Team:McMasterU/Description

Summary


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.


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Figure 1: DNAzyme: DNAzymes are oligonucleotides with catalytic potential. These oligonucleotides are made with a recognition element which binds to a target molecule. In combination with their catalytic activity, this makes DNAzymes a powerful tool for biosensing. In an inactive DNAzyme, the catalytic region is bound to the recognition element and unable to hydrolyze a bond between an adjacent fluorophore and quencher, and thus fluorescence does not occur. In the presence of the target molecule, recognition element preferentially binds to it, opening the catalytic region for activity. The bond between the fluorophore and quencher is cleaved and fluorescence can occur.


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 2, 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


Figure 1: Structure of DNAzyme<sup>1</sup>
Figure 2


Project Structure


Our project focused on 2 main prongs of DNAzyme applications:

DNAzyme Plate Optimization: Optimizing a plate-based medium for the DNAzyme biosensors using the placeholder model of a common lab strain of Escherichia coli K12 as a proof of concept for the applicability of DNAzymes diagnostics in a solid plate medium. Lab members Julie Fothergill-Robinson, Audrey Jong, Sean Leung and Angela Dong worked on this, ultimately optimizing the characteristics of the plate, proving our self-manufactured DNAzyme's efficacy and specificity, and obtaining proof of concept for the effectiveness, user-friendliness and feasibility of our design.


C. Difficile Project: Generating a novel cis-acting DNAzyme using in vitro selection for the detection of pathogenic strains of C. difficile. Lab members Jessica Chee and Neda Pirouzmand dedicated their summer to this, cloning NAP1, NAP7, and wild type TcdC into pET15 plasmids and working on expressing the genes.


How we Ensure Specificity: In-Vitro Selection


In vitro selection: The recognition element of DNAzymes provide them with the crucial specificity required for a biosensor. To generate a selective and specific recognition element, in vitro selection or SELEX is used. A library of ~20bp long oligonucleotides is made, containing all possible permutations. That's up to 1 x 10^15 oligonucleotides in the library! In positive selection, the ECM of the target microbe is used for selection as it contains the proteins that the microbe secretes and is therefore indicative of the target's presence. The oligo-library is exposed to the ECM: the DNA which binds is kept, while the other members of the library are washed away. In negative selection, the ECM of NON-TARGETS (microbes we do not want to detect with the DNAzyme) are used. Here, oligos which bind are washed away and those which do not bind are kept. This cycle of positive and negative selection is continuously repeated, until all that remains from the library are the highly specific and selective recognition elements.

Figure 3: In vitro selection: The recognition element of DNAzymes provide them with the crucial specificity required for a biosensor. To generate a selective and specific recognition element, in vitro selection or SELEX is used. A library of ~20bp long oligonucleotides is made, containing all possible permutations. That's up to 1 x 10^15 oligonucleotides in the library! In positive selection, the ECM of the target microbe is used for selection as it contains the proteins that the microbe secretes and is therefore indicative of the target's presence. The oligo-library is exposed to the ECM: the DNA which binds is kept, while the other members of the library are washed away. In negative selection, the ECM of NON-TARGETS (microbes we do not want to detect with the DNAzyme) are used. Here, oligos which bind are washed away and those which do not bind are kept. This cycle of positive and negative selection is continuously repeated, until all that remains from the library are the highly specific and selective recognition elements.

Genetic evolution by natural selection has guided life as we know it through billions of years of Earth’s harshest environments, giving rise to millions of diverse and steadfast lifeforms that we see around us today. In the lab, we can mimic this grandiose process to our advantage on a microscale using a technique called in-vitro selection3. By following Darwin’s principles of natural selection in a controlled environment, it is possible to artificially evolve large groups of DNA molecules from randomness and select for those with useful functions. This is done by exposing large, “random libraries” of short DNA strands to other molecules of interest, and removing the species in the pool that don’t react to them in the way we desire. These reactions are observed on the macroscale using the tried and true technique of polyacrylamide gel electrophoresis on the radio-labelled, denatured (untied and linearized) DNA libraries after exposure. In the end, an assortment of DNA molecules comprising of more than a quintillion random nucleotide sequences is reduced to a handful of highly specific, functional product species.


If selected successfully to process a substrate on que, these nucleic acids are called DNAzymes. We work with DNAzymes that have been artificially selected to serve as a detection platform for E. coli, one of the most infamous antimicrobial drug-resistant strains of bacteria in the world4,5. Our DNAzymes react in the presence of a cocktail of molecules radiated specifically from the extracellular matrix of the E. coli bacterium. The ensuing process results in the cleavage of an RNA unit bridging a quencher-fluorophore DNA complex, resulting in the emission of a signature green glow. In essence, we’ve used one of nature’s oldest solutions to generate something innovative, creative, and potentially revolutionary to the field of medical point-of-care testing.



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.
  3. Wilson, D. S. & Szostak, J. W. In Vitro Selection of Functional Nucleic Acids. Annual Review of Biochemistry 68, 611–647 (1999).
  4. Aguirre, S., Ali, M., Salena, B. & Li, Y. A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection. Biomolecules 3, 563–577 (2013).
  5. Antimicrobial resistance: global report on surveillance. (World Health Organization, 2014).