.

‘SEQ ID NO: 29 SubSS—FIB: ACCGCACCTCguCCCCAGCTC SEQ 'ID NO: 73 Sub61—FIB: CTCGACCCCguCTCCACGCCA SEQ ID NO: ‘75 sub72-FIBs ATCACGCCTCguCTCCTCCCAG SEQ ID NO: 7.7 ' FIB: IO ATCACTCCCCguCCCCTCCCAG 'SEQ ID No: 78 Sub7'5—FIB: TGACCCTCCTCguCTCCCCACTA SEQ ID NO: 80 Sub79-FIB: 'TCCTCTCCCCguCCCCTTCAACC SEQ ID NO: 81 SubBO—FIB: AACCGCCCTCguCCCGTGAACC SEQ iD NO: 85' Sub85—FIB: ACCGCACCTCguCCCCTCCCAG , 7.3. Reaction Components: Measurement of ic and potential non-specific. cleavage ofuniversal substrates by a DNAzyme at 52°C and 58°C Cleavage of sal substrates was measured by monitoring cent signal caused by the binding and subsequent modification of a substrate by a DNAzyme.

Cleavage of a universal substrate by a DNAzyme will result in the separation of the fluorophOre and quencher producing in an increase in fluorescence. All reactions, as outlined in Table 17, contained 1x PCR Buffer II (Applied Biosystems), 10 mM MgC12, 200 nM Substrate and NF-HZO in total volume of 25 uL. Each reaction was run in duplicate as either a “test” (addition of 10 nM DNAzyme) or “control” (addition of NF-' H20) reaction. Reactions were performed on a M ime PCR ion System (BioRad) at 52 and} 58°C. Fluorescence for each reaction was programmed to be read after 1 second for the first'50 cycles and then programmed to be read after 25 seconds for the next 50 cycles.

' Table 17: DNAzyme-Substrate combinations tested for specific and ecific cleavage test; substrate and DNAzyme arms fully complementary (testing for specific cleavage) -ve; control reactions, substrate and DNAzyme arms not fully complementary (testing for non-specific cleavage) 7.5. Results: Measurement of specific and potential non-specific cleavage of universal substrates by a DNAzyme ‘ There was in increase in fluorescence in each ‘test’ reaction containing DNAaymes and their fully complementary substrates. There was no se in fluorescence in any reaction that did not contain DNAzyme (No DNAzyme added). This demonstrates that the increase in fluorescence ed-i'n the DNAzyme-containing ‘test’ ons was due to the binding and subsequent catalytic cleavage of the reporter substrate by the DNAzyme.

For each substrate data set (test and control ons), the raw fluorescence data points were exported into Excel (Microsoft), duplicate values were averaged and then normalised. Normalisation was performed by dividing each averaged data point by the averaged value of the no-DNAzyme reaction after the first reading of reactions ning the same substrate (eg. the averaged fluorescence for the test reaction for Sub6l was divided by the averaged cycle 1 fluorescence (after the first 8 s) for the Sub61 no- DNAzyme reaction; the averaged fluorescence for the no-DNAzymecontrol reaction for Sub6l was divided by the averaged cycle ] fluorescence for the no-DNAzyme control reaction.) These normalised data were then used to calculate the 'signal to noise ratio at 10 minutes, by dividing the test normalized fluorescence at 10 s by the no-DNAzyme 2S normalized fluorescence at 710 minutes. This calculation of signal to noise was med for each temperature. The signal to noise ratio was then plotted on a bar graph to compare the efficiency of ge of each universal substrate with each DNAzyme at the various temperatures tested (Figure 10, (i) and (ii)).

Some reactions with various combinations of ates and non-complementary DNAzymes showed a slightly raised fluorescence level compared to the paired no— DNAzyme control on. This signal did not increase over time and is therefore not tive of cleavage of the universal substrate by the non-complementary DNAzyme.

Detection plots showing this horizontal ound fluorescence all had a signal to noise of less than 1.2. Any reaction showing a signal to noise above 1.2 was therefore deemed to either (i) indicate cleavage of a universal substrate (either specific or non-specific), or (ii) te that a particular combination of substrate and mplementary DNAzyme produce a level of background noise that would not be distinguishable from specific cleavage when in a multiplex format.

All combinations of universal substrates with their fully matched DNAzymes showed high signal to noise ratios (above the threshold of 1.2) at both temperatures tested (Figure 10, (i) and (ii)).

There was no non-specific cleavage of the universal substrates Sub61, Sub74, Sub75, Sub79 and Sub80 by any non-complementary DNAzyme at either temperature (Figure 10, (i) and (ii)).

At 52°C some universal ates were cleaved by DNAzymes that were not fully matched to the substrate (where cleavage is defined as a signal to noise ratio above the threshold of 1.2 as described above). The combinations showing non-specific cleavage were: Sub85 non—specifically cleaved by D255, Sub72 non-specifically cleaved by D274 and D285, and Sub55 ecifically cleaved by D285 (Figure 10, (i)).

The non—specific cleavage of Sub55 by D285 could be expected as Sub55 and D285 differ by only four bases. Alignment of Sub55 with D285 shows the four base mismatch is at the distal end of the 3’ substrate arm away from the critical region adjacent to the ribonucleotides (Table 18). Further, one of the 4 bases is a G/T mismatch which is known in the art to bind with some affinity, albeit weaker than for NT and G/C matches. The non-specific cleavage of Sub85 by D255 may also be expected as Sub85 and D255 differ by only five bases and this five base mismatch is found at the distal end of the 3’ substrate arm away from the critical region adjacent to the ribonucleotides (Table 18). However ‘30 unlike the reverse scenario above, there are no G/T mismatches present and therefore it s the Sub85 is not cleaved as efficiently as the reverse combination of Sub55 and D285. The non~specific cleavage of Sub72 by D274 could be expected as Sub72 and Sub74 differ by only 3 bases. Alignment of Sub72 with D274 shows the two ches t to the ribonucleotides are G/T mismatches (Table 18). This also explains why Sub74 is not cleaved by D272 as the two mismatches are C/A and occur very close to the ribonucleotides and this is enough to disable cleavage of the substrate by the DNAzyme (Table 18). The ecific cleavage of Sub72 by D285 can be explained by the ent of the sequences in Table 18, which shows that the mismatch bases are primarily G/T and this would therefore lead D285 to bind and cleave Sub72. Again, the reverse situation of D272 ecifically cleaving Sub85 would not be expected as the relevant mismatches become the more destabilising C/A mismatches which are unlikely to lead to strong enough binding between the DNAzyme and the substrate to lead to cleavage.

