Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts, 02138, United States.
J Am Chem Soc. 2013 Jan 9;135(1):98-101. doi: 10.1021/ja311331m. Epub 2012 Dec 26.
We developed a method to translate DNA sequences into densely functionalized nucleic acids by using T4 DNA ligase to mediate the DNA-templated polymerization of 5'-phosphorylated trinucleotides containing a wide variety of appended functional groups. This polymerization proceeds sequence specifically along a DNA template and can generate polymers of at least 50 building blocks (150 nucleotides) in length with remarkable efficiency. The resulting single-stranded highly modified nucleic acid is a suitable template for primer extension using deep vent (exo-) DNA polymerase, thereby enabling the regeneration of template DNA. We integrated these capabilities to perform iterated cycles of in vitro translation, selection, and template regeneration on libraries of modified nucleic acid polymers.
我们开发了一种方法,通过使用 T4 DNA 连接酶介导包含各种附加官能团的 5'-磷酸化三核苷酸在 DNA 模板上的聚合,将 DNA 序列转化为高度功能化的核酸。这种聚合沿着 DNA 模板特异性进行,可以以很高的效率生成至少 50 个构建块(150 个核苷酸)长的聚合物。所得的单链高度修饰的核酸是使用深滚(外切)DNA 聚合酶进行引物延伸的合适模板,从而能够再生模板 DNA。我们整合了这些功能,在修饰的核酸聚合物文库上进行体外翻译、选择和模板再生的迭代循环。