Szeto Kylan, Latulippe David R, Ozer Abdullah, Pagano John M, White Brian S, Shalloway David, Lis John T, Craighead Harold G
School of Applied and Engineering Physics, Cornell University, Ithaca, New York, United States of America.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
PLoS One. 2013 Dec 20;8(12):e82667. doi: 10.1371/journal.pone.0082667. eCollection 2013.
Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.
适体是通过指数富集的配体系统进化技术（SELEX）从DNA或RNA文库中筛选出的高亲和力配体，这是一个包括连续选择和扩增步骤的重复性体外过程。由于额外的转录和逆转录步骤，RNA SELEX比DNA SELEX更复杂。在此，我们报告了一种新的筛选方案，即RAPID-SELEX（通过双循环SELEX分离RNA适体），该方案通过系统地跳过不必要的扩增步骤简化了这一过程。使用亲和微柱，我们能够在使用传统SELEX方法进行类似筛选所需时间的三分之一内完成对蛋白质靶点CHK2和UBLCP1的多重筛选。对RAPID和SELEX富集文库进行高通量测序，发现在5×10¹⁵个序列的起始文库中有许多相同的候选适体。对于CHK2，相同的序列在两种筛选中均作为顶级候选序列被优先富集，并被发现能与其各自的靶点结合。这些结果证明了我们筛选方案的效率，最重要的是其稳健性。RAPID提供了一种通用方法，可与任何筛选技术一起使用，以加快适体发现的速度，而不影响筛选性能。