Table" 18. Alignment of Substrates and DNAzymes where ecific cleavage was observed.

Nameor —(seqsubssm 22> 2 GTGGCGTGGAG AGGGGAGGGTCG GA GG D285 (Seq ID: 150) AC CT GTGGCGTGGAG AGGGGE‘EGM GA GG DNAzyme D255 (Seq ID: 139) AC CT ACATCGA m___=sw2<22_______qm22> GTAGTGAGGGG AGGGGAGGG’I‘CGfl GA GG D274 (Seq ID: 144) l AC CT ACATCGA 22222222225> I GTGGQG‘I‘GGAG AGGGGAGGGTCG GA GG DNAzyme D285 (Seq ID: 150) AC CT ACATCGA Substrate Sub74 (Seq ID 77 ) ATCACTCCCCguCECCTCCCAG GTAGTGQGGQG AGAGGAGGGTCG DNAzyme 13272. (Seq ID: 142) GA GG AC CT ACATCGA A Substrate sequence written 5’ to 3’ and DNAzyme sequence written 3’ to 5’. Mismatched bases are in bold and underlined and mismatched bases that are 0/1” are only in hold. The DNAzyme core sequences are in italics.

Increasing the reaction temperature to 58°C created more stringent conditions for hybridization of oligonucleotides and resulted in the loss of almost all the non-specific cleavage seen at 52°C (Figure 10, (ii)). The only combination of universal substrate and DNAzyme that showed non-specific cleavage at 58°C was Sub55 being cleaved non- specifically by D285. This non-specificity is to be expected as SubSS and D285 differ by l0 only four bases and this four base mismatch is t the distal end" of the substrate arm away from the critical region adjacent to the ribonucleotides and one of the mismatches is a G/T (Table 18). Even though there are only three bases different between Sub72 and Sub74, this three base mismatch occurs in the critical region close to the ribonucleotides and are therefore more critical for specificity and will be more ilising at higher temperatures that create more ent conditions for binding of oligonucleotides.

These s demonstrate that the design guidelines produce universal substrates that can be ively multiplexed at a range of temperatures for applications involving -23 DNAzymes. There is a need for some optimization of reaction temperature to provide sufficient ency of binding for some combinations of substrates, but overall the guidelines e sets of substrates that can be multiplexed. One skilled in the art can appreciate that the length of substrate and DNAzyme binding arms can be ed to create more stringent binding at lower and higher temperatures.

Example 8: Use of universal substrates with multiplex MNAzyme qPCR for the quantification of five different c acid targets in a multiplex reaction with annealing temperatures of 52°C or 58°C.

Multiple targets can be simultaneously ed and detected in real time using in vitro target amplification methods stich as qPCR. Further, the amplification of the targets can be aneously monitored in real-time in one multiplexed reaction that comprises 3O multiple unique MNAzymes. Each MNAzyme can be designed with sensor arms specific for one target and substrate arms specific for a unique member of a series of universal substrates. Each target can be individually detected if each of the series of universal ates is labelled with a different fluorophore. The amplification and detection of the multiple targets are performed in a one-step process, wherein PCR amplification and MNAzyme-mediated ion occur simultaneously in a single tube.

Real-time ring generates an amplification curve that can indicate the efficiency of a reaction by the shape of the curve (steepness and speed to reach plateau).

The annealing/detection temperature for MNAzyme qPCR used in the art is between 50 and 54°C This temperature was dictated by the fact that the universal substrates known in the art had a limitation on the temperature at which they were efficiently cleaved with 54°C being the upper limit for efficient cleavage of the series 1 universal substrates. There is a need for a panel of universal substrates that can be combined in a multiplex reaction'and efficiently cleaved at higher temperatures. Not only does this allow greater flexibility in design of primers and partzymes that anneal at higher temperatures and the targeting of G/C rich templates, but it would also enable MNAzyme qPCR detection to be multiplexed with other ime chemistries well known in the art such as ® in which the standard annealing/detection ature ranges from 60 - 65°C. Utility of universal substrates would be greatly increased if substrates d that worked well together at a r range .of temperatures.

In this example, two multiplex reactions were performed both comprising MNAzymes designed to detect five different targets, namely human TFRC, HPRT, TP53, RPL13a and CYP2C9 genes. In Multiplex 1, each target MNAzyme was ed to cleave one of the series 1 universal substrates, Sub2, Sub3, Sub4, Sub6 and Sub7 and in Multiplex 2 each target e was designed to cleave one of the improved series 2 or 3 universal substrates, Sub55, Sub61, Sub74, Sub79' and Sub80. It will be appreciated that any number of targets can be used in accordance with the method and that those d in the art can design appropriate partzymes to detect any target.

The two multiplex reactions were compared to determine the cleavage efficiency of each set of universal substrates by looking at the Shape of the curve (steepness and speed to reach plateau). In this example, amplification and detection are ed at a temperature favourable to all substrates, 52°C, and a temperature outside of the efficient range for l substrates, 58°C. 8.1. Partzyme Oligonucleotides The sequences of the partzymes A and B for each target are listed below from 5’ to 3’. For each target, the partzymes were ed to be used with one of the original series 1 universal substrates and one of the novel improved substrates from series 3. In the following sequences, the bases underlined hybridize to the substrate. The “—1?” indicates 3’ phosphorylation of the oligonucleotide.

