Key Laboratory of Analytical Science, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
Anal Chem. 2012 Jan 3;84(1):350-5. doi: 10.1021/ac2026942. Epub 2011 Dec 9.
We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.
我们开发了一种新的方法，通过单分子乳液聚合酶链反应（PCR）基于琼脂糖液滴微流控技术，从复杂的单链 DNA（ssDNA）文库中高效筛选亲和配体（适体）。在典型的指数富集配体系统进化（SELEX）过程中，首先对富集文库进行测序，然后通过生物信息学方法分析数十到数百个适体候选物。然后通过化学合成可能的候选物，并单独测量它们的结合亲和力。这个过程既耗时又费力，效率低下，成本高昂。为了解决这些问题，我们开发了一种使用我们的琼脂糖液滴微流控技术的高效单分子适体筛选方法。通过常规 SELEX 针对癌症生物标志物 Shp2 蛋白进化的预富集文库的统计稀释 ssDNA 被包裹在单个均匀的琼脂糖液滴中进行液滴 PCR 以生成克隆琼脂糖珠。然后通过高通量荧光细胞仪筛选来自每个克隆珠的扩增 ssDNA 的结合能力。选择具有高结合能力和低 K(d)的扩增 ssDNA 克隆作为适体，可直接用于下游生物医学应用。我们已经鉴定出一种 ssDNA 适体，其选择性识别 Shp2 的 K(d)为 24.9 nM。与传统的测序-化学合成-筛选工作流程相比，我们的方法避免了大规模 DNA 测序和昂贵、耗时的大量 DNA 候选物的合成。因此，琼脂糖液滴微流控方法对于分子进化方法非常高效且具有成本效益，并将在分子进化技术中得到广泛应用，包括 mRNA 显示、噬菌体显示等。
