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.
Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America ; Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
Yongping
Y
Wang
Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
Jacqueline S
JS
Speier
Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Jessica
J
Engram-Pearl
Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Robert B
RB
Wilson
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D007377:Interleukin-3; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D000097573:Red Fluorescent Protein