SEQ ID NO: 157 partzyme A RPL13aA/6—P .AATTGACAAATACACAGAGGTCACAACGAGAGGCGTGAT SEQ ID NO: 163 partzyme B RPLl3aB/6-P CTGGGAGGAAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 120 partzyme A RPLl3aA/80—P AATTGACAAATACACAGAGGTCACAACGAGAGGGCGGTT SEQ ID NO: 121 partzyme B RPL13aB/80-P' GGTTCACGGGAGGCTAGCTCTCAAGACCCACGGACTCCT SEQ ID NO: 96 partzyme A CYP2C9A/3—P GGGAAGAGGAGCATTGAGGAACAACGAGGTTGTGCTG SEQ.ID NO: 97- .partzymé B CYP2C9B/3—P CGGTTGGTGAGGCTAGCTCCGTGTTCAAGAGGAAGC SEQ ID NO: 98 partzyme A CYP2C9A/61aP GGGAAGAGGAGCATTGAGGAACAACGAGGGGTCGAG SEQ ID NO: 99 partzyme B CYP2C9B/61~P TGGCGTGGAGAGGCTAGCTCCGTGTTCAAGAGGAAGC SEQ ID NO: 164 partzyme A 4—P GACGGAACAGCTTTGAGGTGACAACGAGTGCGCCATG SEQ ID NO: 165 partzyme B TP53B/4—P TACTTCTCCCAAGGCTAGCTCGTCTTTGTGCCTGTCCTGG SEQ ID No; 106 partzyme A TP53A/79-P: ACAGCTTTGAGGTGACAACGAGGGGAGAGGA SEQ ID NO: 107 partzyme B TP53B/79—P: GGTTGAAGGGGAGGCTAGCTCGTGTTTGTGCCTGTCCTGG SEQ ID NO: 34 me A TFRCA/Z—P:’ TGGAAGGAGACTGTCACAACGAGAGGAAACCTT SEQ ID NO: 35 partzyme B TFRCB/Z—P: TGCCCAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 58 partzyme A TFRCA/74—P: GGAATATGGAAGGAGACTGTCACAACGAGGGGAGTGAT 'SEQ ID NO: 59 . partzyme B TFRCB/74—P: CTGGGAGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 166 partzyme A HPRTA/7-P AGAAATAGTGATAGATCACAACGAGTGCCATGTTAA SEQ ID NO: 167 partzyme B HPRTB/7—P TATCACAGCCAAGGCTAGCTCATTCCTATGACTGTAGATTTTA SEQ ID NO: 168 partzyme A HPRTA/SS—P CTGAATAGAAATAGTGATAGATCACAACGAGAGGTGCGGT SEQ ID NO: 169 partzyme B HPRTB/SS-P GAGCTGGGGAGGCTAGCTCATTCCTATGACTGTAGATTTTA ' 8.2. Reporter ates ‘ For this example, in each multiplex five different universal substrates were used together in the one reaction chamber. Each universal substrate in each multiplex was labelled with one of five different fluorophores. In the current example, the substrates were 5’ end labelled with a fluorophore and 3’ end ed with a quencher moiety (Table 19). Cleavage of the substrates was monitored at various emission and excitation Wavelengths (Table 19).

Table 19. Substrates and their fluorescent ing Multi - lex 1 ' Sub2-70582 672-684 705--730 _—_II_—— Multi u [ex 2 Sub74 .-Sub74-‘70532 B 672-684 , 30 Sub80 Sub80—O67OBZ. Quasar 670 620 650 675-690 Sub79 Sub79-TRIBR Texas Red |E_ _560-590 610-650 Sub55 SubSS-HIB - _II_ 515 535 560-580 Sub6l Sub61-FIB _|]_ 450-490. 510-530 .

‘ A BHQI; black hole quencher l, BHQZ; black hole quencher 2, IB; Iowa black® FQ, IBR; Iowa black® RQ *CFX96 Real-Time PCR Detection System-(Biomd) excites, and measures on of, each fluorophore over a range of wavelengths for each channel. ' . The reporter substrates tested in this example are shown below with the sequence, ’ to 3’. The lower case bases represent RNA and the upper case bases ent DNA. 1-SEQ ID No; 21 SubZ—Q7OSBZ: AAGGTTTCCTCguCCCTGGGCA SEQ ID NO: 22 Sub3i-FIB: AACCguCACCAACCG SEQ ID NO: 23 Sub6-Q67OB2: ATCACGCCTCguTCCTCCCAG 'SEQ ID NO: 24 B: TTAACATGGCACguTGGCTGTGATA SEQ ID NO: 171 Sub4—TRBZ: CATGGCGCACguTGGGAGAAGTA SEQ ID NO: 73 Sub6l-FIB: CTCGACCCCguCTCCACGCCA SEQ ID NO: 77 Sub74—Q7OSB2: ATCACTCCCCguCCCCTCCCAG' IO SEQ ID NO: 80 Sub79—TRIBR: TCCTCTCCCCguCCCCTTCAACC SEQ ID NO: 81 Sub80—Q670B2: AACCGCCCTCguCCCGTGAACC SEQ ID NO: 29 SubSS—HIB: ACCGCACCTCguCCCCAGCTC 8.3 Tdrget sequences and PCR primersfor amplification ofthe CYP2C9, TP53, HPRT, TFRC and RPL13a genes i The ' target PCR amp'lic-ons for all five genes. were generated by in vitro amplification of Human DNA extracted‘ from 1M9 cell line (Promega). The amplicons were generated using the oligonucleotide ‘PCR primers listed 5’ to 3’ below. The sequence in bold corresponds to a universal tag (U1, U2 or U3) that increases the Tm of the primer without affecting the specificity of the primer to the gene target. This tag improves cation efficiency in PCR ons.

