The development of nuclease-mediated signal amplification technology for analyte detection

Abstract

This research project aimed to develop novel isothermal signal amplification chemistries for the detection of nucleic acid in a point-of-care (POC) format. Such detection could offer rapid diagnosis for clinical and in-the-field applications, where immediate answers may be required and/or access to central laboratory facilities is limited. The methods utilised various oligonucleotide switches that could be cleaved by Exonuclease III (Exo III) and Restriction Endonucleases (REases) only in the presence of the target sequence. The cleavage produced signal corresponding to the amount of the target.

The Exo III switches were devised to be cleavable when the target changed the conformation of the oligonucleotide complexes. On the other hand, the REases switches were developed by modifying the availability of functional recognition sites for MnlI and TspDTI. The switches became cleavable when partial recognition sites were completed by Driver Fragment (DF) molecules. During development, novel properties of REases were explored such as cleavage with recognition sites containing nicks and inhibition of activity by the presence of overhang fragments at the recognition sites. The REase switches were then incorporated into a feedback cascade of cleavage between two partially complementary switches to further amplify the signal. The cleavage of a first complex produced a first DF which completed a second complex, which is then cleaved to produce a second DF which, in turn, completed another first complex thus creating a circular feedback cascade. Since signal is produced at each step, the process which is known as EzyAmp, results in amplification of the output signal.

The universality of the cascade was demonstrated by linking DF production to various target initiation steps, including a MNAzyme or 4-way junction step for nucleic acid detection, and an aptamer step for the detection of protein. The MNAzyme initiation allowed a rapid and sensitive detection of synthetic nucleic acid targets. The cascade also performed well in multi-chamber format, and in the presence of serum and wholeblood samples. These features showcase the technologyâ s advantageous capabilities and potential for use in a POC setting.