Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing
Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing
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DOI：
10.1073/pnas.1009331107
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发表时间：
2010-08-31
影响因子：
11.1
通讯作者：
Soh, H. Tom
中科院分区：
文献类型：
--
作者：
Cho, Minseon;Xiao, Yi;Soh, H. Tom
我们描述了微流控选择与高通量DNA测序技术的集成，用于快速有效地发现核酸适体。通过测序方法定量选择适体通过多轮选择跟踪超过1000万个单个序列的拷贝数和富集倍数，从而能够识别高亲和力适体，而无需将池完全收敛到少量序列。重要的是，该方法允许区分由实验偏差而不是真正的高亲和力靶结合引起的序列。作为证明，我们已经鉴定了在3轮内以K（d）< 3 nM特异性结合PDGF-BB蛋白的适体。此外，我们表明，通过测序定量选择适体鉴定的适体与通过常规克隆方法发现的适体相比，亲和力高出3-8倍，特异性高出2-4倍。鉴于许多生物组合文库是用核酸编码的，我们推断我们的方法可以扩展到其他类型的文库，用于一系列分子功能。
We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have similar to 3-8-fold higher affinity and similar to 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.