Shortening Pairing of the 3′ Tail of shRNA with Target mRNA Reduces the Off-Target Effect
-
摘要: 基于对microRNA和短发夹RNA（shRNA）的3′尾功能的理解，提出了一种仅通过缩短shRNA的3′尾与靶标序列的互补长度来降低脱靶效应的方法。此方法可以在不损伤shRNA基因沉默效率的前提下达到降低shRNA脱靶效应的目的，从而有效提高shRNA的基因沉默特异性。此策略不受反义链3′区域序列的限制，可以显著改进RNA干扰设计的规则，一定程度上简化shRNA药物设计中的序列限制，拓宽其作为治疗和诊断工具在医疗中的用途和前景。Abstract: The use of RNA interference (RNAi) is becoming a routine method of scientific discovery and treatment of human diseases. However, its application is hindered by adverse effects, especially the off-target effects. In this work, we established an shRNA test platform based on the plasmid to test the silencing ability of short hairpin RNA (shRNA) targeting survivin gene designed according to shRNA online software and then screened out two shRNAs with excellent silencing ability. The off-target effects were measured by testing the silencing ability of two selected shRNAs against targets with single-nucleotide mutations at different locations. Based on our understanding of the 3' tail function of microRNA (miRNA) and small interfering RNA (siRNA), we propose a method to reduce the off-target effect of short hairpin RNA by shortening the complementary length of the 3' tail and target sequence of siRNA only. This method can reduce the off-target effect of shRNA without damaging the silencing efficiency of shRNA gene, so as to effectively improve the specificity of shRNA gene silencing. Compared to previous studies of the weak base pairing of “seed” and 3' regions to reduce the off-target effect, our method is simpler and more efficient, without being limited by the sequence of 3' regions of the antisense strand. This method of reducing the off-target effects provides new ideas for the design of shRNA and siRNA, simplifies the sequence restriction in the design of shRNA and siRNA drugs to a certain extent, and broadens their use and prospects as therapeutic and diagnostic tools in medical treatment.
-
Key words:
- short hairpin RNA /
- RNA interference /
- gene silencing /
- 3' tail /
- off-target effect
-
图 1 shRNA 在 HEK293T 细胞中的靶标沉默能力：（a）含有 shRNA 靶标的 EGFP mRNA 的荧光图像（其中Control为元靶标shRNA）；（b）shRNA 对照组对 EGFP 表达水平的影响；（c）shS1～shS6 对靶标的沉默能力（误差条表示3次生物重复数 据的标准差（s.d.））
Figure 1. Silencing ability of shRNAs in HEK293T cells: (a) Fluorescent images of the EGFP expression levels treated with shRNAs or non-targeting shRNA plasmid. EGFP/mCherry ratio of the EGFP mRNA treated with (b) non-targeting shRNA control or (c) corresponding shRNA plasmid (The error bars indicate the standard deviation (s.d.) of the data from three biological replicates)
图 2 shS3（左）和 shS5（右）的脱靶效应：（a，b）不同位置单核苷酸突变的靶序列（红色标记）；（c，d）为 shRNA 对照样品处理的对应单核苷酸突变靶序列的 EGFP 表达水平；（e，f）对应 shRNA 处理的含单碱基突变靶序列 EGFP 表达水平( C为不含突变靶标)
Figure 2. The off-target effect of shS3 (Left panel) and (Right panel): (a, b) Target sequences with single nucleotide mutation at different locations (marked in red); (c, d) Expression level of EGFP mRNAs with corresponding target sequence with single nucleotide mutation, treated with the shRNA control sample; (e, f) shRNAs silencing ability of the EGFP mRNAs with corresponding target sequence with single nucleotide mutation(C means target without mismatch)
图 3 从 3'端缩短 shRNA 的反义链对其靶沉默能力的影响：（a，b）shRNA 的序列（与靶标匹配的反义序列用蓝色标记，不匹配的核苷酸用红色标记）；（c，d）HEK293T 细胞中 shRNA 沉默的结果（ C1 为对照 shRNA 处理样品；误差条表示3次生物重复数据的标准差（s.d.））
Figure 3. Influence of shorted antisense strand of shRNA in silencing ability: (a, b) The sequence of shRNAs (The antisense sequence matching with targets is blue, and the mismatched nucleotides are marked in red); (c, d) shRNAs silencing results in HEK293T cells (C1 means non-targeting shRNA-treated control. The error bars indicate the standard deviation (s.d.) of the data from three biological replicates)
图 4 shS3系列shRNA对含不同位置单碱基突变的靶标的沉默能力（(C为不含突变靶标））
