Yoshiaki Kobayash
读完需要
速读仅需 11 分钟
👀
RNA interference mediated by small interfering RNA (siRNA) has been widely used as a procedure to knock down the expression of an intended target gene with perfect sequence complementarity. However, siRNA often exhibits off-target effects on genes with partial sequence complementarities. Such off-target effect is an undesirable adverse effect for knocking down a target gene specifically. Here we describe the powerful strategy to avoid off-target effects without affecting the RNAi activity by the introduction of DNA or 2′-O-methyl modifications in the siRNA seed region. These two types of chemical modifications repress off-target effects through different molecular mechanisms.
RNA 干扰 (RNAi) 介导的小干扰 RNA (siRNA) 已被广泛用于特异性敲减目标基因表达。然而，siRNA 经常对具有部分序列互补性的非靶基因产生脱靶效应，这会影响实验结果的准确性。本文介绍了通过在 siRNA 种子区引入 DNA 或 2'-O-甲基修饰来抑制脱靶效应的策略。这两种修饰通过不同的分子机制提高 siRNA 的特异性。
👀
RNA interference (RNAi) is induced by small interfering RNA (siRNA), a duplex composed of the guide and passenger strands of 21-nucleotide (nt)-long RNAs with 2-nt 3′ overhangs. The siRNA is loaded onto Argonaute (AGO) protein , which is a core component of the RNA-induced silencing complex (RISC) . Subsequently, one of the two strands (passenger strand) is ejected and the other strand (guide strand) remains loading on AGO. Then, the guide strand base pairs with its target mRNA with perfect sequence complementarity, leading to repression of gene expression through the cleavage by AGO (Fig. 1). Synthetic siRNAs have become a powerful tool not only for studying gene function but also for applying therapeutics. Because the first siRNA drug was approved by FDA and EMA in 2018, the developmental research for therapeutic applications expected to become popular.
RNA 干扰 (RNAi) 由小干扰 RNA (siRNA) 介导，siRNA 是一种双链 RNA 分子，由两条 21 个核苷酸长度组成，其中包含 2 个核苷酸的 3'突出端。siRNA 会被加载到 Argonaut (AGO) 蛋白上，AGO 蛋白是 RNA 诱导沉默复合物 (RISC) 的核心组成部分。加载后，siRNA 的两条链中会有一条 (过客链) 被去除，剩下的一条链 (引导链) 继续结合在 AGO 蛋白上。然后，引导链会与靶 mRNA 序列完全匹配并结合，通过 AGO 蛋白的切割作用抑制靶基因的表达 (见图 1)。合成的 siRNA 不仅是研究基因功能的利器，还被用于治疗领域。由于首个 siRNA 药物在 2018 年获得 FDA 和 EMA 的批准，siRNA 在治疗领域的开发研究正日益受到关注。
Fig. 1 Schematic presentation of the mechanisms of siRNA-mediated RNAi and sequence-dependent off-target effects. SiRNA, a duplex composed of the guide and passenger strands, is loaded onto AGO protein. Passenger strand is ejected and guide strand remains loading on AGO. The mRNA with exact complementary sequence with siRNA guide strand is repressed by RNAi through its cleavage by AGO protein. On the other hand, the sequence-dependent off-target effect is induced for the unintended mRNAs with sequence complementarities with siRNA seed region (2–8 nucleotides from the 5′ end of the guide strand) in their 3′UTRs through translational repression.siRNA 介导 RNAi 及序列依赖性脱靶效应示意图。siRNA 是由引导链和乘客链组成的双链分子，加载到 AGO 蛋白上。乘客链被去除，引导链则保持在 AGO 蛋白上。与 siRNA 引导链序列完全互补的 mRNA 会通过 AGO 蛋白的切割而被 RNAi 抑制表达。另一方面，对于具有序列互补性的非靶 mRNA，siRNA 引导链的种子区 (引导链 5' 端的 2-8 个核苷酸) 会与其 3' 非翻译区 (3'UTR) 结合，从而导致序列依赖性脱靶效应，引起翻译抑制。
RNAi efficiencies of siRNAs are widely different depending on their sequences, and only a limited fraction of siRNAs, which satisfy the functional siRNA sequence rules, is effective in mammalian cells. Meanwhile, the siRNA often exhibits off-target effects when the siRNA seed region (2–8 nts from the 5′ end of the guide strand) base pairs with the unintended genes (Fig. 1). The off-target effect is considered to be induced by the similar molecular mechanism with miRNA-mediated RNA silencing, which differs from the mechanism of RNAi. Furthermore, the degree of off-target effect is correlated positively with the thermodynamic stability in base-pairing between the seed region of the siRNA guide strand and the unintended mRNA. Therefore, siRNA with low seed-target stability may be a promising tool for target-specific RNAi with little off-target effect. However, the number of the functional siRNA sequences with lower seed-target stability is substantially small.
