Capillary Electrophoresis鈥揝ystematic Evolution of Ligands by Exponential Enrichment Selection of Base- and Sugar-Modified DNA Aptamers: Target Binding Dominated by 2鈥?O,4鈥?C-Methylene-Br

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• 作者：Yuuya Kasahara ; Yuuta Irisawa ; Hiroto Fujita ; Aiko Yahara ; Hiroaki Ozaki ; Satoshi Obika ; Masayasu Kuwahara
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：May 21, 2013
• 年：2013
• 卷：85
• 期：10
• 页码：4961-4967
• 全文大小：528K
• 年卷期：v.85,no.10(May 21, 2013)
• ISSN：1520-6882

文摘

Chemically modified DNA aptamers specific to human 伪-thrombin were obtained from oligodeoxyribonucleotide (ODN) libraries by using a capillary electrophoresis鈥搒ystematic evolution of ligands by exponential enrichment (CE鈥揝ELEX) method. These libraries contained 2鈥?O,4鈥?C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the primer region and/or C5-modified thymidine bearing N⁶-ethyladenine (t) in the nonprimer region. Modified DNA aptamers showed high binding affinities to the target, with dissociation constants (K_d) values in the range of subnanomolar to several ten nanomolar levels. The introduction of base modification significantly suppressed the frequency of G-quadruplex motifs, which are often seen in thrombin-binding DNA aptamers. The resulting alternatives contained the 10-mer consensus sequence t₅Gt₂G₂, which is frequently found in modified DNA aptamers with subnanomolar protein binding affinities. Furthermore, some base- and sugar-modified DNA aptamers with the 12-mer consensus sequence t₂G₂tC(A/G)A₂G₂t displayed binding activities that were dependent on the presence of B/L nucleotides in the primer region. Such aptamers were interestingly not recovered from a natural DNA library or from DNA libraries modified with either B/L nucleotides or t鈥檚. This emerging characteristic binding property will enable the creation of a direct selection methodology for DNA-based molecular switches that are triggered by chemical conversion of B/L nucleotides introduced to constant sequence regions in ODN libraries.