Eveleigh, Australia
Eveleigh, Australia

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Bone S.M.,SpeeDx Pty Ltd | Bone S.M.,University of New South Wales | Todd A.V.,SpeeDx Pty Ltd | Todd A.V.,University of New South Wales
Chemical Communications | Year: 2014

Novel methods were developed to isothermally detect target nucleic acids and initiate signal amplification cascades. The methods utilize target-specific MNAzymes to activate universal primer molecules. These primers can subsequently promote the autonomous synthesis of DNAzymes capable of cleaving nucleic acid substrates and generating signal, which further creates the potential for circular feedback. This journal is © the Partner Organisations 2014.

Mokany E.,SpeeDx Pty Ltd. | Todd A.V.,SpeeDx Pty Ltd.
Methods in Molecular Biology | Year: 2013

Multicomponent nucleic acid enzymes (MNAzymes) are nucleic acid enzymes composed of multiple oligonucleotide partzymes that only associate to form catalytic complexes in the presence of a target nucleic acid. Once assembled, MNAzymes cleave a separate substrate (probe) between fluorophore and quencher labels to produce a fluorescent signal indicative of the presence of the target. MNAzymes are particularly useful as tools for monitoring the accumulation of amplicons during real-time quantitative PCR (qPCR). The partzyme pairs have sensor domains that are complementary to adjacent regions in the amplicons such that their partial catalytic core domains form a complete active MNAzyme core. The probe-binding domain of the partzymes can be complementary to any one of a series of well-characterized universal probes. Since there is no need to synthesize and optimize new target-specific probes for each new target, MNAzyme qPCR provides a flexible alternative which allows target-specific interrogation with a generic readout. A series of universal probes have been designed that perform with high reliability, yielding consistent and reproducible results for any target, making the development of multiplex qPCR assays faster, cheaper, and simpler. This chapter describes a 5plex MNAzyme RT-qPCR method which simultaneously quantifies five mRNA transcripts with high efficiency and specificity using five unique universal probes, each labeled with a different fluorophore. © 2013 Springer Science+Business Media, New York.

Mokany E.,SpeeDx Pty Ltd. | Tan Y.L.,SpeeDx Pty Ltd. | Bone S.M.,SpeeDx Pty Ltd. | Fuery C.J.,SpeeDx Pty Ltd. | Todd A.V.,SpeeDx Pty Ltd.
Clinical Chemistry | Year: 2013

BACKGROUND: MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. METHODS: We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. RESULTS: A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted singletarget assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r2 values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. CONCLUSIONS: MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper. © 2012 American Association for Clinical Chemistry.

Mokany E.,SpeeDx Pty Ltd. | Bone S.M.,SpeeDx Pty Ltd. | Young P.E.,Victor Chang Cardiac Research Institute | Doan T.B.,Victor Chang Cardiac Research Institute | Todd A.V.,SpeeDx Pty Ltd.
Journal of the American Chemical Society | Year: 2010

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines. © 2010 American Chemical Society.

Speedx Pty Ltd | Date: 2013-02-20

The present invention provides modified oligonucleotides and methods for their use in the detection of nucleic acids. The oligonucleotides and methods find particular application in amplifying and/or detecting areas of genetic variation in target nucleic acid sequences.

SpeeDx Pty Ltd. | Date: 2011-11-21

The present invention relates to compositions and methods for the use of enzymes composed of nucleic acid and/or protein enzymes to generate and amplify a signal indicative of the presence of a target. More particularly, the invention relates to compositions comprising nucleic acid structures that serve as partial or complete enzyme substrates and methods for using these structures to facilitate detection of targets.

Speedx Pty Ltd | Date: 2013-01-15

The present invention relates to Multicomponent Nucleic Acid Enzymes (MNAzymes) and methods for their use. MNAzymes comprise two or more oligonucleotide components which self-assemble in the presence of one or more MNAzyme assembly facilitator molecules to form a catalytically active structure. Compositions for making MNAzymes, and collections of MNAzymes are provided. Also provided are methods for using MNAzymes for the detection, identification and/or quantification of one or more targets. The methods can be practiced in solution-based assays or in assays where one or more reaction components are attached to a support structure. The methods allow for multiplexing the MNAzyme detection to detect multiple targets in a single reaction. Also provided are kits for making the compositions, and for practicing the methods provided herein.

Virco and Speedx Pty Ltd | Date: 2011-11-09

The invention concerns a method for the extraction of nucleic acids from biological samples e.g. tissue material or sputum derived from human or animal species and the quantitative detection thereafter of said nucleic acids e.g. in terms of viral load, more specifically RSV viral load detection.

Speedx Pty Ltd | Date: 2012-09-10

The invention relates to nucleic acid substrates for catalytic nucleic acid enzymes and methods utilising the substrates.

SpeeDx Pty Ltd | Date: 2013-06-18

The present invention relates to compositions and methods for the detection of target molecules, and the amplification of detectable signals generated by detection assays. More specifically, the present invention relates to methods utilizing catalytic nucleic acid enzymes to generate and/or amplify a signal indicative of the presence of target molecules (e.g. nucleic acids and proteins), and compositions for use in the methods.

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