Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety
In addition to their evolutionary optimized functions, the extraordinary physical and functional properties of nucleic acids provide the opportunity for a plethora of new bio-molecular devices and methods. Designer nucleic acids have been contemplated for therapeutic entities, biosensors, nano-scale devices and tools for molecular computation. The methods exploit the characteristics of DNA self-assembly, electro-conductivity, information elements, amplification, switching, molecular detection and catalytic activity. Further, since DNA is robust, stable and thermostable it provides an ideal material for molecular engineering of mechanical or computational devices.
Single stranded nucleic acids, such as DNA and RNA, have the ability to fold into complex three-dimensional structures that can function as highly specific receptors (e.g. aptamers) and catalysts (e.g. ribozymes, DNAzymes). Further, the requirement for complementarity between nucleic acid strands for hybridization forms the basis for a wide range of techniques, which allow target detection (e.g. microarray analysis, Northern blotting or Southern blotting), and/or target amplification (e.g. the polymerase chain reaction). Further, hybridization provides the basis for nucleic acid nano-scale construction and for DNA based computational strategies.
A wide variety of nucleic acid molecules, with enzymatic or catalytic activity, have been discovered in the last 20 years. RNA enzymes (“ribozymes”) occur in nature but can be engineered to specifically recognize and modify a target RNA substrate (Haseloff and Gerlach, 1988). In vitro evolution techniques have facilitated the discovery and development of many more catalytic nucleic acids, including deoxyribonucleic acids often referred to as “DNA enzymes” or “DNAzymes” (reviewed Emillson and Breaker, 2002). In vitro evolved DNAzymes and/or ribozymes have been discovered which have the capacity to catalyse a broad range of reactions including, but not limited to, cleavage of nucleic acids, ligation of nucleic acids, phosphorylation of nucleic acids, nucleic acid capping, amino acid adenylation, cofactor synthesis, RNA polymerization, template-directed polymerization, RNA-protein conjugation, aldol reaction, alcohol oxidation, aldehyde reduction, purine and pyrimidine nucleotide synthesis, alkylation, amide synthesis, urea synthesis, formation of peptide bonds, peptidyl-RNA synthesis, acyl transfer, aminoacylation, carbonate hydrolysis, phosphorothioate alkylation, porphyrin metallation, formation of carbon-carbon bonds, Pd nanoparticle formation, biphenyl isomerization, formation of ester bonds, formation of amide bonds, DNA deglycosylation, thymine dimer photoreversion or phosphoramidate cleavage (reviewed Silverman, 2007)
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 cleaving either RNA (Breaker and Joyce, 1994; Santoro and Joyce, 1997; Santoro and Joyce, 1998) or DNA (Carmi et al., 1996) molecules. Ribozymes are also able to cleave both RNA (Haseloff and Gerlach, 1988) and DNA (Raillard and Joyce, 1996) target sequences.
The “10:23” and “8:17” DNAzymes are capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds. These DNAzymes cleave native 3′-5′ phosphodiester linkages to create reaction products which have 2′,3′-cyclic phosphate and 5′-hydroxyl groups (Santoro and Joyce, 1997; reviewed Emilsson and Breaker, 2002).
DNAzymes which specifically ligate distinct nucleic acid sequences after hybridizing to two independent substrate nucleic acids have also been characterized. Specific deoxyribozymes (DNAzymes) can ligate 2′,3′-cyclic phosphate and 5′-hydroxyl products and create non-native 2′-5′ linkages. Examples of such DNAzymes include the “7Z81” and “7Z48” ligases (Prior et al, 2004) as well as the “7Q10” DNAzyme ligase (Flynn-Charlebois et al, 2003). Other DNAzymes have been described (Coppins and Silverman, 2004) which can ligate 2′,3′ diol and 5′-triphosphate products and create native 3′-5′ linkages.
Several catalytic nucleic acids have similar basic structures with multiple domains including a conserved catalytic domain (“catalytic core”) flanked by two non-conserved substrate-binding domains (“arms”), which specifically recognize and hybridise to the substrate. Examples of nucleic acids with this basic structure include, but are not limited to, the hammerhead ribozyme, the 10:23 and 8:17 DNAzymes, the “7Z81”, “7Z48” and “7Q10” DNAzyme ligases, the “UV1C” thymine dimer photoreversion DNAzyme and the “DAB22” carbon-carbon bond forming DNAzyme. To date catalytic nucleic acids are typically uni-molecular although examples of catalytic nucleic acids with more than one component are known but these required extensive engineering (Tabor et al, 2006, Kurata et al, 2000).
Catalytic nucleic acids have been shown to tolerate only certain modifications in the area that forms the catalytic core (Perreault et al., 1990; Perreault et al., 1991; Zaborowska et al., 2002; Cruz et al., 2004; Silverman, 2004). Depending on the stringency of the reaction conditions, some degree of mismatch may be tolerated within the substrate arms. However, the requirement for Watson Crick base pairing is sufficiently strict so as to have enabled the development of protocols that use catalytic nucleic acids to facilitate the discrimination of closely related sequences (Cairns et al., 2000) (WO 99/50452).
Target amplification and detection technologies, such as PCR, have been widely used in research and/or in clinical diagnostics. However, despite their power, each has inherent disadvantages. They all require the use of protein enzymes (e.g. DNA polymerase, RNA polymerase, reverse transcriptase, and or ligase). The inclusion of protein enzymes increases the complexity and cost of reagent manufacture and decreases the shelf life of kits containing reagents. Other associated technical challenges include contamination by replicons (target amplicons) from previous reactions leading to false positive signal, and/or background signal caused by replication of primer sequences (primer-dimers) or background caused by target-independent ligation.
Nucleic acid enzymes and nucleic acid enzyme cascades have been considered for a range of biotechnological applications, especially in diagnostics. They could allow detection of proteins and nucleic acids for disease diagnosis by facilitating signal amplification.
Several groups have reported using catalytic nucleic acids for the detection of nucleic acid targets, and other analytes with colourimetric readouts (Elghanian et al., 1997, Mirkin et al, 1996, and Liu and Lu, 2004). Examples of signal amplification cascades, which use uni-molecular catalytic nucleic acids, are known in the art. For example, Paul and Joyce (2004) described a replication cascade mediated by a ribozyme with ligase activity. In another approach, a signal amplification cascade used two inactive, circularized 10:23 DNAzymes which were capable of activating each other by cross cleavage resulting in linearisation (Levy and Ellington, 2003).
There is an ongoing need in the art for the detection of targets, in particular detection using amplification systems such as cascades, for example, detection systems involving cascades which comprise a plurality of nucleic acid enzymes and in particular at least one multicomponent nucleic acid enzyme (MNAzyme). The present invention provides detection methods involving nucleic acid enzyme cascades which incorporate at least one nucleic acid enzyme with ligase activity. Moreover, the use of nucleic acid enzymes with ligase activity allows formation of components for nucleic acid complexes, such as assembly facilitators, partzymes, substrates, DNAzymes or components thereof.