Target detection and signal amplification

A method for detecting the presence or absence of a first target molecule in a sample, the method comprising:

• (a) contacting the sample with at least one molecular complex comprising a BL and a first catalytic nucleic acid enzyme, the BL comprising first and second segments hybridised to the first enzyme by complementary base pairing, and an intermediate segment between the first and second segments comprising a first substrate for a catalytic nucleic acid enzyme, wherein at least a segment of the first substrate is not hybridised to the first enzyme;
• a first initiator enzyme, and a reporter substrate;
• wherein,
• the first initiator enzyme has binding specificity for the first target and the first substrate of said intermediate segment and hybridisation of the target to the first initiator enzyme induces catalytic activity of the first initiator enzyme thereby facilitating cleavage of the first substrate when hybridised to the first initiator enzyme;
• the cleavage of the first substrate causes dissociation of hybridised strands of the blocker oligonucleotide and the first catalytic nucleic acid enzyme of said molecular complex;
• and
• the first enzyme of said molecular complex has binding specificity for the reporter substrate and, after said dissociation, is capable of hybridising to the reporter substrate and cleaving it to thereby provide a detectable signal indicative of the presence of the first target in the sample;
• and
• (b) determining whether a detectable signal is generated by said contacting, wherein detection of the signal indicates the first target molecule is present, and failure to detect the signal indicates the first target molecule is absent.

A method for detecting the presence or absence of a target molecule in a sample, the method comprising:

• (a) contacting the sample with:
• a molecular complex comprising a first catalytic nucleic acid enzyme, or component thereof, hybridised to a blocker oligonucleotide by complementary base pairing;
• an initiator catalytic nucleic acid enzyme;
• a precursor substrate,
• a reporter substrate;
• a first restriction endonuclease;
• and
• a strand displacing polymerase or component thereof;
• wherein
• the initiator enzyme has binding specificity for the target, and hybridisation of the target to the initiator enzyme induces catalytic activity of the initiator enzyme facilitating catalytic modification of the precursor substrate when hybridised to the initiator enzyme, to thereby provide a primer oligonucleotide;
• the blocker oligonucleotide provides a single-stranded overhang segment extending from the molecular complex which comprises a first partial recognition site for the first restriction endonuclease;
• the overhang segment comprises nucleotides which form a binding site for the primer;
• the primer is capable of hybridising with the binding site, thereby providing a free 3′ hydroxyl group for the strand displacing polymerase or component thereof to initiate synthesis of a second catalytic nucleic acid enzyme having base pair complementarity with the blocker oligonucleotide;
• said synthesis of the second catalytic nucleic acid enzyme causes disassociation of hybridised segments of the first catalytic nucleic enzyme and the blocker oligonucleotide, and the first catalytic nucleic enzyme, after said disassociation, is capable of hybridising to and cleaving the reporter substrate to thereby provide a detectable signal;
• said hybridising of the primer and/or said synthesis completes the first partial recognition site, and the first restriction endonuclease is capable of introducing a single-strand nick at a junction between the primer and the second catalytic nucleic acid enzyme, thereby providing a free 3′ hydroxyl group for the strand displacing polymerase or component thereof to initiate synthesis of a third catalytic nucleic acid enzyme having base pair complementarity with the blocker oligonucleotide;
• and
• said synthesis of the third catalytic nucleic acid enzyme causes disassociation of hybridised segments of the second catalytic nucleic enzyme and the blocker oligonucleotide, and the second catalytic nucleic enzyme, after said disassociation, is capable of hybridising to and cleaving the reporter substrate to thereby provide a detectable signal;
• and
• (b) determining whether a detectable signal is generated by said contacting, wherein detection of the signal indicates the target molecule is present, and failure to detect the signal indicates the target molecule is absent.

A method for detecting the presence or absence of a target molecule in a sample, the method comprising:

• (a) contacting the sample with:
• a molecular complex comprising a first catalytic nucleic acid enzyme, or component thereof, hybridised to a blocker oligonucleotide by complementary base pairing;
• an initiator catalytic nucleic acid enzyme;
• a precursor substrate,
• a reporter substrate;
• an enzyme having phosphatase activity;
• and
• a strand displacing polymerase or component thereof;
• wherein
• the initiator enzyme has binding specificity for the target, and hybridisation of the target to the initiator enzyme induces catalytic activity of the initiator enzyme facilitating catalytic modification of the precursor substrate when hybridised to the initiator enzyme, to thereby provide an inactive form of a primer oligonucleotide comprising a 2′3′cylic phosphate group at its 3′ end;
• the enzyme having phosphatase activity is capable of removing the 2′3′cylic phosphate group from said inactive form of the primer oligonucleotide, to thereby provide an active form of the primer oligonucleotide;
• a portion of the blocker oligonucleotide of said molecular complex is single-stranded and comprises nucleotides which form all or a portion of a binding site for the active form of the primer oligonucleotide;
• the active form of the primer oligonucleotide is capable of hybridising with the binding site by complementary base pairing, thereby providing a free 3′ hydroxyl group for the strand displacing polymerase or component thereof to initiate synthesis of a second catalytic nucleic acid enzyme having base pair complementarity with the blocker oligonucleotide;
• said synthesis of the second catalytic nucleic acid enzyme causes disassociation of hybridised segments of the first catalytic nucleic enzyme and the blocker oligonucleotide, and the first catalytic nucleic enzyme, after said disassociation, is capable of hybridising to and cleaving the reporter substrate to thereby provide a detectable signal;
• and
• (b) determining whether a detectable signal is generated by said contacting, wherein detection of the signal indicates the target molecule is present in the sample, and failure to detect the signal indicates the target molecule is absent in the sample.