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Rapid Detection of Pathogens Using Novel Ribonucleic Acid-Based Technologies

Wower, Jack
Auburn University
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The goal of this project is to develop novel RNA-based technologies --complementary RNAs and programmable RNA aptamer networks -- that can be used to advance rapid, specific and sensitive detection of pathogens. These technologies could also be used as vehicles for the delivery of multiple drugs on-demand and over specific periods of time. We will synthesize complementary RNAs (cRNAs) for incorporation into RNA sensors and evaluate their effectiveness for the detection of bacteria. These sensors will target tmRNA, an RNA molecule that is present in all bacteria. Because tmRNA is absent in eukaryotic cells, the possibility that RNAs derived from humans, animals and plants interfere with the detection process is low.

The development of RNA-based technology into a simple inexpensive sensor for detection of bacterial tmRNAs will involve (i) identification of tmRNA segments suitable for cRNA synthesis, (ii) cRNA synthesis using IRA, (iii) optimization of the base pairings between cRNA and gold-attached DNAs, (iv) assembly of the cRNA/DNA module into an acoustic sensor suitable for wireless monitoring, (v) synthesis and characterization of self-assembling RNA aptamer networks for enhancing detection levels, and (vi) packaging of the RNA sensors into a single microfluidic chip for the automated purification of bacterial RNAs, synthesis of cRNA as well as its detection. This work brings to the forefront a better realization of the important but currently insufficiently understood role of RNAs in numerous biological processes. Building RNA sensors allows for detection of infectious RNAs at the source for the immediate protection of humans, domestic animals and crops. Our nucleic acid-based technology, when perfected and commercialized, is expected to have a significant impact in food agriculture, medical, national security and research industries.

Although in this proposal we target only one type of non-coding RNA molecule (tmRNA), our technology is applicable for detecting other RNA molecules. Because many non-coding RNA are essential for controlling vital cellular processes, we expect that our biosensing technology will speed up cancer research and provide tools for advancing our insights into the origins of many developmental defects. Inexpensive and user-friendly sensors that can detect bacteria or viruses in minutes (rather than in days) will revolutionize the way food production and distribution are monitored. One can predict that if we are able to detect bacterial contaminations in the food chain quickly and efficiently, costly recalls of food can be limited and many food-borne diseases avoided.

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Non-Technical Summary: We propose to develop novel types of flexible and programmable ribonucleic acid-based sensors in order to detect bacterial RNAs at the sub-micro scale. We expect that RNA-based sensors will be inexpensive, user-friendly and detect bacteria in minutes (rather than in days). By amplifying a complementary RNA (cRNA), a non-biological derivative of the target, contamination is avoided and high specificity and sensitivity are readily achieved. To boost the sensitivity of RNA detection, we will use RNA aptamer networks that can bind signaling dyes such as Malachite Green (MG) and a fluorescently labeled antibiotic Neomycin. RNA aptamer networks could also be used as programmable platforms for the on-demand delivery of drugs and therefore contribute to the advancement of personalized medicine.

Approach: We will use advanced biochemical methods for the in vitro synthesis and derivatization of complementary RNAs (cRNAs) and programmable RNA aptamer networks. We will apply bioinformatics tools to identify segments of Escherichia coli and Salmonella enterica tmRNAs that can serve as templates for the synthesis of cRNAs suitable for the specific identification and detection of each bacterium. These cRNAs will be synthesized using an isothermal amplification approach (IRA) in the presence of 2'-fluoropyrimidine triphosphates to produce cRNA derivatives that are RNase A-resistant. To enhance pathogen detection, we will construct RNA aptamer networks composed of multiple copies of two types of RNA aptamers. One type of aptamer forms a chain of loop-receptor (LR) inter-molecular interactions (or RNA network) to increase the stability and sensitivity of the enhancer. The other binds the signaling dyes such as Malachite Green (MG) and a fluorescently labeled antibiotic Neomycin. We will integrate RNA sensors into a robust microfluidic chip. These microfluidic chips will sequentially process nanoliter volumes of liquids to isolate bacterial cells, lyse them, isolate target RNA, produce multiple copies of its cRNA, and then use RNA aptamer networks associated with fluophores to detect the bacterial RNA. The chip will be fabricated by multilayer soft lithography that provides robust and accurate micromechanical valves.

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Nat'l. Inst. of Food and Agriculture
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