A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution
A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution
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DOI：
10.1021/ja808558a
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发表时间：
2009-01-28
影响因子：
15
通讯作者：
Hansen, Nils Jakob Vest
中科院分区：
文献类型：
--
作者：
Hansen, Margit Haahr;Blakskjaer, Peter;Hansen, Nils Jakob Vest
DNA三向连接的中心，构成约10（~ 24）L体积，被用作有效的反应器来创建化学产物的DNA编码文库。氨基酸和短肽通过可裂解和不可裂解的接头与寡核苷酸连接。寡核苷酸序列在中心含有两个通用组装结构域和对所连接的结构单元特异性的远端密码子序列。这些缀合物以组合方式的逐步自组装和化学反应在单个反应容器中在DNA三通接头中产生五肽文库。我们证明了一个均匀分布的100肽库的形成。每个文库成员含有一个短的合成肽，该合成肽连接到一个独特的遗传密码上，产生了体外分子进化过程所必需的“基因型-表型”连接。[Leu]-脑啡肽肽的选择性富集从模型库中的1/10百万的原始频率到仅在两轮亲和选择中的1.7%的最终频率，并证明了非天然系统的成功分子进化。
The center of DNA three-way junctions, constituting a yoctoliter (10(-24) L) volume, is applied as an efficient reactor to create DNA-encoded libraries of chemical products. Amino acids and short peptides are linked to oligonucleotides via cleavable and noncleavable linkers. The oligonucleotide sequences contain two universal assembling domains at the center and a distal codon sequence specific for the attached building block. Stepwise self-assembly and chemical reactions of these conjugates in a combinatorial fashion create a library of pentapeptides in DNA three-way junctions in a single reaction vessel. We demonstrate the formation of an evenly distributed library of 100 peptides. Each library member contains a short synthetic peptide attached to a unique genetic code creating the necessary "genotype-phenotype" linkage essential to the process of in vitro molecular evolution. Selective enrichment of the [Leu]-enkephalin peptide from an original frequency of 1 in 10 million in a model library to a final frequency of 1.7% in only two rounds of affinity selection is described and demonstrates successful molecular evolution for a non-natural system.