DNA-based molecular circuits for diagnostics and therapeutics

Nucleic acids are a uniquely flexible and multi-faceted class of molecules that fulfill fundamental and defining tasks such as replication and determination of heritable characteristics in every living organism. From the microscopic to the gigantic, from the most primitive to the most complex, life has been both molded and served by nucleic acids. Nucleic acid circuits straddle the realm of nature and technology. The elegance of interaction between nucleic acid molecules invites us to gain a deeper understanding of the naturally occurring systems they compose and to apply our ingenuity and foresight toward developing ever more complex synthetic systems. Nature has provided these basic building blocks, which we can now arrange – and augment – for the purpose of creating molecular-level machinery. Here we describe some ways in which we have rationally harnessed nucleic acids. In preparation for outbreaks of novel and deadly avian influenza viruses, we used quantitative polymerase chain reaction (qPCR) to track the number of flu virus particles surviving in the presence of potential antiviral drugs. We engineered tunable on/off switches that can be used to evaluate a series of conditions for diagnostic applications or to enable ‘smart’ drugs that sense, analyze, and respond to their microenvironment. We optimized the conditions for, and used, a unique set of guanine-rich DNA sequences called G-quadruplexes, whose enzymatic and structural properties make them prime effector candidates in diagnostic platforms. G-quadruplex folding powers isothermal DNA amplification, and the small organic molecules they bind endow G-quadruplexes with expanded catalytic abilities. We genotyped drug resistance mutations in tuberculosis via visually detectable color changes in the reaction buffer. We developed a paper fluidics assay that employs soluble and bead-immobilized nucleic acids to scan for genes in tuberculosis, and upon detection, to generate a readily observable discoloration on the paper strip. Finally, we probed the boundary of nucleic acid circuitry by attempting to expand its language via the incorporation of unnatural nucleobases into oligonucleotide components of a catalytic hairpin assembly (CHA) circuit. We subsequently evaluated the resilience of the unnatural CHA circuit to contamination by random DNA species, such as may be encountered in clinical samples.