Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries

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.体外筛选（in vitro selection）技术已助力发现诸多有望开发为抑制剂与生物传感器的适配体（aptamer），但针对特定靶点分离具备预期亲和力与特异性的适配体仍存在诸多难题。一种可行的优化思路是使用针对适配体分子中反复检出的基序（motif）所强化的文库。其中一类高频出现的基序为双层鸟嘌呤四链体（two-tiered guanine quadruplex）。本研究旨在探究能否通过设计，使DNA文库（DNA library）中包含大量可折叠为双层鸟嘌呤四链体的分子。借助全面的圆二色谱（circular dichroism）分析，我们发现可通过设计让DNA文库中占比极高的序列采取鸟嘌呤四链体结构。对小型文库中的单条序列进行分析后发现，不同拓扑结构的四链体混合存在，可提供体外筛选所需的多样性。我们还发现引物结合位点（primer-binding site）会对四链体的形成产生不利影响，并据此开发了一种无需引物的四链体文库筛选后扩增方法。随着鸟嘌呤四链体富集型DNA文库的开发，未来有望提升分离以四链体为骨架的适配体的成功率，进而优化体外筛选实验的整体成效。