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We propose to develop novel aptamer-based technology for naked eye detection of bacterial pathogens. Development of this technology into simple and fast assays for on site detection of pathogens will involve (i) identification of DNA segments best suitable for visualization of the target gene on a filter, (ii) synthesis of both monomeric and contacamerized green fluorescent RNA (GFR) aptamers, (iii) construction of supramolecular assemblies of streptavidin that are able to bind multiple copies of GFR aptamers, (iv) selection and synthesis of DNA aptamers that specifically bind to bacterial proteins, (v) preparation of Sepharose beads derivatized with DNA aptamers that specifically recognize bacterial proteins, (vi) selection of RNA aptamers that specifically recognize and bind to bacterial proteins, (vii) synthesis of RNA molecules composed of one copy the bacterial protein-specific aptamer and multiple copies of GFR aptamer.Specifically, we propose to:1. Synthesize concatamers of GFR aptamers for use in enhanced target detection. We will synthesize derivatives of the GFR aptamer that upon binding 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) emits strong fluorescent light. To increase the optical signal, we construct concatamers of GFR molecules containing 2-4 GFR aptamer. Our preliminary data indicate, that fluorescent RNA concatamers could be used for detection of bacteria and bacteriophage proteins.2. Construct supramolecular assemblies of streptavidin and fluorescent RNA aptamer. Alternatively, to enhance target detection, we will use supramolecular streptavidin assemblies that are able to bind 5-7 copies of the GFR aptamer. We have already successfully used this approach for detection of MS2 bacteriophage coat proteins.3. Select and synthesize DNA and RNA aptamers that specifically recognize and bind to bacterial proteins and live bacteria. The enzyme-linked immunosorbent assay (ELISA) is a diagnostic tool that uses antibodies for detection of protein antigens. Suitable antibodies are not easily available, could be easily inactivated and are costly to produce. Therefore, in our assay, we will use aptamers instead of antibodies. Using SELEX approach, we will select and synthesize two types of aptamers against either bacterial proteins or live bacteria. DNA aptamers will be used to capture and attach bacterial protein (or live bacteria) to a solid medium (e.g. Sepharose) while RNA aptamers will be used to mediate visualization of bacterial proteins (or live bacteria).4. Use the new assays for naked eye detection of the target genes and proteins. Methods for extraction of genomic DNA and proteins from bacteria are well established. To facilitate detection of a selected target gene, genomic DNA will be broken into approximately 500-700 nucleotide long fragments, deposited on a nylon filter and chemically denatured. To detect bacterial proteins, protein extracts will be filtered through a disposable column pre-packed with Sepharose that is derivatized with DNA aptamers specifically binding target proteins. This step is essential for capturing bacterial proteins and removing molecules that otherwise could interfere with the detection of the target protein.5. Engage undergraduate and graduate students, and K-12 teachers from surrounding communities in interdisciplinary and collaborative research. We will recruit undergraduate and graduate students from our respective colleges to participate in all aspects of the proposed research projects. In addition, we will take advantage of existing programs at Auburn University to recruit K-12 science and math teachers and their students to participate in this research project during the summer term. Moreover, we will recruit outstanding students from underrepresented groups with the help of the NSF-EPSCoR funded Center for Environmental and Cellular Signal Transduction (CECST) that works closely with historically black colleges and universities.
Cost-efficient monitoring of food borne pathogens is essential for maintaining the security of our foods. Conventional assays for the detection of pathogens employ polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Recently developed approaches use DNA biosensors that convert the hybridization of two DNA strands into electrical or optical signals. We propose to develop novel assays for naked eye detection of bacterial pathogens. In our approach, biotinylated small DNA and RNA probes bind first to the target DNA and proteins, respectively. The formation of the probe:target complexes is detected by RNA molecules emitting green fluorescent light that is readily observable by the naked eye. Because the optical signal is very strong, amplification of target molecules is not required. To develop this novel nucleic acid-based technology, we specifically propose to (1) synthesize concatamers of green fluorescent RNA (GFR) aptamers for use in enhanced target detection, (2) construct supramolecular assemblies of streptavidin and GFR aptamer, (3) synthesize RNA and DNA aptamers that specifically recognize and bind to common foodborne bacterial pathogens including Salmonella enterica, Listeria monocytogenes and Escherichia coli 0157:H7, (4) use the new assays for naked eye detection of these bacterial pathogens. Preliminary experiments have already demonstrated that our technology is suitable for naked eye visualization of pathogenic bacteria and bacteriophage proteins. This inherently interdisciplinary project provides excellent opportunities for education and training in a fascinating cutting-edge technology.Our work brings to the forefront a better realization of the important but insufficiently utilized roles of RNA in nanotechnologies. Building RNA sensors allows for detection of infectious agents at the source for the immediate protection of humans, domestic animals and foods. Our nucleic-acid-based technology, when perfected and commercialized, is expected to have a significant social impact in food industries, agriculture and medicine. One can predict that if we are able to detect pathogens quickly and efficiently, costly recalls of food can be limited and many food-borne diseases avoided. Moreover, state-of-the-art training of undergraduate and graduate students in molecular biology and nanoengineering will be a strong motivator for their pursuit of careers in the socially responsible life sciences and biotechnology.