Figure 4. Silencing ability of shS3-derived shRNAs to targets with single nucleotide mismatch at different locations (C means target without mismatch)
图 5 shS5系列shRNA对含不同位置单碱基突变的靶标的沉默能力(C为不含突变靶标）
Figure 5. Silencing ability of shS5-derived shRNAs to targets with single nucleotide mismatch at different locations (C means target without mismatch)
表 1 靶向生存素mRNA的shRNA
Table 1. shRNAs targeting survivin mRNA
Name Target sequence (DNA) GC/% Antisense sequence (DNA) shS1 GCATCTCTACATTCAAGAACT 38.10 AGTTCTTGAATGTAGAGATGC shS2 GCCCAGTGTTTCTTCTGCTTC 52.39 GAAGCAGAAGAAACACTGGGC shS3 GCGCTTTCCTTTCTGTCAAGA 47.62 TCTTGACAGAAAGGAAAGCGC shS4 GGACAGAGAAAGAGCCAAGAA 47.62 TTCTTGGCTCTTTCTCTGTCC shS5 GCAAAGGAAACCAACAATAAG 38.10 CTTATTGTTGGTTTCCTTTGC shS6 GAAGAAAGAATTTGAGGAAAC 33.34 GTTTCCTCAAATTCTTTCTTC 
下载: 导出CSV
表 2 用于shRNA载体构建的DNA序列
Table 2. DNA sequence for shRNA vector construction
Strand Sequence (5′ to 3′) shS1-T caccGCATCTCTACATTCAAGAACTTTCAAGAGAAGTTCTTGAATGTAGAGATGC shS1-B AAAAGCATCTCTACATTCAAGAACTTCTCTTGAAAGTTCTTGAATGTAGAGATGC shS2-T caccGCCCAGTGTTTCTTCTGCTTCTTCAAGAGAGAAGCAGAAGAAACACTGGGC shS2-B AAAAGCCCAGTGTTTCTTCTGCTTCTCTCTTGAAGAAGCAGAAGAAACACTGGGC shS3-T caccGCGCTTTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAAAGCGC shS3-B AAAAGCGCTTTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAAAGCGC shS4-T caccGGACAGAGAAAGAGCCAAGAATTCAAGAGATTCTTGGCTCTTTCTCTGTCC shS4-B AAAAGGACAGAGAAAGAGCCAAGAATCTCTTGAATTCTTGGCTCTTTCTCTGTCC shS5-T caccGCAAAGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCTTTGC shS5-B AAAAGCAAAGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCTTTGC shS6-T caccGAAGAAAGAATTTGAGGAAACTTCAAGAGAGTTTCCTCAAATTCTTTCTTC shS6-B AAAAGAAGAAAGAATTTGAGGAAACTCTCTTGAAGTTTCCTCAAATTCTTTCTTC shS3-20-T caccGCGCTTTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAAAGCG shS3-20-B AAAACGCTTTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAAAGCGC shS3-19-T caccGGGCTTTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAAAGCC shS3-19-B AAAAGGCTTTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAAAGCCC shS3-18-T caccGGCCTTTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAAAGGC shS3-18-B AAAAGCCTTTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAAAGGCC shS3-17-T caccGGCGTTTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAAACGC shS3-17-B AAAAGCGTTTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAAACGCC shS3-16-T caccGGCGATTCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGAATCGC shS3-16-B AAAAGCGATTCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGAATCGCC shS3-15-T caccGGCGAATCCTTTCTGTCAAGATTCAAGAGATCTTGACAGAAAGGATTCGC shS3-15-B AAAAGCGAATCCTTTCTGTCAAGATCTCTTGAATCTTGACAGAAAGGATTCGCC shS5-20-T caccGCAAAGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCTTTG shS5-20-B AAAACAAAGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCTTTGC shS5-19-T caccGGAAAGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCTTTC shS5-19-B AAAAGAAAGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCTTTCC shS5-18-T caccGGTAAGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCTTAC shS5-18-B AAAAGTAAGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCTTACC shS5-17-T caccGGTTAGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCTAAC shS5-17-B AAAAGTTAGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCTAACC shS5-16-T caccGGTTTGGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCCAAAC shS5-16-B AAAAGTTTGGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCCAAACC shS5-15-T caccGGTTTCGAAACCAACAATAAGTTCAAGAGACTTATTGTTGGTTTCGAAAC shS5-15-B AAAAGTTTCGAAACCAACAATAAGTCTCTTGAACTTATTGTTGGTTTCGAAACC
下载: 导出CSV
表 3 用于EGFP表达载体构建的DNA序列
Table 3. DNA sequence for construction of EGTP expression vector
