Functionalized Aptamers for Lead (Pb (II)) Biosensing and Therapeutic Applications

Ramis de Ayreflor Reyes, Solimar

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Functionalized Aptamers for Lead (Pb (II)) Biosensing and Therapeutic Applications

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Lead is a heavy metal that is highly toxic, and damage done by lead to human cells and tissues is irreversible. The build-up of lead in the human body can affect the brain, kidneys, liver, blood, reproductive system, and nervous system. Current methods to detect lead in human blood are accurate and sensitive, however at home test kits to detect lead in the environment are not sensitive enough to detect lead at the cause of concern levels of 35 ppb, and sometimes require the consumer to wait a week for results. The primary treatment option for lead poisoning is chelation therapy, which can allow for the redistribution of lead from tissues into the brain. One new possibility for both lead detection and treatment of lead poisoning is the use of aptamers. Aptamers are short, single-stranded DNA or RNA sequences that bind with high specificity to target ions, molecules, or proteins. Single-stranded DNA aptamers that bind specifically to lead have already been identified and used in a fluorescence-based assay for lead detection. However, the performance of the system under real environmental conditions, including varying pH and temperature, has not been studied. Aptamers have undergone clinical evaluation for ocular diseases, cancer, and hematological diseases, but there are no studies of aptamers being used to treat acute lead poisoning. This project had two main goals: (1) to determine the capacities and limitations of using aptamers for detecting lead, and (2) to discover if the aptamers could be used as therapeutics for acute lead poisoning. To achieve the first goal, we performed various assays with known lead- binding aptamers to compare different fluorescent molecules and quencher pair aptamer complexes and determine the best performance conditions and limitations for a fluorescent aptamer-based lead detection system. Our results reveal that the fluorescent reporter system may not be ideal as a signal output due to interference from environmental factors including pH and calcium. To achieve our second goal, we developed an in vivo lead toxicity assay using the model organism C. elegans and measured the effect of aptamer exposure on lead related phenotypes. We hypothesized that these aptamers in extracellular fluid or in the blood stream would bind free lead ions, thereby protecting the organism from lead toxicity. We compared brood size, length, and movement phenotypes of the C. elegans to determine the impact of lead exposure. We then exposed the C. elegans to various aptamers to determine if treatment with aptamer recues the affected phenotype. Our results show a dramatic, full protection of aptamer- treated animals from the lead-related phenotypes we examined. The results suggest that aptamer- based therapies have enormous potential for the treatment of heavy metal toxicity and therefore warrant further investigation.

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