Guide oligonucleotide-dependent DNA linkage that facilitates controllable polymerization of microgene blocks.
Faster and more efficient searches of a huge protein sequence space for the purpose of conducting experiments in protein evolution can be achieved through the development of a block shuffling-based evolution system. One of the key components of such a system is the accurate and efficient linkage of gene units. Here we introduce a new method that allows accurate and controllable linkage of microgene blocks. This method employs a thermostable DNA ligase that links two single-stranded microgene blocks when they hybridize a complementary guide oligonucleotide. At high temperature, the ligation of the microgene units is fully dependent on the guide oligonucleotide, which can exclude undesired polymer formation, including the incorporation of microgenes having illegitimate sizes and "head-to-head" and "tail-to-tail" ligation of blocks. We were also able to assemble three microgene units using two guide oligonucleotides. Using this method of controllable linkage should facilitate further development of a step-by-step system for the polymerization of gene blocks, leading to a versatile block shuffling-based protein evolution system.
Shiba
Kiyotaka
K
Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Toshima, Tokyo 170-8455. kshiba@jfcr.or.jp
Hatada
Tomoko
T
Takahashi
Yuki
Y
Noda
Tetsuo
T
D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D058727:RNA, Small Untranslated
D011994:Recombinant Proteins; D004247:DNA; D011088:DNA Ligases; D058727:RNA, Small Untranslated