siRNA 的 RNAi 效率取决于其序列，只有符合功能性 siRNA 序列规则的少部分 siRNA 才可以在哺乳动物细胞内发挥有效作用。然而，当 siRNA 的种子区 (位于引导链 5' 端的 2-8 个核苷酸) 与非靶基因序列碱基配对时，通常会产生脱靶效应 (见图 1)。siRNA 引起的脱靶效应被认为与 microRNA 介导的 RNA 沉默具有相似的分子机制，这种机制不同于 RNAi 机制。此外，脱靶效应的严重程度与 siRNA 引导链种子区和非靶 mRNA 之间碱基配对的热力学稳定性呈正相关。因此，种子区-靶标稳定性较低 (结合力较弱) 的 siRNA 可能成为具有高特异性和低脱靶效应的 RNAi 工具。然而，功能序列库中这种低稳定性 siRNA 的数量非常少。
Thus, we have established the methods to overcome such sequence-dependent limitations using two types of chemical modifications (Fig. 2): One type of chemical modification is DNA and the other is 2′-O-methyl (2′-OMe). The results of reporter assay (Fig. 3), structural simulation (Fig. 4), and microarray (Fig. 5) reveal that the seed-dependent off-target effect is able to be reduced by the introduction of DNA and 2′-OMe modifications in the siRNA seed region without reducing RNAi activity through different molecular mechanisms: DNA modification in the siRNA seed region decreased the thermodynamic stability in the seed-target base-pairing stability, and 2′-OMe modification induced steric hindrance in the duplex formation on the AGO protein. These chemical modifications may be useful tools for potential therapeutic applications.
为了克服 siRNA 序列依赖性的限制，我们采用了两种类型的化学修饰方法 (见图 2): 一种是 DNA 修饰，另一种是 2'-O-甲基 (2'-OMe) 修饰。报告基因检测 (见图 3)、结构模拟 (见图 4) 和微阵列分析 (见图 5) 的结果表明，在 siRNA 种子区引入 DNA 和 2'-OMe 修饰能够通过不同的分子机制降低种子依赖性脱靶效应，同时保持 RNAi 的活性。siRNA 种子区的 DNA 修饰降低了种子区与靶标 mRNA 的碱基配对热力学稳定性；而 2'-OMe 修饰则会在 AGO 蛋白上结合的双链 siRNA 中引入空间位阻效应。这些化学修饰方法有望成为未来 siRNA 治疗应用的利器。
Fig. 2 Flow for selecting functional siRNAs with reduced sequence-dependent off-target effect by two types of chemical modifications. (a) From all of human mRNA sequences, 56,375,087 of 23-mer sequences are selectable. This number of siRNAs is set as 100%. (b) Functional siRNA sequences can be selected by using the four rules described in Note 1. (c) The siRNAs with sequences containing low seed–target Tm values (see Note 2) for both strands are selected for reduction of off-target effects. (d) Using DNA or 2′-OMe modification in the siRNA seed region makes it possible to overcome the sequence limitations of siRNA for reducing seed-dependent off-target effects through different molecular mechanisms: reduction of base-pairing stability by DNA modification, and steric hindrance by 2′-OMe modification.两种化学修饰方法降低 siRNA 序列依赖性脱靶效应的功能 siRNA 选择流程。从所有的人类 mRNA 序列中，可以选择 56,375,087 个 23 核苷酸序列。该 siRNA 数量设置为 100%。b） 可以使用注釋 1 中描述的四条规则来选择功能性 siRNA 序列。c） 选择双链序列中种子区与靶标结合稳定性 (Tm 值，见注釋 2) 较低的 siRNA，以减少脱靶效应。d） 在 siRNA 种子区引入 DNA 或 2'-OMe 修饰，可以通过不同的分子机制克服 siRNA 序列限制以降低种子依赖性脱靶效应：DNA 修饰降低碱基配对稳定性，2'-OMe 修饰引入空间位阻。
