Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries
Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries
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DOI：
10.1093/protein/gzab017
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发表时间：
2021-02-15
影响因子：
2.4
通讯作者：
Whitehead, Timothy A.
中科院分区：
文献类型：
--
作者：
Kirby, Monica B.;Medina-Cucurella, Angelica, V;Whitehead, Timothy A.
产生特定突变集合的组合文库对于解决涉及分子进化中的偶然性、突变之间的上位关系以及功能性抗体和酶工程的蛋白质工程问题是必不可少的。在这里，我们提出了涉及基于模板的切口诱变的组合诱变方法的优化，其允许产生对于数万个用户定义的变体具有>99%覆盖率的文库。非优化方法导致低文库覆盖率，这可以通过由诱变寡核苷酸和模板之间的核苷酸错配自由能差异引起的寡核苷酸退火偏倚模型来合理化。优化的方法使用更长的引物组和更快的退火条件减轻了这种热力学偏差。我们的更新方法应用于两个抗体片段，在2天内以约140倍的测序覆盖深度递送了我们所需文库的99.0%（32451/32768个文库成员）至>99.9%覆盖率（32757/32768）。
Generating combinatorial libraries of specific sets of mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well as functional antibody and enzyme engineering. Here we present optimization of a combinatorial mutagenesis method involving template-based nicking mutagenesis, which allows for the generation of libraries with >99% coverage for tens of thousands of user-defined variants. The non-optimized method resulted in low library coverage, which could be rationalized by a model of oligonucleotide annealing bias resulting from the nucleotide mismatch free-energy difference between mutagenic oligo and template. The optimized method mitigated this thermodynamic bias using longer primer sets and faster annealing conditions. Our updated method, applied to two antibody fragments, delivered between 99.0% (32451/32768 library members) to >99.9% coverage (32757/32768) for our desired libraries in 2 days and at an approximate 140-fold sequencing depth of coverage.