Functional nucleic acids as bacterial and viral sensors
Jan 21, 2022
2:30PM to 3:30PM
Date(s) - 21/01/2022
2:30 pm - 3:30 pm
Pathogens have long presented a significant threat to human lives and hence rapid detection of infectious pathogens is vital for improving human health. Current detection methods oftentimes lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms. In this presentation, I will discuss the work completed in my group, in collaboration with several other groups at McMaster, towards developing FNA-based biosensors for detecting pathogenic bacteria, and most recently the SARS-CoV-2 viruses that are responsible for the COVID-19 pandemic. We have conducted in vitro selection experiments to derive RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori and Legionella pneumophila. In most cases, a “many-against-many approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections to ensure high specificity towards the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also isolated for detection of bacteria such as Clostridium difficile. Mostly recently, we have developed multiple aptamers that recognize the spike proteins of the original SARS-CoV-2 virus and several variants of concern. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly, have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, saliva and stool, has now been demonstrated with an outstanding sensitivity, highlighting the tremendous potential of using FNA-based sensors in clinical applications. I will further describe ways to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases, strategies to maintain the functionality of FNAs in clinical samples, and strategies to deal with issues like signal loss caused by non-specific binding and biofouling. Lastly, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.
Yingfu Li is a Professor in the Department of Biochemistry and Biomedical Sciences and the Department of Chemistry and Chemical Biology at McMaster University. He earned a PhD in Biochemistry from Simon Fraser University in 1997, where he discovered a DNA molecule that catalyzes porphyrin metalation, which won him Governor General Academic Gold Medal and Natural Sciences and Engineering Research Council of Canada Doctoral Prize, both in 1998. He was awarded a Medical Research Council of Canada postdoctoral fellow in 1999 and carried out his postdoctoral research at Yale University between 1997-1999, where he studied a series of catalytic DNA molecules for DNA phosphorylation, DNA capping and DNA ligation. In November 1999, he joined McMaster University as an Assistant Professor in the Department of Biochemistry and Biomedical Sciences and the Department of Chemistry and Chemical Biology, was promoted to Associate Professor in 2005 and Full Professor in 2010. At McMaster, he has established a research group focusing on artificial nucleic acid molecules with catalytic and/or binding properties. He has published extensively in the fields of chemistry, biochemistry and molecular evolution of nucleic acids, including over 200 research and review articles, over 20 book chapters, and 1 book. He has filed over 30 patents on functional nucleic acids and diagnostic tests. He has also served as an Associate Editor of Journal of Molecular Evolution and as a member of editorial board of Scientific Reports, and Analysis and Sensing. He has received several recognitions, including Canada Research Chair, New Investigator Award from the Canadian Institute of Health Research, Premier Research Excellent Award from Ontario Government, and McBryde Medal from Canadian Society of Chemistry.