Fig. 3 Measurement of RNAi and off-target activities by dual luciferase reporter assays . (Top) Structures of the constructs for luciferase reporter assays. psiCHECK-gCM_VIM270 contains a CM sequence of the siRNA guide strand in the downstream of Renilla luciferase CDS (CDS). psiCHECK-gSM_VIM270, psiCHECK-pCM_VIM270, or psiCHEK-pSM_VIM270 contains three tandem repeats of the SM sequences of the guide strand, the CM and SM sequences of the passenger strand, respectively. The reporter assays were performed as follows: HeLa cells were co-transfected with each concentration of unmodified, DNA- or 2′-OMe-modified siVIM-270, with each psiCHECK construct and pGL3 control vector. Cells were harvested 24 h after transfection and relative luciferase activities were measured. (Bottom) Unmodified, DNA- or 2′-OMe-modified siVIM-270 sequences (left). Results of RNAi activities on the guide CM target using psiCHECK-gCM_VIM270, off-target activities on the guide SM target using psiCHECK-gSM_VIM270, RNAi activities on the passenger CM target using psiCHECK-pCM_VIM270 and off-target activities on the passenger SM target using psiCHECK-pSM_VIM270 by the unmodified or modified siVIM-270s. We used siGY441 as siRNA control (siCont). The data were averaged from three independent experiments, and the bar indicates the standard deviation双萤光素酶报告检测 siRNA 的 RNAi 活性和脱靶效应。上图 为荧光素酶报告检测的载体结构图。psiCHECK-gCM_VIM270 在 Renilla 荧光素酶 (Renilla luciferase) 的编码区 (CDS) 下游包含 siRNA 引导链的 CM 序列。psiCHECK-gSM_VIM270、psiCHECK-pCM_VIM270 或 psiCHEK-pSM_VIM270 分别包含三个串联重复的引导链 SM 序列、过客链的 CM 序列和乘客链的 SM 序列。报告检测过程如下：HeLa 细胞与等量未修饰、DNA 修饰或 2'-OMe 修饰的 siVIM-270、每个 psiCHECK 载体和 pGL3 控制载体一起转染。转染 24 小时后收集细胞，并测量相对荧光素酶活性。下图 为未修饰、DNA 修饰或 2'-OMe 修饰的 siVIM-270 序列 (左侧)。使用 psiCHECK-gCM_VIM270 检测靶向引导链 CM 序列的 RNAi 活性，使用 psiCHECK-gSM_VIM270 检测靶向引导链 SM 序列的脱靶效应，使用 psiCHECK-pCM_VIM270 检测靶向乘客链 CM 序列的 RNAi 活性，以及使用 psiCHECK-pSM_VIM270 检测未修饰或修饰的 siVIM-270s 靶向乘客链 SM 序列的脱靶效应。我们使用 siGY441 作为 siRNA 对照 (siCont)。数据来自三个独立实验的平均值，柱状图表示标准偏差。
Fig. 4 Computational prediction of the steric hindrance of 2′-OMe-modified siRNAs loaded on the AGO protein. (a) Crystal structure of unmodified 5′-AAA-3′ RNA with K566 and R792 of AGO protein shown by Schirle et al., and the optimized structures of unmodified (b) and 2′-OMe-modified (c) RNAs with K566 and R792. (d) Superposition at C4′-C3′-O3′ of A3 of unmodified RNA with 2′-OMe-modified RNA.计算预测 2'-OMe 修饰 siRNA 加载到 AGO 蛋白上时的空间位阻。a） Schirle 等人展示的未修饰 5'-AAA-3' RNA 与 AGO 蛋白 K566 和 R792 的晶体结构，以及未修饰 (b) 和 2'-OMe 修饰 (c) 的 RNA 与 K566 和 R792 的优化结构。d） 未修饰 RNA 的 A3 的 C4'-C3'-O3' 与 2'-OMe 修饰 RNA 的叠加。
Fig. 5 Microarray analysis of RNAi activities and off-target effects of unmodified, and DNA- and 2′-OMe-modified siRNAs. (a) Expression levels of the target vimentin gene in the cells transfected with the unmodified and modified siRNAs relative to that in the mock-transfected cells, respectively. (b) MA plots of microarray data of the cells transfected with unmodified, and DNA- and 2′-OMe-modified siRNAs, respectively. The vertical axis indicates the log2 fold change of signal intensity of each gene in each type of siRNA-transfected cells relative to that of mock (M value), and the horizontal axis indicates the averaged log10 signal intensity of each gene in mock and siRNA transfection (A value). The dark blue dots indicate the transcripts with seed-matched (SM) sequences in the 3′UTRs of the mRNAs, and the light blue dots indicate the other transcripts. (c) Cumulative distribution of microarray data of the cells transfected with unmodified, and DNA- and 2′-OMe-modified siRNAs, respectively. The horizontal axis indicates the M value of (b), and the vertical axis indicates the cumulative fraction of transcripts. The red line indicates the cumulative curve of SM transcripts, and the black line indicates the cumulative curve of the other non-SM transcripts. (d) Mean log2 fold-change of the downregulated SM transcripts compared to those of non-SM transcripts. The horizontal axis indicates the mean fold-change of off-target transcripts in the cells transfected with unmodified, and DNA- and 2′-OMe-modified siRNAs, respectively.未修饰、DNA 修饰和 2'-OMe 修饰 siRNA 的 RNAi 活性和脱靶效应的芯片分析。a） 分别转染未修饰和修饰 siRNA 的细胞中，靶向中间 filament 蛋白 (vimentin) 基因的表达水平相对于 mock (空载) 转染细胞的表达水平。b） 未修饰、DNA 修饰和 2'-OMe 修饰 siRNA 转染细胞的芯片数据 MA 图。纵轴表示每种类型的 siRNA 转染细胞中每个基因信号强度 (强度) 的 log2 fold 变化 (相对于 mock 的 M 值)，横轴表示 mock 和 siRNA 转染细胞中每个基因平均的 log10 信号强度 (A 值)。深蓝色圆点表示 mRNA 的 3'UTR 中具有种子匹配序列 (SM) 的转录本，浅蓝色圆点表示其他转录本。c） 未修饰、DNA 修饰和 2'-OMe 修饰 siRNA 转染细胞的芯片数据累积分布图。横轴表示 (b) 的 M 值，纵轴表示转录本的累积分数。红色曲线表示 SM 转录本的累积曲线，黑色曲线表示其他非 SM 转录本的累积曲线。d） 下调调控的 SM 转录本与非 SM 转录本相比的平均 log2 fold 变化。横轴表示未修饰、DNA 修饰和 2'-OMe 修饰 siRNA 转染细胞中脱靶转录本的平均 fold 变化值。
👀
2.1 Design and Preparation of siRNAs siRNA 的设计与制备
siDirect2.0 software: http://siDirect2.RNAi.jp
Guide strand sequence of siRNA against human vimentin gene 针对人波形蛋白基因的 siRNA 的引导链序列 (siVIM-270): 5′-UUGAACUCGGUGUUGAUGGCG-3′.
Passenger strand sequence against human vimentin gene 针对人波形蛋白基因的过客链序列(siVIM-270): 5′-CCAUCAACACCGAGUUCAAGA-3′.
siRNA against green fluorescent protein (siGY441), which does not have perfectly complementary sequences in complete-match (CM) and seed-match (SM) reporter constructs (see Subheading 2.2), is used as a negative control.
针对绿色荧光蛋白 (siGY441) 的 siRNA 用作阴性对照，该蛋白在完全匹配 (CM) 和种子匹配 (SM) 报告构建体中不具有完全互补的序列（参见小标题 2.2）。
Guide strand of siGY441siGY441 的引导链序列: 5′-AUGAUAUAGACGUUGUGGCUG-3′.
Passenger strand of siGY441siGY441 的过客链序列: 5′-GCCACAACGUCUAUAUCAUGG-3′.
Annealing buffer 退火缓冲液 (2×): 40 mM NaCl, 20 mM Tris–HCl, pH 7.5.
19% Polyacrylamide gel 聚丙烯酰胺凝胶.
TBE buffer.
