Poster abstracts
Poster number 21 submitted by Jessica Bush
Synthesis and Characterization of Nucleic Acid Aptamers Targeted at Aspergillus Surface Carbohydrates
Jessica Bush (Department of Chemistry, Albion College), Megan Sheridan (Department of Chemistry, Albion College), David Engelke (Department of Chemistry, University of Michigan ), Christopher Rohlman (Department of Chemistry, Albion College)
Abstract:
Aspergillus is a common fungus found naturally throughout the world. Normally these spores are inhaled 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). The high specificity and affinity binding capabilities of aptamers make them a potent diagnostic and therapeutic tool. Aptamers can be selected to target and identify a wide variety of infectious diseases (McKeague and DeRose, 2012). By selecting for specific nucleic acid aptamers and fluorescently labeling these molecules, we aim to develop a new detection method for Aspergillosis that is more sensitive and less invasive. 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 target carbohydrates. 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. In parallel, F-modified RNA aptamers targeted at the Aspergillus cell surface carbohydrate beta-D-glucan were selected from an N40 randomized template pool. RNA aptamers were generated using in vitro transcription of the template pool utilizing F-NTPs. Successful aptamers were selected through nine successive rounds of binding to beta-D-glucan, reverse transcription, and cDNA template pool amplification. Binding characteristics as well as characterization of the aptamer pool sequence diversity are currently in progress.
References:
Challenges and Opportunities for Small Molecule Aptamer Development
Maureen McKeague and Maria C. DeRosa (2012) Journal of Nucleic Acids volume 2012 doi:10.1155/2012/748913
Detection of Aspergillus DNA in Cerebrospinal Fluid from Patients with Cerebral Aspergillosis by a Nested PCR Assay M. Hummel, B. Spiess, K. Kentouche, S. Niggemann, C. Bohm, S. Reuter, M. Kiehl, H. Morz, R. Hehlmann, and D. Buchheidt (2006) Journal of Clinical Microbiol 44(11): 3989–3993.
Keywords: Aptamer, Carbohydrate