Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing

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• 作者：Stefania Valenzano ; Annalisa De Girolamo ; Maria C. DeRosa ; Maureen McKeague ; Roberto Schena ; Lucia Catucci ; Michelangelo Pascale
• 刊名：ACS Combinatorial Science
• 出版年：2016
• 出版时间：June 13, 2016
• 年：2016
• 卷：18
• 期：6
• 页码：302-313
• 全文大小：599K
• 年卷期：0
• ISSN：2156-8944

文摘

Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (K_d) values in the range of 0.2–152 μM with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages.