An Optimized microRNA Backbone for Effective Single-Copy RNAi

被引：534

作者：

Fellmann, Christof ^{[1
]}

Hoffmann, Thomas ^{[2
]}

Sridhar, Vaishali ^{[1
]}

Hopfgartner, Barbara ^{[2
]}

Muhar, Matthias ^{[2
]}

Roth, Mareike ^{[2
]}

Lai, Dan Yu ^{[1
]}

Barbosa, Ines A. M. ^{[2
]}

Kwon, Jung Shick ^{[1
]}

Guan, Yuanzhe ^{[1
]}

Sinha, Nishi ^{[1
]}

Zuber, Johannes ^{[2
]}

机构：

[1] Mirimus Inc, Cold Spring Harbor, NY 11724 USA

[2] Res Inst Mol Pathol IMP, A-1030 Vienna, Austria

来源：

CELL REPORTS | 2013年 / 5卷 / 06期

关键词：

SHRNA LIBRARIES; MOLECULAR-BASIS; IN-VIVO; INTERFERENCE; RECOGNITION; EXPRESSION; VECTORS; MIRNAS; SYSTEM; CELLS;

D O I：

10.1016/j.celrep.2013.11.020

中图分类号：

Q2 [细胞生物学];

学科分类号：

071013 [干细胞生物学];

摘要：

Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs'' often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 30 of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E'', which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.

引用

页码：1704 / 1713

页数：10

共 38 条

[1]

Molecular basis for target RNA recognition and cleavage by human RISC [J].