SEQ ID NO: 91 Forward primer SITFRC_U1 GCTAAAACAATAACTCAGAACTTACG SEQ ID NO: 92 e primer . 3TFRC_U2 TQTGAGGTTACCATCCTA , SEQ ID NO: 125 Forward primer 5TP53__U3 CTEACTTACTGCCTCTTGCTTCTC SEQ ID NO: 126 Reverse primer ~3TP53_U2 CAGCTCTGTGCGCCGGTCTCTC SEQ ID NO: 127 Forward primer 5RPL13a_U3 CTAAACCGGAAGAAGAAACAGCTCA SEQ ID NO: 128 Reverse primer 3RPLl‘3a__U2 CAGGAGGAATTAACAGTCTTTATTGG SEQ ID NO: 129 Forward primer .5CYP2C9_U3 CTAACCTCATGACGCTGCGGAA SEQ ID NO: 130 Reverse primer 3CYP2C9_’U2 CAGATATGGAGTAGGGTCACCCA SEQ ID NO: 176 Forward primer 5HPRT_U3 CTAACTTTGCTGACCTGCTGGATTA SEQ ID. NO: 177 Reverse primer 3HPRT_U2 CAGCAATAGCTCTTCAGTC’I‘GATAA 8.4. Reaction Components: Amplification and detection of target sequences in a multiplex MNAzyme qPCRformat Real-time amplification and'detection of the target sequences was performed in total reaction volume of 25 uL. All reactions were conducted in a CFX96 Real-Time PCR ' Detection System (Bio-Rad). The cycling parameters were either, 1) 95°C for 2 minutes, 40 cycles of 95°C for 15 seconds and ”52°C for 60 seconds or 2) 95°C for 2 minutes, 40 cycles of 95°C for 15 seconds and 58°C for 60 seconds. Fluorescent data were ted at either the 52°C or 58°C step. Each lex reaction was run in duplicate and contained 10 mM MgC12 10 units Ribosafe RNase inhibitor , 200 MA of each dNTP, . ne), 1 x Immobuft‘er (Bioline), 2 units MyTaqHS (Bioline). The identity of the partzymes, primers and substrates and their respective concentrations were as listed in Table 20. ons contained either DNA te (100 ng or 391 pg ) or no target control (NF-H20).

Multiplex reactions were set up with primers, substrates. and their associated part'zymes as in Table 20>(Multiplex l or lex 2). The same PCR primers were used for both multiplex reactions and all partzymes had the same -sensing portions. Any differences in efficiency of reactions detecting the same target will therefore be attributable to differences in the ncy of cleavage of the substrates.

Table 20. Oligonucleotide ations used for each multiplex on ' primer 3'Primer (20 nM (400 nM ate i PzA & P23 (200 nM each) except except (200 nM each) * = 80 nM) * = 200 nM) Multiplex l TFRCA/Z-P TFRCBS/Z-P TFRC Sub2-Q705B2 5TFRC_U3 3TFRC~U2 SE O ID NO: 34 SE O ID NO: 35 SEO ID NO: 2] HPRTA/‘l-P HPRTBS/7-P Sub7-JB HPRT 5HPRT_U3* 3HPRT_U2 V SEO ID NO: 166 SEO lD NO: 167 SEO ID NO: 24 ' 5TP53__U1 TPS3A/4-P TPS3BS/4-P Sub4-TRBZ TP53 3TP53_U2“ SEO 10 NO: 164 SEO ID NO: 165 VSEO ID NO: 171 A/6-P RPLlSaB/6-P RPL13a Sub6Q67OBZ SRPL13a_Ul 3RPLl3a _U2 SE O lD NO: 157 SEO lD NO: 163 SEO ID NO: 23 CYP2C9A/3-P CYP2C9B/3-P Sub3~FIB CYP2C9 5CYP2C9_U1 3CYP2C9_U2 SE O ID NO: 96 SEO ID NO: 97 SE O ID NO: 22 Multiplex 2 TFRCA/74-P TFRCB/74-P TFRC Q70532 5TFRC~U3 3TFRC_U2 SEO ID NO: 58 SEO ID NO: 59 SEO ID NO: 77 HPRTA/SS-P HPRTB/SS—P SubSS-HIB HPRT 5HPRT_U3* 3HPRT_U2 SEO ID NO: 168 SEO ID NO: 169 SEO ID NO: 29 TP53N79~P TP53B/79-P Sub79-TRIBR TP53 5TP53__U1 3TP53_U2* SEO ID NO: 106 SEO ID NO: 107 SEQ ID NO: 80 RPLl3aA/80-P RPLlSaB/SO-P Sub80-Q67OBZ RPLl3a SRPLI 3a_u1 3RPLl3a _U2 SEO ID NO: 120 SEO ID NO: 121 SEO ID NO: 8] 9_U2 CYP2C9A/6 l -P CYP2C9B/6 l -P Sub6l-FIB CYP2C9 5CYP2C9flU1 SEQ ID NO: 98 SEQ ID NO: 99 SEQ ID NO: 73 8.5. Results: Amplification of target and cleavage of er substrate in ‘- multiplex MNAzyme qPCRformat Each multiplex reaction containing human genomic DNA showed an increase in fluorescence over time for the real-time detection of the genes CYP2C9, TP53, HPRT, RPL13a and TFRC. For all reactions, the fluorescence of the no-DNA target control was lower than that in the DNA target~containing ons. This trates that the increase in fluorescence produced in target-containing reactions is due to target dependent l0. assembly of catalytically active MNAzymes that then d one of the universal substrates.

The amplification plots of the CYP2C9, TP53, HPRT, RPL13a and TFRC genes at 52°C demonstrated that Multiplex 2 (Figure 11, (i)), which used the new improved series 2 and 3 sal substrates, had steeper curves that reached plateau faster than those [5 observed for Multiplex 1 (Figure. 11, (i)), which used series 1 universal substrates. There was only a small difference in the amplification plots for detection of TFRC with Sub2 versus Sub74 at 52°C. One skilled in the art would appreciate that all of the amplification plots of Multiplex 1, using series 1 vs series 2 and 3 substrates, are of a sufficiently good quality to be readily acceptable in the field for detection of these targets.

The amplification plots of the CYP2C9, TP53, HPRT,,RPL13a and TFRC genes at 58°C demonstrated that Multiplex 2 e 11, (ii)), which used the new improved Series 2 and 3 universal substrates, had conSiderably steeper curves and reached plateau substantially faster than plex 1 (Figure 11, (ii)), which used series 1 universal substrates. Further, one'skilled in the art would acknowledge that the amplification curves produced by use of Sub2, Sub3, Sub6 and Sub7 in Multiplex 1 may not be of sufficient quality for robust detection of target and thus may not be lly acceptable in the field. lO l, the new series 2 and 3 substrates show improved y, and therefore robustness, of multiplex data at temperatures tly in use with MNAzyme qPCR reactions and enable multiplex detection at a temperature much higher than previously capable. The new design guidelines increase the probability of designing universal substrates that extend the ability to multiplex at temperatures currently used for l5 MNAzyme qPCR and that produce robust data at higher reaction temperatures.

