Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
PLoS One. 2013 May 24;8(5):e64131. doi: 10.1371/journal.pone.0064131. Print 2013.
The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.
体外选择过程导致了许多具有开发成抑制剂和生物传感器潜力的适体的发现，但仍然存在一些问题，即难以分离出具有所需亲和力和特异性的某些靶标的适体。一种可能的改进方法是使用经过重复分离的适体分子中的基序增强文库。一个经常观察到的基序是双层鸟嘌呤四链体。在这项研究中，我们研究了是否可以设计 DNA 文库以包含大量能够折叠成双层鸟嘌呤四链体的分子。使用全面的圆二色性分析，我们发现可以设计 DNA 文库以包含大量采用鸟嘌呤四链体结构的序列。从小文库中分析单个序列发现了不同拓扑结构的四链体混合物，为体外选择提供了所需的多样性。我们还发现引物结合位点不利于四链体的形成，并设计了一种方法来对无引物四链体文库进行选择后扩增。随着富含鸟嘌呤四链体的 DNA 文库的发展，有可能提高分离利用四链体支架的适体的机会，并提高体外选择实验的成功率。
