Poster W1 - Ad-hoc Designed DNA Probes for Detection of Bacterial Infections and Contaminations using Diverse Transducing Systems

Introduction: Bacterial infections, from foodborne pathogenic Escherichia coli and sexually transmitted Neisseria gonorrhoeae, can cause severe diseases in patients and cost billions of dollars to the US healthcare system every year. Current testing methods for bacterial infections display major limitations such as lack of sensitivity, specificity, reusability and/or cost and accessibility to the user. Here, we report a new sensing strategy for bacterial detection, which exploits restriction enzymes (RE) as specific biomarkers. RE are highly conserved and cleave specific DNA sequences in a bacterial-specific and predictive fashion. We designed nuclease-resistant DNA probes (REProbe) containing recognition sequences for the EcoRI and NgoMIV endonucleases, which are expressed by E. coli and N. gonorrhoeae, respectively. These duplexed and hairpin DNA probes can be functionalized with linkers, reporting molecules and modified bases to improve versatility.

Materials and

Methods: REProbes were conjugated to different substrates and the addition of either purified restriction enzyme or bacterial lysate produced a detectable signal. The REProbes were tested for sensitivity and specificity with assays using spectrophotometry, surface plasmon resonance spectroscopy, and electrochemistry. For spectrophotometry, biotin streptavidin conjugation was used to bind the fluorescent DNA probes to a 96-well plate. The plate was then measured in a spectrophotometer before and after incubation of an array of restriction enzymes, bacterial lysates and buffers. For electrochemical data, gold-thiol conjugation was used to bind DNA probes to a gold screen printed electrode. Measurements were then taken using electrochemical impedance spectroscopy, cyclic and square wave voltammetry with the addition of buffer and restriction enzymes. For surface plasmon resonance spectroscopy, biotin-streptavidin bioconjugation was used to capture a biotinylated REProbe on a streptavidin SPR chip. After measuring the capture value of the mass bound to the surface; buffer, restriction enzymes and bacterial lysates were flowed through the cell. The angle shift in the instrument was then measured again to obtain the relative change in baseline recorded before and after cleavage of DNA.

Results, Conclusions, and Discussions: Spectrophotometry assays demonstrate that purified EcoRI and lysed RTX strain of E. coli, which expresses EcoRI, can cut the desired REProbe to produce a detectable signal. The REProbes exhibit specificity from a lack of cleavage by purified PvuII, SbfI and XhoI. Additionally, pOpen strain of E. coli, which does not express EcoRI, produced no cleavage in the DNA probes. Having even a single base-pair of the restriction site changed causes there to be no cleavage of DNA. These experiments were also performed with a duplex containing the restriction site for NgoIV, from N. gonorrhoeae; cultured and lysed N. gonorrhoeae was able to selectively cleave the probe to produce a signal. The electrochemical assays were performed only on the EcoRI REProbe. Electrochemical impedance spectroscopy showed a selective decrease in impedance when EcoRI was added to the system. Cyclic and square wave voltametric assays exhibited a selective increase in current across the electrode with the addition of EcoRI. The surface plasmon resonance assays that were done with the EcoRI REProbe showed a significant and selective shift in the baseline corresponding to the loss of mass at the surface when EcoRI and RTX lysate were flown through.. Collectively, our data shows that REProbes can be used to develop new diagnostics tests that are highly sensitive (due to signal amplification) and specific, making it possible to build new sensing platforms that are robust, affordable, and stable. Not least, the REProbe technology can also be used to develop functional assays for rapid and high-throughput identification of new restriction enzymes and screening of new nuclease inhibitors.

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