Strand Sequence (5′ to 3′) E-shS1-T ctagGCATCTCTACATTCAAGAACT E-shS1-B gatcAGTTCTTGAATGTAGAGATGC E-shS2-T ctagGCCCAGTGTTTCTTCTGCTTC E-shS2-B gatcGAAGCAGAAGAAACACTGGGC E-shS3-T ctagGCGCTTTCCTTTCTGTCAAGA E-shS3-B gatcTCTTGACAGAAAGGAAAGCGC E-shS4-T ctagGGACAGAGAAAGAGCCAAGAA E-shS4-B gatcTTCTTGGCTCTTTCTCTGTCC E-shS5-T ctagGCAAAGGAAACCAACAATAAG E-shS5-B gatcCTTATTGTTGGTTTCCTTTGC E-shS6-T ctagGAAGAAAGAATTTGAGGAAAC E-shS6-B gatcGTTTCCTCAAATTCTTTCTTC E-shS3-M5-T ctagGCGCTTTCCTTTCTGTGAAGA E-shS3-M5-B gatcTCTTCACAGAAAGGAAAGCGC E-shS3-M10-T ctagGCGCTTTCCTTACTGTCAAGA E-shS3-M10-B gatcTCTTGACAGTAAGGAAAGCGC E-shS3-M13-T ctagGCGCTTTCCATTCTGTCAAGA E-shS3-M13-B gatcTCTTGACAGAATGGAAAGCGC E-shS3-M15-T ctagGCGCTTTGCTTTCTGTCAAGA E-shS3-M15-B gatcTCTTGACAGAAAGCAAAGCGC E-shS3-M17-T ctagGCGCTATCCTTTCTGTCAAGA E-shS3-M17-B gatcTCTTGACAGAAAGGATAGCGC E-shS3-M18-T ctagGCGGTTTCCTTTCTGTCAAGA E-shS3-M18-B gatcTCTTGACAGAAAGGAAACCGC E-shS5-M5-T ctagGCAAAGGAAACCAACATTAAG E-shS5-M5-B gatcCTTAATGTTGGTTTCCTTTGC E-shS5-M10-T ctagGCAAAGGAAACGAACAATAAG E-shS5-M10-B gatcCTTATTGTTCGTTTCCTTTGC E-shS5-M13-T ctagGCAAAGGAATCCAACAATAAG E-shS5-M13-B gatcCTTATTGTTGGATTCCTTTGC E-shS5-M15-T ctagGCAAAGGTAACCAACAATAAG E-shS5-M15-B gatcCTTATTGTTGGTTACCTTTGC E-shS5-M17-T ctagGCAAACGAAACCAACAATAAG E-shS5-M17-B gatcCTTATTGTTGGTTTCGTTTGC E-shS5-M18-T ctagGCATAGGAAACCAACAATAAG E-shS5-M18-B gatcCTTATTGTTGGTTTCCTATGC
下载: 导出CSV
-
[1] FIRE A, XU S, MONTGOMERY M K, KOSTAS S A, DRIVER S E, MELLO C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature,1998,391(6669):806-811. doi: 10.1038/35888 [2] KIM V N, HAN J, SIOMI M C. Biogenesis of small RNAs in animals [J]. Nat Rev Mol Cell Biol,2009,10(2):126-139. [3] MEISTER G, TUSCHL T. Mechanisms of gene silencing by double-stranded RNA [J]. Nature,2004,431(7006):343-349. doi: 10.1038/nature02873 [4] XIE X, LU J, KULBOKAS E J, GOLUB T R, MOOTHA V, LINDBLAD-TOH K, LANDER E S, KELLIS M. Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals [J]. Nature,2005,434(7031):338-345. doi: 10.1038/nature03441 [5] JACKSON A L, BARTZ S R, SCHELTER J, KOBAYASHI S V, BURCHARD J, MAO M, LI B, CAVET G, LINSLEY P S. Expression profiling reveals off-target gene regulation by RNAi [J]. Nat Biotechnol,2003,21(6):635-637. doi: 10.1038/nbt831 [6] JACKSON A L, BURCHARD J, SCHELTER J, CHAU B N, CLEARY M, LIM L, LINSLEY P S. Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity [J]. RNA,2006,12(7):1179-1187. doi: 10.1261/rna.25706 [7] CHEN J, PENG Y, ZHANG H, WANG K, ZHAO C, ZHU G, REDDY PALLI S, HAN Z. Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA [J]. RNA Biology,2021,18(11):1747-1759. doi: 10.1080/15476286.2020.1868680 [8] BARTOSZEWSKI R, SIKORSKI A F. Editorial focus: Understanding off-target effects as the key to successful RNAi therapy [J]. Cellular and Molecular Biology Letters,2019,24:69. [9] MENG Z, LU M. RNA interference-induced innate immunity, off-target effect, or immune adjuvant? [J]. Frontiers in Immunology,2017,8:331. [10] JACKSON A L, BURCHARD J, LEAKE D, REYNOLDS A, SCHELTER J, GUO J, JOHNSON J M, LIM L, KARPILOW J, NICHOLS K, MARSHALL W, KHVOROVA A, LINSLEY P S. Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing [J]. RNA,2006,12(7):1197-1205. doi: 10.1261/rna.30706 [11] CAFFREY D R, ZHAO J, SONG Z, SCHAFFER M E, HANEY S A, SUBRAMANIAN R R, SEYMOUR A B, HUGHES J D. siRNA off-target effects can be reduced at concentrations that match their individual potency [J]. PLOS One,2011,6(7):e21503. doi: 10.1371/journal.pone.0021503 [12] PETRI S, MEISTER G. siRNA Design Principles and Off-Target Effects [M] Totowa, NJ: Humana Press, 2013: 59-71. [13] GIBSON D G, YOUNG L, CHUANG R Y, VENTER J C, HUTCHISON C A, SMITH H O. Enzymatic assembly of DNA molecules up to several hundred kilobases [J]. Nat Methods,2009,6(5):343-345. doi: 10.1038/nmeth.1318 -
点击查看大图
计量
- 文章访问数: 106
- HTML全文浏览量: 21
- PDF下载量: 20
- 被引次数: 0
