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3.1 Aptamer-siRNA Design 适配体-siRNA 设计
Choice of an appropriate aptamer. Choose the aptamer based on the cell system you want to target and on its ability to rapidly internalize within the cells overexpressing its target in a receptor-dependent manner. To generate the construct, extend the aptamer sequence at the 3′-end with the stick sequence.
选择合适的适配体。根据您想要靶向的细胞系统以及其在受体依赖性方式下对其靶标的快速内化能力来选择适配体。为了生成构建物，将适配体序列在 3'端延伸至棒-桥序列。
Choice of an appropriate siRNA. Choose the siRNA based on experimentally tested sequences or by using siRNA tools available online (see Note 11). The sequences reported here will be 19–21 nt in length. Extend the antisense strand at the 3′-end with the stick sequence. We recommend checking: (a) the correct annealing of the stick-antisense oligo to the corresponding siRNA sense strand by structure prediction (see Note 12) and gel electrophoresis (see below Subheading 3.6, Note 13); (b) the annealing with the stick-antisense sequence does not alter aptamer folding by using common structure prediction software (see Note 12); (c) the therapeutic efficacy of the generated siRNA duplex upon cell transfection (see Subheading 3.7, Note 14). The general aspects of aptamer attachment to different siRNA strands or the optimization of used siRNA sequences have been well discussed in several studies.
选择合适的 siRNA。根据实验测试的序列或使用在线 siRNA 工具来选择 siRNA（详见注 11）。这里报告的序列长度为 19-21 个核苷酸。将反义链在 3'端延伸至棒-桥序列。我们建议检查：(a)通过结构预测（参见注 12）和凝胶电泳（参见下文 3.6 节，注 13）验证棒-桥-反义寡核苷酸与相应 siRNA 的正义链的正确退火；(b)使用常见的结构预测软件（参见注 12）检查与棒-桥-反义序列的退火是否改变了适配体的折叠；(c)在细胞转染后检查生成的 siRNA 双链的治疗效力（参见 3.7 节，注 14）。关于适配体如何附着到不同 siRNA 链或优化所用 siRNA 序列，已经在多项研究中得到了广泛讨论。
3.2 Construct Preparation (Fig. 1) 构建体制备
Determine the concentration of each RNA oligo dissolved in sterile RNAse-free water (see Note 15) spectrophotometrically, assuming that one A260 unit is equal to 40 mg/ml of RNA.
将 RNA 寡核苷酸溶解于无菌 无 RNAse 酶水中，使用紫外分光光度计测定每种浓度（参见注 15），假设一个 A260 单位等于 40 mg/ml RNA。
To prepare constructs, anneal 5 μM antisense-stick siRNA strand to 5 μM of complementary siRNA guide (ratio 1:1) in 1× annealing buffer by incubating at 95 °C for 10 min, 55 °C for 10 min, and 37 °C for 20 min. Then, subject an equal amount of stick aptamer to short denaturation-renaturation steps (85 °C for 5 min, snap-cooling on ice for 2 min and warming up to 37 °C) and add it to the generated stick-siRNA duplex. Transfer the mixture at 37 °C for 30 min.
为了制备构建体，将 5 μM 反义链棒-桥 siRNA 与 5 μM 互补 siRNA 正义链在 1× 退火缓冲液中以 1:1 的比例退火。 退火步骤为：95 °C 孵育 10 分钟，55 °C 孵育 10 分钟，37 °C 孵育 20 分钟。 然后，对等量的棒-桥适配体进行短暂的变性-复性步骤（85 °C 孵育 5 分钟，立即冰上急冻 2 分钟，然后升温至 37 °C），并将其添加到生成的棒桥-siRNA 双链中。 将混合物在 37 °C 孵育 30 分钟。
After annealing procedures, immediately use or store the constructs at −20 °C (see Note 16).
退火程序完成后，构建体可以立即使用，也可以保存在 -20 °C 的冰箱里（参见注 16）
Fig. 1 Stick-based conjugate. Schematic representation of constructs generated by stick-end annealing. 4XC3 indicates the 4X((CH2)3) carbon spacer 图 1 基于棒桥末端退火的构建体。构建体的示意图。4XC3 表示 4X((CH~2~)~3~)碳间隔
3.3 Control of the Correct Annealing by Gel Electrophoresis 通过凝胶电泳检测正确退火
Control the correct annealing on a 12% non-denaturing PAGE gel. Load unconjugated RNAs or annealed constructs (10–15 pmol each) on the gel and run it in 1× TBE (see Note 17).
使用 12% 非变性 PAGE 胶进行电泳来检测退火是否正确。在凝胶上加载未结合的 RNA 或已退火的构建物（每个 10-15 pmol），并在 1× TBE 缓冲液中运行电泳（见 注释 17）。
Visualize gel bands by ethidium bromide staining and GEL.DOC XR gel camera.