Example 9: Use of universal substrates, designed to be onal at s temperatures, with MNAzymes in real-time PCR.

A new series of highly active substrates has been invented, using a novel set of design guidelines, for use with the 10-23 DNAzyme or MNAzyme based on the 10-23 DNAzyme.

MNAzymes can be tailored to produce a detectable effect, via cleavage or ligation of a substrate, at s temperatures. The efficiency and stringency of catalytic activity of an MNAzyme or DNAzyme can be manipulated by changing the reaction temperature.

Another way to optimise efficiency and stringency of catalytic ty is to modify the Tm and/or length of the substrate(s) and matching partzymes or matching DNAzyme. The Tm of a substrate can be increased by adding nucleotides on to the 5’ and/or 3’, ends'of the substrate sequence. These s can be made within the deSign guidelines. The tides chosen for this extension can also have an effect on the dition of extra G or C bases to the 3’ or 5’ end will have a greater impact on the Tm than the addition of extra A or T bases. Similarly, the Tm of substrates can be reduced by removing nucleotides from the 3’ or 5’ end. MNAzyme partzymes and DNAzymes can be truncated or extended "to match the adjusted substrate sequence.

In this example, the length and base composition of the series 2 substrate, Sub55, was modified to produce a range of derivative substrates with a variety of Tm’s (Table 21). Modifications included truncating the substrate by removal of one to three nucleotides from each of the 5’ and 3’ ends; extending the substrate by the addition of nucleotides to both of the 5’ and 3’ ends. The effect of adding different nucleotides to produce this extension was also tested by designing substrates with either additional A or C tides at the 5” and 3’ ends. The resulting ates were then tested in a MNAzyme qPCR reaction to assess the lity of design of derivatives of substrates and their utility at a range of temperatures. The PCR amplification and MNAzyme- mediated detection were med in a one-step process,‘ wherein PCR amplification and MNAzyme-mediated detection occurred simultaneously in a single tube. The efficiency of cleavage of substrates can be measured by the Ct value generated at several annealing temperatures. Reactions that produce a lower Ct value are tive of more efficient cleavage of a specific ate since such ons reach the threshold cycle .

Table 21: SubSS and derivatives ' T[11* GCACCTCguCCCCAGC S‘“b55(16) . 16 52 SEQ ID no: 172 j - CGCACCTCguCCCCAGCT Sub55,(18) SEQ ID NO: 173 -18 5 8 SW55 ACCGCACCTCguCCCCAGCTC 21 SEQ ID NO: 29' .

AACCGCACCTCQ'UCCCCAGCTCA - (23A) 23 ID NO: 72 SEQ 174 . CACCGCACCTCguCCCCAGCTCC Sub55(23C) SEQ ID NO: 175. -23 76 " All sequences written 5’ to 3’; uppercase represents DNA and lowercase represents RNA * Tm given here equates to the melting temperature of the bases bound to the two partzymes calculated using the Wallace rule. When the substrate is bound to the e based on the 10-23 DNAzyme, or the -23 DNAzyme itself, the “g” cleoti'de remains unbound therefore does not contribute to the overall bound Tmi ‘ 9.1. Partzyme Oligonucleotides In the experiments conducted to measure the rate of catalytic activity of SubSS and its’ derivatives described in Table 21, the partzyme oligonucleotides A and B were designed with target sensor arms complementary to the human TFRC_ gene. The sequences of the A and B partzymes are listed below from 5’ to 3’, where the bases underlined hybridize to the substrate. The “-P” indicates 3’ phosphorylation of the oligonucleotide.

SEQ ID NO: 179 . partzyme A TFRCA/55(18)—P:- GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCG SEQ ID NO: 180 partzyme B TFRCB/55(18)-P: AGCTGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 181 partzyme A TFRCA/55(l6)eP: GGAATATGGAAGGAGACTGTCACAACGAGAGGTGC SEQ ID NO: 182 partzyme.B TFRCB/55(l6)-P: GCTGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 183 , partzyme A TFRCA/55(23A)—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCGGTT SEQ ID NO: 184 partzymé B TFRCB/55(23A)—P: TGAGCTGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: 185 partzyme B 55(23C)—P: .GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCGGTG SEQ ID NO: 186 partzyme B TFRCB/55(23C)—P: GGAGCTGGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT SEQ ID NO: ' 46 partzyme A TFRCA/SS—P: GGAATATGGAAGGAGACTGTCACAACGAGAGGTGCGGT SEQ ID NO: 45 partzyme B TFRCB/55~P: GAGCTGGGGAGCCTAGCTCCTCTGACTGGAAAACAGACT 9.2. Reporter Substrates In the current example, the substrates were end labelled with a Quasar 670 moiety at the 5’ end and a BHQ2 moiety at the 3’ end. ge of the substrates Was monitored ' at 665 nm (Quasar 670 emission wavelength) with excitation at635 nm (Quasar 670' excitation ngth). The reporter substrates tested in this example are shown below with the ce, 5’ to 3’. The lower" case bases ent RNA and the upper case bases represent DNA. , SEQ ID NO: 29' SubSS-Q67OB2: ACCGCACCTCguCCCCAGCTC SEQ ID NO: 172 sdb55(16)~Q670B2: GCACCTCguCCCCAGC SEQ ID No; 173_ Sub55(18)—Q67OBZ: CGCACCTCguCCCCAGCT SEQ ID NO: 174 Sub55 (23A) ~Q67OB2: ACCTCguCCCCAGCTCA SEQ ID NO: 175 sub55(23C)—-Q67OB2.

ACCTCguCCCCAGCTCC 9.3. Target sequence and PCR primersfor amplification. of TFRC The target sequence for this example was ‘a PCR amplicon generated by in vitro amplification of human genomic DNA, extracted from the 1M9 cell line (Promega), using the oligonucleotide PCR primers listed below. Primer sequences are listed 5’ to 3’.

