Synthesis and Characterization of Nucleic Acid Aptamers Targeted at Aspergillus Surface Carbohydrates

Aspergillus is a common fungus that is found naturally throughout the world. These spores are inhaled on a daily basis and processed within the body without any negative consequences, however prolonged exposure to high quantities of Aspergillus can cause allergic or toxic symptoms, and infection in immuno‐suppressed individuals (Hummel et al., 2006). Traditional detection methods for Aspergillus are difficult and invasive including biopsies of cerebral lesions and extraction of cerebrospinal fluid, and frequently yield negative results (Hummel et al., 2006). These detection methods are often not feasible for immunocompromised patients. Aptamers present a new potential detection method for Aspergillosis. Aptamers are single stranded DNA or RNA sequences, which bind to a target molecule with high affinity and specificity (Navani et al., 2009). These sequences can be selected bind to ligands associated with Aspergillosis. Due to the high specificity and affinity binding capabilities of aptamers, they have become an emerging diagnostic and therapeutic tool. With research applications including artificial gene synthesis, DNA sequencing, library construction, molecular probes, and polymerase chain reaction, aptamers can be selected to target and identify a wide variety of infectious diseases (McKeague and DeRose, 2012). Such application allows us to target and identify a wide variety of infectious diseases including fungal infections caused by the mold, Aspergillus. By selecting for specific nucleic acid aptamers and fluorescently labeling these molecules, we can develop a new detection method for Aspergillosis that is more sensitive and less invasive. Once selected, fluorescent tagging can be used to observe their interaction with the target molecule. As a means for developing and evaluating the selection process several carbohydrate targets were studied. DNA aptamers were synthesized by binding a randomized N15 DNA sequence to cassettes to a target carbohydrate found on the Aspergillus surface. Once bound washing processes eliminated nonbinding DNA segments followed by an elution process and asymmetric PCR to produce a single stranded pool of aptamer candidates for the next round of selection. Mobility shift assays have also been used to further refine the aptamer pool. The potential of mobility shift assays opens the possibility to expand our aptamer research to investigate a new variety of ligands for future study as well as optimize the selection process. In parallel, RNA aptamers targeted at the Aspergillus cell surface carbohydrate beta‐D‐glucan were selected from an N40 randomized template pool. PCR amplification was then used to generate the initial DNA template pool. RNA aptamers were generated via in vitro transcription of the template pool utilizing modified NTPs. Successful aptamers were selected through nine successive rounds of binding to beta‐D‐glucan, reverse transcription, and cDNA template pool amplification. This work will describe binding characteristics as well as the RNA aptamer pool sequence diversity.