Introduction of Mismatches in a Random shRNA-Encoding Library Improves Potency for Phenotypic Selection

RNA interference (RNAi) is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA) encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3) dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.

RNA干扰（RNA interference, RNAi）是一类通过小型非编码RNA介导、干扰基因表达的分子机制。我们此前构建了一套核苷酸水平完全随机化的短发夹RNA（short-hairpin-loop RNA，shRNA）编码文库[1]。该文库中，发夹结构的茎区序列完全互补。为提升初始命中序列的效能，优化文库筛选过程中的信噪比，并简化通过聚合酶链式反应（PCR）进行的命中序列检索流程，我们在随机模板背景下，于每个发夹茎区的两个半段之间引入随机错配，由此构建了第二代文库。在一项针对可使白介素3（interleukin-3, IL3）依赖型细胞系抵抗IL3撤除的shRNA的筛选实验中，第二代文库获得的命中序列效能显著优于同筛选体系内第一代文库的命中序列。我们的随机诱变方法在随机模板中展现出良好的有效性，因此理论上可适用于任意目标DNA模板。第二代文库的效能提升，拓展了小型RNA疗法及生物工具相关的无偏筛选的应用范畴。