Selection of DNA Aptamers for Mycotoxins

Abstract

Mycotoxins deoxynivalenol (DON) and patulin (PAT) are toxic fungal metabolites that contaminate food commodities worldwide. They pose health risks for humans and animals, and have a negative impact on the economy. Mycotoxin detection is vital to ensure public safety.Although traditional detection techniques for mycotoxin detection (e.g. LC-MS) have proven to be highly selective and sensitive, simpler, more rapid, and more cost-effective approaches are required. Aptamer-based techniques are a possible solution. Aptamers are oligonucleotides that can bind to molecules with high affinity and specificity, generated via an in vitro process known as SELEX. We attempted to generate aptamers for DON and PAT. The selection process for DON was challenging and it would be beneficial to continue SELEX, whereas the process for PAT was simpler thus sequences for potential aptamers were obtained. This work has contributed to the production of aptamer-based assays and biosensors for mycotoxin detection.ii Acknowledgements I made it to the end of my Master's degree but not without a lot of hard work, determination, and most importantly, the continued support from a long list of people.First and foremost, I must acknowledge my supervisor Dr. Maria DeRosa. I know she had to exercise a lot of patience with me as I learned, and continue to learn, how to do research like a scientist. She brought out my strengths and helped me overcome my weaknesses. In addition to all that, she has provided me with learning and research opportunities that I will never forget.Her passion and enthusiasm are truly inspiring, and I am very grateful to have had the chance to work for her! My lab mates -Eman, Erin, Phepa, Nadine, and Emily (in order of desk arrangement). Spending every day with you girls made those hard days in the lab livable. Our lunchtime breaks rarely passed without a good laugh. When my SELEX would go wrong for no apparent reason or when I would forget to clamp my gels before pouring, you would also get angry alongside me or give me encouraging words so I wouldn't quit. Of course, I can't forget the men of the lab; I've always enjoyed a good laugh with Christopher, Selvan, and Dr.Velu. If I ever needed a hand, they always willingly offered their time to help. A special bond was formed and I'm grateful for meeting all of you.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion

“…First, since starting oligonucleotide libraries can typically encompass anywhere from 10 18 to 10 40 possible unique sequences (for libraries containing 30-70 random nucleotides), it is unrealistic to screen every possible sequence in a SELEX experiment, and this will inevitably exclude high-quality aptamer candidates. 6,7 Second, due to the low copy number of each sequence in the starting library, oligonucleotides with desirable binding properties can easily be lost during the early rounds of SELEX. 8,9 Finally, these aptamers can be eliminated if they have low PCR amplification efficiency due to their sequence and/or structure.…”

Domain-Based Fuzzing for Supervised Learning of Anomaly Detection in Cyber-Physical Systems

“…Python scripts are used to create the fuzzer. The fuzzer uses the Pycomm library developed by Ruscito [24] to communicate with the Allen-Bradley ControlLogix PLC of SWaT (specifically, in this experiment, the PLC1 which controls the raw water process). Pycomm is used to emulate realistic attacks against level sensor LIT 101, motorized valve MV 101, and the flow rate indicator F IT 101, by systematically modifying the sensor readings A few preliminary attacks were launched by changing the LIT 101 readings to a constant (randomly chosen) anomalous value (between LL to L or H to HH ) for 30 seconds, restoring it to the actual value, and giving the system time to recover before launching another attack.…”

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

“…The difficulty in selecting small molecules may be attributed to the large difference in size between the aptamer and the target, which can cause crowding during selections, thereby decreasing selection efficiency (Ruscito & DeRosa, 2016). Furthermore, binding of the aptamer to a small molecule target does not result in a substantial mass difference, which makes partitioning more challenging.…”

“…In fact, in the past two decades, very few changes have been made to the classical small molecule aptamer selection protocol. Reviews on aptamer selection strategies as applied to small molecules can be found in the works of Ruscito and McKeague (McKeague & DeRosa, 2012;Ruscito & DeRosa, 2016).…”

“…Both antibodies and aptamers have proven to be effective as molecular recognition probes, displaying selective and high-affinity target binding. However, aptamers, which are single-stranded, synthetic oligonucleotides, offer several advantages over antibodies (Ruscito & DeRosa, 2016). First, aptamers are selected via an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk & Gold, 1990).…”