Biological System Analysis Using a Nanopore Transduction Detector: from miRNA Validation, to Viral Monitoring, to Gene Circuit Feedback Studies

The system biologist is currently lacking a general-use method for a gene circuit ‘voltmeter’ or gene system algorithm ‘print statement’. What is needed is a non-destructive, carrier non-modifying, means of testing ‘live’ biological systems at the single-molecule level. A method using the nanopore transduction detector (NTD) is demonstrated for single-molecule characterization in some situations, so may provide what is lacking. An important aspect of this approach is that use can be made of inexpensive antibody, protein, aptamer, duplex nucleic acid, or nucleic acid annealing molecules (for miRNA and viral monitoring) that have specific binding to the system component of interest. The NTD transducer’s specific binding can also be designed to have low affinity binding as needed, such that there can be a ‘catch and release’ on low copy-number molecular components, such that there is not a disruption to the molecular system under study. NTD transducers are typically constructed by linking a binding moiety of interest to a 14 Stephen Winters-Hilt nanopore current modulator, where the modulator is designed to be electrophoretically drawn to the channel and partly captured, with its captured end distinctively modulating the flow of ions through the channel. Using inexpensive (commoditized) biomolecular components, such as DNA hairpins, this allows for an easily constructed, versatile, platform for biosensing. High specificity high affinity binding also allows a very versatile platform for assaying at the single molecule level, even down to the single isoform level, including molecular substructure profiling, such as glycosylation profiling in antibodies. An inexpensive commoditized pathway for constructing nanopore transducers is demonstrated. Nanopore transduction detector based reporter/event-transducer molecules may serve as a means to perform multicomponent mRNA-miRNA-protein and protein-protein systems analysis in general settings.