Abstract

The increasing prevalence of multidrug-resistant and hypervirulent bacterial strains represents a growing global healthcare concern. However, early detection of pathogenic microbes allow for timely care of patients and the prevention of infectious strains proliferation. In the face of the current challenges in profiling bacterial infections, we are designing a fast and user-friendly detection assay using fluorogenic DNAzymes as the molecular probe. Our fluorogenic DNAzymes are single-stranded functionalized DNA capable of cleaving a fluorophore-quencher construct specifically in the presence of E. coli. Upon cleavage, the quencher can no longer suppress the fluorophore, resulting in intense fluorescence. This fluorescence intensity can be also used to quantify the amount of E. coli, and potentially achieve strain-specific recognition. Our novel approach to early pathogen detection technology can potentially enhance our ability to respond to disease outbreaks from infectious bacteria.

Background

The increasing prevalence of multidrug-resistant and hypervirulent bacterial strains represents a growing global healthcare concern. Early detection of pathogenic microbes is essential for timely care of patients and for preventing the spread of infectious diseases, particularly in hospital settings with immunocompromised patients.

The need for rapid pathogen detection can be addressed using biosensors. Biosensors have two key components: a recognition element for the target/pathogen, and a signal that is dependent upon target/pathogen detection. Typically, the recognition element has been made of proteins such as antibodies or enzymes. A recent advancement in recognition elements is the applicability of nucleic acids for this role. Functional nucleic acids with defined catalytic function, termed DNAzymes, provide a promising approach toward biosensing. DNAzymes are inherently more stable than proteins, and they can be created to recognize a broad range of targets using in vitro directed evolution. As well, DNAzymes can be chemically synthesized at a low cost and without the need for animals (as is the case for antibodies).¹

McMaster iGEM has set out this year to design a fast and user-friendly detection assay using fluorogenic DNAzymes as the molecular probe. Our fluorogenic DNAzymes are single-stranded functionalized DNA capable of cleaving a fluorophore-quencher construct specifically in the presence of E. coli. Fluorescence was chosen for its high detection sensitivity and capability for real-time detection. Upon cleavage, the quencher can no longer suppress the fluorophore, resulting in intense fluorescence. This fluorescence intensity can be used to quantify the amount of E. coli. DNAzymes can be created specifically for various bacterial species, and potentially for strain-specific recognition.

In Figure 1, the fluorophore (F) and quencher (Q) are attached to the nucleotides flanking the cleavage site (R). The black strand is the DNAzyme which specifically recognizes a bacterial target (such as E. coli) and cleaves its substrate (red strand), resulting in a fluorescence signal.²

References

1. Willner, I., B. Shlyahovsky, M. Zayats, and B. Willner. 2008. DNAzymes for sensing, nanobiotechnology and logic gate applications. Chemical Society Reviews 37: 1153.
2. Tram, K., P. Kanda, and Y. Li. 2012. Lighting Up RNA-Cleaving DNAzymes for Biosensing. Journal of Nucleic Acids 2012: 1-8.