SEQ ID NO; 187 Forward primer STFRC: AACAATAACTCAGAACTTACG SEQ ID NO: 188 Reverse primer 3TFRC: CTTTCTGAGGTTACCATCCTA 9.4. Reaction Components:Amply‘ication and detection oftarget sequence Real—time amplification and detection of the target sequence was med111 a total on volume of 25 uL. All reactions were conducted in a P QPCR system (Stratagene/Agilent) The cycling parameters were varied by the annealing temperature, (underlined as folloWs), and were either, 1) 95°C for 10 minutes, 5 cycles of 95°C for 15 seconds and 55°C for 30 seconds, 50 cycles of 95°C for 15 seconds and EC. for 60 seconds, or 2) ' 95°C for 10 minutes, 5 cycles of 95°C for'15 seconds and 55°C for 30 seconds, 50 cycles of95°C for 15 seconds and 52°C for 60 s, or 3) . 95°C for 10 minutes, 5 cycles ‘of 95°C for, 15 seconds and 55°C for 30 seconds, 50 cycles of 95°C for 15 seconds andm for 60 s, or 4) 95°C for 10 minutes, ’5 cycles of 95°C for 15 seconds and 55°C for 30 seconds, 50 cycles of 95°C for 15 seconds and @219 for 60 seconds.

All fluorescent data were collected at the annealing temperature. Reactions were set up with ates. and their associated partzymes as in Table 22. Each set of reaction conditions were run in duplicate and contained 40 nM STFRC, 200'nM of 3TFRC, 200 nM each of partzyme A and partzyme B, 200 11M of substrate, 8 mM MgC12, 200 uM of. each dNTP, 10 units Rnasin (Promega), 1 x Immobuffer (Bioline), 1 unit of Immolase (Bioline) and either genomic DNA template (100' ng) or no target (NF-H20). Separate ons were set up to test each substrate with its matched'partzymes. The same PCR primers were used for all reactions and all partzymes had the same target-sensing portions. Any differences in efficiency of reactions will therefore be attributable to ences in the efficiency of cleavage of the substrates at various temperatures.

Table 22: Partzyme combinations used for each universal substrate Substrate Partzyme B Sub55 TFRCB/SS-P SEO ID NO: 29 SEQ ID NO: 46 SEQ ID NO: 45 Sub55(16) TFRCB/55(16)-P SEQ ID NO: 172 SEQ ID NO: 181 SEO ID NO: 182 Sub55(l8) TFRCA/55(18)-P 55(18)-P SEQ ID NO: 173 SEO ID NO: 179 SEQ ID NO: 180 Sub55(23A) TFRCB/55(23A)-P SEQ ID NO: 174 SEQ ID NO: 183 SEQ ID NO: 184 Sub55(23C) TFRCB/55(23C)-P SEIIDN02175 SEO ID NO: 185 SEO ID NO: 186 9.5. Results: Amplification et and cleavage of er substrate Each reaction ning human genomic DNA showed an increase in fluorescence over time for real time ion of the TFRC gene using Sub55 and various derivatives.

The fluorescence of the no-DNA target control was lower than that in the DNA target- containing reactions. This trates that the increase in fluorescence produced in target-containing reactions is due to target dependent assembly of catalytically active MNAzymes that then d one of the universal substrates.

The efficiency of the reactions, measured by the Ct, was dependent on the compatibility of the reaction temperature (annealing/cleavage temperature) and the Tm of the substrate used in the reaction. The various Sub55 derivatives have different lengths and nucleotide compositions and thus different melting temperatures (Tm) and are therefore ed to perform differently at the various annealing temperatures tested (50, 52, 55, and 60°C). The results, in Table 23, ShOW the Ct for each Substrate at the different reaction temperatures. The Ct values in bold indicate the substrates(s) which performed most efficiently at the atures indicated.

At lower temperatures the shorter substrate (Sub55(18)) performed better (had the lowest Ct value). As the annealing temperature increased, substrates with increased length and therefore Tm, performed optimally. One skilled in the art could produce derivatives of substrates that could be efficiently cleaved at a chosen on temperature by lengthening or shortening the substrate arms from the 5’ and/or 3’ ends, and/or changing the nucleotide composition at the 5’ and 3’ ends of the substrate.

Table 23: Ct values for e qPCR med using SuhSS and derivatives at various annealing temperatures Example 10: Use of universal substrates with MNAzymes qPCR at an annealing temperature of 58°C.

MNAzymes can be used to monitor amplification of target nucleiciacids in real-time using in vitro target amplification methods such as PCR. Furthermore, ime monitoring during qPCR using MNAzyme substrates labelled with fluorophore and er pairs generates a curve on which a threshold line, of an arbitrary level of l0 cence, can be placed over the exponential phase of the reactions, producing a value which can be known as a Ct (cycle threshold). Reactions that produce a lower Ct value are indicative of more efficient cleavage of a specific ate since such reactions reach the threshold cycle faster. In this example, amplification and detection are performed in a one-step process, wherein PCR amplification and MNAzyme-mediated detection occur IS simultaneously in a single tube. Where all other reaction conditions are the same the Ct value can be influenced by the sequence of the universal ate. The annealing/detection temperature for MNAzyme qPCR used in the art is between 50 and 54°C. This temperature was dictated by the fact that the universal substrates known in the art had a limitation on the ature at which they were efficiently cleaved with 54°C being the upper limit for the series 1 universal substrates. There is a need for universal substrates that cleave at higher temperatures to allow greater flexibility in design of primers and partzymes that anneal at higher temperatures. This design flexibility for primers and partzymes could be of great benefit for many applications such as c targets of interest that have high tages of G and C bases in their sequence, requiring higher reaction temperatures and hence partzymes and primers with higher Tms for specific detection.

Investigation into efficiency of cleavage of substrates based on the performance of the series 1 and 2 ates, lead to the development of guidelines. to aid in a third round of substrate designs, resulting in the series 3 substrates. These guidelines included but were not limited to (i) seven or more cytosine nucleotides in the ten bases surrounding the ribonucleotides (Nil—Nu), (ii) bases ately adjacent to the ribonucleotides are cytosines (N3 and N9) (iii) total content of substrate has >64% pyrimidines and (iv) total Tm of the oligonucleotide is 66°C or greater (where this latter guideline is only applicable if the reaction temperature for substrate ge is above 50°C).