2.2 Luciferase Reporter Vector and Oligonucleotides with CM and SM Sequences 荧光素酶报告载体和具有 CM 和 SM 序列的寡核苷酸
psiCHECK-1 vector 载体 (Promega).
Oligonucleotides with CM sequences of siVIM-270 guide strand (gCM) with cohesive XhoI/EcoRI ends 具有 siVIM-270 引导链 (gCM) 的 CM 序列（具有粘性 XhoI/EcoRI 末端）的寡核苷酸: 5′-tcgaGCGCCATCAACACCGAGTTCAAGAG-3′ and 5′-aattCTCTTGAACTCGGTGTTGATGGCGC-3′.
Oligonucleotides with three tandem repeats of SM sequences of siVIM-270 guide strand (gSM) with cohesive XhoI/EcoRI ends 具有 siVIM-270 引导链 (gCM) CM 序列的三个串联重复序列和粘性 XhoI/EcoRI 末端的寡核苷酸: 5′-tcgaGAATGATGCACCAGGAGTTCAAGAAATGATGCACCAGGAGTTCAAGAAATGATGCACCAGGAGTTCAAGAG-3′ and 5′-aattCTCTTGAACTCCTGGTGCATCATTTCTTGAACTCCTGGTGCATCATTTCTTGAACTCCTGGTGCATCATTC-3′.
Oligonucleotides with three tandem repeats of CM sequences of siVIM-270 passenger strand (pCM) with cohesive XhoI/EcoRI ends 具有 siVIM-270 过客链 (pCM) CM 序列的三个串联重复序列和粘性 XhoI/EcoRI 末端的寡核苷酸: 5′-tcgaTCTTGAACTCGGTGTTGATGGCGAATCTTGAACTCGGTGTTGATGGCGAATCTTGAACTCGGTGTTGATGGCGAA-3′ and 5′-aattTTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGA-3′.
Oligonucleotides with three tandem repeats of CM sequences of siVIM-270 passenger strand (pSM) with cohesive XhoI/EcoRI ends 具有 siVIM-270 过客链 (pSM) CM 序列的三个串联重复序列和粘性 XhoI/EcoRI 末端的寡核苷酸: 5′-tcgaAATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGG-3′ and 5′-aattCCATCAACTCCTGGTGCATCATTCCATCAACTCCTGGTGCATCATTCCATCAACTCCTGGTGCATCATT-3′.
2.3 Cell Culture 细胞培养
Human HeLa cells or other culture cells 人 HeLa 细胞或其他培养细胞。
24-Well plate (Sumitomo Bakelite) 24 孔板 (Sumitomo Bakelite)
2.4 Transfection and Luciferase Reporter Assay Reagents 转染和荧光素酶报告基因检测试剂
pGL3-Control vector (Promega).pGL3-对照载体（Promega）
Lipofectamine 2000 reagent (Thermofisher). Lipofectamine 2000 试剂(Thermofisher)。
Dual-Luciferase Reporter Assay System (Promega).双荧光素酶报告基因检测系统(Promega)
1× passive lysis buffer (Promega).1×被动裂解缓冲液（Promega）。
2.5 Calculation of T~m~ T~m~（解链温度）计算
The melting temperature (T~m~) values are calculated using the following formula using ΔH and ΔS thermodynamic parameters shown in Xia et al. and Sugimoto et al..
解链温度（T~m~）使用下式计算，热力学参数ΔH和 ΔS采用夏以及杉本等人文章中的值。
ΔH (kcal/mol), sum of nearest neighbor enthalpy change最近邻焓变之和。
A, helix initiation constant (−10.8)螺旋起始常数（−10.8）。
ΔS, sum of nearest neighbor entropy change最近邻熵变之和。
R, gas constant (1.987 cal/deg/mol)气体常数 (1.987 cal/deg/mol)。
Ct, total molecular concentration of strand (100μM)链的总分子浓度（100μM）。
Fix [Na+] at 100 mM.将 [Na+] 固定在 100 mM。
2.6 Microarray Analysis 微阵列分析
RNeasy kit (Qiagen).
NanoDrop 2000 spectrophotometer (Thermo Scientific).
Bioanalyzer (Agilent).
Agilent one-color spike mix kit (Agilent).
Agilent SurePrint G3 human GE microarray (8 × 60 K, ver. 2.0, 3.0) (Agilent).👀
