Functionalized Aptamers for Protection Against Cadmium Toxicity

O'Connor, Abigail

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Functionalized Aptamers for Protection Against Cadmium Toxicity

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Cadmium is a toxic heavy metal that is commonly ingested with contaminated food and water and inhaled in cigarette smoke. Cadmium affects several organs in the body, including the heart, pancreas, liver and kidneys, causing diseases such as cardiovascular disease, kidney disease and osteoporosis. There are currently no specific treatments for cadmium poisoning, but an exciting new therapeutic possibility has emerged, aptamers. Aptamers are short, 20 to 100 nucleotides long, oligonucleotides of single-stranded DNA or RNA that bind to a wide variety of specific targets with high selectivity and specificity through three-dimensional shape-dependent interactions. A fluorescent cadmium aptamer has been generated that detects cadmium in a dose-dependent manner, showing promise for the accurate detection of cadmium in our food, water and environment. However, no therapeutic applications of cadmium aptamers for cadmium toxicity have been tested. This project has two main goals: 1. Assess the efficacy of cadmium aptamers to protect against cadmium toxicity in vivo and 2. Assess the efficacy of cadmium aptamers to protect against cadmium toxicity in vitro. To accomplish the first goal, several assays in C. elegans were performed to examine the effects of cadmium aptamers on cadmium toxicity in vivo. We completed multiple approaches of brood size assays, showing no rescue by Cd-2-2 and Cd-4 aptamers of cadmium-induced brood size reduction. However, the Cd-4 aptamer may have shown signs of protection of the egg laying ability of adult C. elegans, but this needs further investigation. We also saw no effect of the Cd-4 aptamer on the behavior of C. elegans in response to a copper repellent post cadmium exposure. The behavior assay did produce a hyper stimulatory phenotype in C. elegans in response to copper post cadmium exposure, which was recorded for the first time. To accomplish the second goal, I performed assays in WT U2OS cells to examine the effects of cadmium aptamers on cadmium toxicity in vitro. First, the binding ability of cadmium aptamers to Cd2+ ions was examined using circular dichroism spectroscopy (CD spec). Although consistent with previous research, the CD spec results were inconclusive regarding the binding of the aptamer to Cd2, as the scramble sequence control produced almost identical results. An assay was then performed to test any protective effect of the Cd-4 aptamer on cadmium-induced stress granule formation in WT U2OS cells, but no protective effect was observed. A neutral red uptake assay was also conducted to assess any alleviating effects of the Cd-4 aptamer on reduced cell viability of WT U2OS cells in the presence of cadmium. The results showed no effect of the Cd-4 aptamer on cell viability. Taken together, these results call for further investigation of cadmium aptamers, specifically their binding ability and interactions with Cd2+ ions, any specific cadmium toxicity mechanisms hindering potential protective effects of the aptamers and the possible protective effect of the Cd-4 aptamer on egg laying ability in adult C. elegans exposed to Cd.

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