In this example, the series 2‘ universal substrate, Sub59 is compared to the series 3 substrate, Sub77 to compare the cleavage efficiency in real-time PCR at 58°C to ensure that the design guidelines produce universal substrates with a high probability of elevated temperature. The level of cleavage . applicability to MNAzyme qPCR at an efficiency was ined by measuring the Ct value for reactions containing ent universal substrates. .1. Partzyme Oligonucleotides In the experiments conducted to measure the efficiency of cleavage of the series 2 and series 3 sal substrate in real-time PCR, all the partzyme oligonucleotides A and B were designed with sensor arms complementary to the same sequence of the human TFRC gene. The sequences of the A and B partzymes are listed below from 5’ to 3’, where the bases underlined ize to their matched universal substrate. The “—P” indicates 3’ orylation of the ucleotide. .

SEQ ID NO: 47 me A TFRCA/59-P: GGAATATGGAAGGAGACTGTCACAACGAAGGGAGGAGG SEQ ID NO: 62 partzyme A TFRCA/77-’P: GGAATATGGAAGGAGACTGTCACAACGAGAGGGAGGAC SEQ ID NO: 63 partzyme B TFRCB/77—P: AGGAGGAGGGAGGCTAGCTCCTCTGACTGGAAAACAGACT 10.2. Reporter Substrates The reporter substrates for this example are shown below with the sequence, 5’ to 3’. The lower case bases represent RNA and the upper case bases represent DNA. In the t example,ythe substrates were end labelled with a 6-FAM moiety at the 5’ end (indicated by a “F” in the name of the substrates below) and an Iowa Black® FQ quencher moiety at the 3’ end'(indicated by a “IB” in the name of the substrates below). Cleavage of the substrates was monitored at 530 um (FAM emission wavelength on Mx3005P (Stratagene)) with excitation at 485 nm (PAM excitation wavelength on Mx3005P (Stratagene)).

SEQ ID NO: 33 Sub59—FIB: CCTCCTCCCTguCCCTCCTCCT ' SEQ ID NO: 79 Sub77—FIB: CTCCTCCCTCguCCCTCCTCCT .3. Targetsequence and PCR primersfor amplification of TFRC The target sequence for this example was a PCR amplicon from'the TFRC gene generated by in vitro amplification of human genomic DNA, extracted from the lM9 cell line (Promega), using the oligonucleotide PCR primers listed below. Primer sequences are listed 5’ to 3’.

SEQ ID NO: 187 Forward primer 5TFRC: AACAATAACTCAGAACTTACG SEQ ID NO: 188 Reverse primer 3TFRC: CTTTCTGAGGTTACCATCCTA .4. Reaction Components: Amplification and quantification oftarget sequence is Real-time PCR amplification and detection of the target sequence was performed in a total reaction volume of 25 ML. All reactions were conducted in an P QPCR system (Stratagene). Reactions were set up with substrates and their associated partzymes as in Table 24. The g'parameters were, 95°C for 2 minutes, 40 cycles of 95°C for seconds and 58°C for 60 seconds (data collected at the 58°C step). Each set of reaction conditions were run in ate and ned 40 nM -5TFRC, 200 nM of 3TFRC, 200 nM each of partzyme A and partzyme B, 200 nM substrate, 8 mM MgC12, 200 pM of each dNTP, 10 units RNasin (Promega), l x Immobuffer (Bioline), 2' units of STM DNA polymerase (Bioline) and either genomic DNA template (50 ng) or no target (NF-H20).

Table 24: Partzyme combinations used for each universal substrate .

Subfinue Iliflfiflfiilflllllll Par IneB ‘ Sub59 59-P TFRCB/77-P SEI ID NO: 33 SEI ID NO: 47 . SEI ID NO: 63 Sub77 TFRCA/77-P TFRCB/77-P SEQ ID NO: 79 SEI ID NO: 62 SEI ID NO: 63 .5. Results: Amplification et and cleavage ofreporter substrate Each MNAzyme qPCR reaction ning human genomic DNA showed an se in fluorescence over time for the real-time detection of TFRC from human genomic DNA. For all reactions the fluorescence of the no—DNA target control was lower than that in the DNA target-containing reactions This demonstrates that the increase in fluorescence produced in target-containing reactions is due to target dependent ly of catalytically active MNAzymes that then cleaved one of the universal reporter substrates.

The reactions with the series 2, substrate Sub59, showed an averaged Ct value of 28.5 while the reactions with the series 3 substrate Sub77 showed an averaged Ct value of 26.5.

Of note is that Sub59 and Sub77 have the same Tm and %C/T but differ in the number of cytosines in the central region N4-N13 and the composition of N3. In the central region surrounding the cleotides Sub77 has a cytosine in position N3 which s to have led to improved cleavage efficiency over Sub59 indicated by a lower Ct value (26.5). Sub59 contains .a thymine at position N3 and an added cytosine at the distal end of' the 5’ arm which has led to a reduced cleavage reaction and hence a higher Ct value (28.5). 115' Of note is the importance of the nature of the nucleotide sequence of the efficiently - cleaved substrate and the proximity of c nucleotides to the ribonucleotides of the substrates. These featuresform the basis of a set-of guidelines that result in universal substrates With a higher probability of being cleaved efficiently at ed temperatures.

These design guidelines include but are not limited to (not all may be necessary): (i) seven or more cytosine nucleotidesin the ten bases surrounding the ribonucleotides (N4- Nis); (ii) the bases immediately adjacent to the ribonucleotides are cytosines (Ng and N9); (iii) total t of substrate has >64% pyrimidine’s; (iv) total Tm of the oligonucleotide is 66°C or greater (where this latter guideline is only applicable ‘if the reaction temperature for substrate ge is above 50°C) (Table 25). The earlier Ct value for the series 3 substrate; Sub77, is expected as this substrate es with all of these design ines, while Sub59 complies with only two of these design guidelines (Table 25).

Table 25: Efficiency of cleavage of universal substrates (listed in order of cleavage efficiency based on Ct) Sub77 CTCCTCCCTCguCCCTCCTCCT SEQ ID NO: 79 CCTCCTCCCT uCCCTCCTCCT Sub59 SEQ ID NO: 33 3of A uppercase bases ent DNA and lowercase bases represent RNA and pOSlthD 0f baseIn a substratels represented by (Nx)’N ;'N2~N3‘NrNstg‘NrNg‘l’R-T_Y-N9-N]o-N 1'l"N lz-N 13'N [4‘N | 5-(Nx) i % C/T (pyrimidines) of sequence length shown above for each substrate, does not include ribonucleotides * Tm given here equates to the melting temperature of the bound bases calculated using the Wallace rule only calculated for bases that hybridize to their complement. When the substrate is bound to the MNAzym‘e based on the 10—23 DNAzyme the “g” ribonucleotide remains unbound therefore does not contribute to .the l bOund Tm.

~ The number of the design guidelines (1), (ii), (iii) and/or (iv) that have been met bythe ate sequence.

REFERENCES - PCT International ation No. WO/2007/041 774 lo PCT Internationali Publication No. WO/2008/040095 PCT International Publication No. WO/2008/122084 US. Patent No. 4,683,202 .8. Patent No. 4,683,195 us. Patent No. 4,800,159 us. Patent No. 4,965,188 US. Patent No. 5,176,995 US publication number 2007-0231810 "L's publication number 136536.

US publication number 201 1-0143338 Cairns, M.J, Hopkins, T;M._, Witherington, G., Wang, L. and Sun, L (1999) Target site selection for an RNA-cleaving catalytic DNA: Nat. Biotechal7: 480-486.

Cruz, R.P., Withers, J.B. and .Li, Y. (2004) Dinucleotide junction cleavage versatility of 8-17 deoxyribozymmChem Biol. Jami 1(1): 57—67. ult, J ., Labuda, D., ’Usm’an, N., Yang, J. and Cedergren, R. (1991) .Relationship between 2'-hydr0xyls and magnesium binding in'the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry 30(16): 4020-5. ult, J., Wu, T., Cousineau, B., Ogilvie,.K. and Cedergren, R. (1990) Mixed 1 deoxyribo- and ribo-oiigonucleotides with catalytic activity. Nature 344(6266): 5654.

Silverman, S. (2004) ng up is easy to do (if you're a DNA enzyme that s RNA). Chem Biol. Jan;11(l): 7-8.Wallace, R.B.,' Shaffer, J., Murphy, R.F., Bonner, J., Hirose, T. and Itakura K. (1979) Hybridization of tic oligodeoxyribonucleotides to ¢;174 DNA: the effect of single base pair mismatch. Nucl. - Acids Res. 6(11): 3543- 3558.

Zaborowska, Z., , J., Erdmann, V. and k, J. (2002) Sequence requirements in the catalytic core. of the "10-23" DNA enzyme. J Biol Chem. 277(43): ' l 240617-22.

Claims (17)

CLAIMS :

1. An isolated polynucleotide substrate for a tic nucleic acid enzyme, said polynucleotide substrate comprising a sequence N1-N2-N3-N4-N5-N6-N7-N8-rR-rY-N9-N10-N11- 3-N14-N15 wherein: rR is a purine ribonucleotide; rY is a pyrimidine ribonucleotide; each of N1-N15 are nucleotides; six or more of N5-N13 are ne nucleotides; N9 is a cytosine nucleotide; the pyrimidine ribonucleotide comprises uracil; and less than three of N9-N15 are guanine nucleotides.

2. The isolated polynucleotide substrate according to claim 1, wherein the cleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 25-27, 29-30, 33, 72-90, or 172-175.

3. The isolated polynucleotide substrate ing to claim 1 or claim 2, wherein seven or more, or eight or more of N5-N13 are cytosine nucleotides.

4. The isolated polynucleotide substrate according to claim 3, wherein seven or more of N5- N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 29, 73, 76-80, 82-83, 85-90, or 172-175.

5. The ed polynucleotide substrate according to claim 3, wherein eight of N5-N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 76, 77, 80, 83 or 87.

6. The ed cleotide substrate according to claim 1 or claim 2, wherein seven or more, or eight or more of N4-N13 are cytosine nucleotides.

7. The isolated polynucleotide substrate ing to claim 6, wherein seven or more of N4- N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 27, 29, 73, 76-83, 85-90, or 172-175. 11013114_1

8. The isolated cleotide substrate according to claim 6, wherein eight of N4-N13 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 76, 77, 79-80, 82-83, 87, 88 or 90.

9. The isolated polynucleotide substrate according to claim 1 or claim 2, wherein six or more, seven or more, or eight or more of N4-N12 are cytosine nucleotides.

10. The isolated cleotide substrate according to claim 9, wherein seven or more of N4- N12 are cytosine nucleotides and the polynucleotide substrate ses or consists of a ce defined by any one of SEQ ID NOs: 27, 29, 73, 76, 77, 79-83, 85-88, 90 or 172-175.

11. The isolated polynucleotide ate according to claim 9, wherein eight of N4-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 76, 77, 80, 83, 87, or 88.

12. The isolated polynucleotide substrate according to claim 1 or claim 2, wherein six or more, seven or more, or eight or more of N5-N12 are ne nucleotides.

13. The isolated polynucleotide substrate according to claim 12, n seven or more of N5-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 29, 73, 76, 77, 80, 83, 85-88 or 172-175.

14. The isolated polynucleotide substrate according to claim 12, wherein eight of N5-N12 are cytosine nucleotides and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 76, 77, 80, 83, or 87.

15. The isolated polynucleotide substrate according to any one of claims 1 to 14, wherein any one or more of N1, N2, and/or N8 is a ne nucleotide.

16. The isolated cleotide substrate according to claim 15, wherein N8 and N9 are cytosine nucleotides, and the polynucleotide substrate comprises or consists of a sequence defined by any one of SEQ ID NOs: 25-26, 29-30, 72-90, or 172-175.

17. The isolated polynucleotide substrate according to any one of claims 1 to 16, wherein two, one or none of N9-N15 are guanine nucleotides. 11013114