用溴化乙锭染色后，使用 GEL.DOC XR 胶凝电泳图像分析仪观察胶上条带。
Monitor the correct annealing by the presence of a shifted band of migration as compared to the individual components (Fig. 2).
正确的退火会使条带的位置移动，与单个组分的条带位置相比有所不同 (见图 2)。
Fig. 2 Electrophoresis of annealed AsiC. Representative gel electrophoresis to control the correct annealing of Gint4.T-STAT3 AsiC containing the anti-PDGFRβ aptamer (Gint4.T) conjugated to STAT3 siRNA by the stick-based approach. M, DNA base pair (bp) ladderGint4.T-STAT3 AsiC 的电泳。代表性凝胶电泳图，用于控制通过基于棒-桥的方法将抗 PDGFRβ抗体（Gint4.T）连接到 STAT3 siRNA 的 Gint4.T-STAT3 AsiC 的正确退火。M，DNA 碱基对（bp） ladder
3.4 Cell Culture (For Binding and Internalization or Functional Activity Assays) 细胞培养（用于结合和内化或功能活性检测）
Seed cells in 6-well plates the day before treatments.
在进行处理的前一天，将细胞接种于 6 孔板中。
For binding and internalization analysis, seed 2 × 10^5^ cells per well.
用于结合和内化分析，每个孔接种 2 × 10^5^个细胞。
For functional activity assays, seed 1.4 × 10^5^ cells/well (about 60% confluence). Treat cells with 400 nM aptamer or aptamer-siRNA construct (see Notes 18 and 19) or transfect with 100 nM siRNA duplex as control using Lipofectamine 2000 according to manufacturer’s instructions.
用于功能活性检测，每个孔接种 1.4 × 10^5^ 个细胞 (融合度约为 60%)。用 400 nM 适配体或适配体-siRNA 构建体处理细胞 (见注 18 和 19)。作为对照，使用 Lipofectamine 2000 转染 100 nM siRNA 双链 (按照制造商的说明进行操作)。
Following 3 days, process cells for functional analyses (either by RT-qPCR or by immunoblotting as detailed below).
对细胞进行 3 天处理后，进行后续的功能分析 (RT-qPCR 或免疫印迹，见下文详细说明)。
3.5 Binding and Internalization Analysis by RT-qPCR
Add RNAs (aptamers or constructs) to the cells at 200 nM for different incubation times (ranging from 15 min to 2 h) at 37 °C in the presence of 100 mg/ml tRNA used as a nonspecific competitor. At selected times, wash cells three times with PBS (to remove unbound sequences and recover total bound sequences) or with PBS 0.5 M NaCl (to remove cell surface-bound sequences and measure amount of internalized RNA). Then, recover RNAs with 1 ml/sample TRIzol containing 0.5 pmol/ml of the CL4 oligo used as reference control (see Note 20) and extract.
将 RNA (适配体或构建体) 以 200 nM 的浓度加入到细胞中，并在 37 °C 孵育不同时间 (15 分钟到 2 小时)，加入 100 mg/ml tRNA 作为非特异性竞争物。 在选定的时间点，用 PBS 洗涤细胞三次 (去除未结合的序列并回收所有结合的序列) 或用含 0.5 M NaCl 的 PBS 洗涤 (去除细胞表面结合的序列并测量内化 RNA 的量)。 然后，使用含 0.5 pmol/ml 参考控制寡核苷酸 CL4 的 1 ml/样品 TRIzol 提取 RNA (见注 20)。
Determine the amount of recovered RNA by performing a two-step RT-qPCR protocol. In step 1, RNA is reverse transcribed using specific 3′ primers and the following protocol: heating step at 65 °C for 5 min, annealing step at 22 °C for 5 min, extension at 42 °C for 30 min followed by end extension at 48 °C for 30 min and enzyme inactivation at 95 °C for 5 min. In step 2, the RT products are PCR amplified with iQ SYBR Green Supermix (Bio-Rad) by heating at 95 °C for 2 min, followed by 40 cycles of heating at 95 °C for 30 s, annealing at 55 °C for 30 s, and extending at 60 °C for 30 s. A melt curve stage by heating at 60–95 °C is performed.
通过执行两步 RT-qPCR 实验来测定回收的 RNA 量。 第一步是使用特异性 3' 末端引物进行 RNA 逆转录，具体步骤为：65 °C 加热 5 分钟，22 °C 退火 5 分钟，42 °C 延伸 30 分钟，然后在 48 °C 延伸 30 分钟，最后 95 °C 灭活酶 5 分钟。 第二步，使用 iQ SYBR Green Supermix (Bio-Rad) 对 RT 产物进行 PCR 扩增，具体步骤为：95 °C 加热 2 分钟，然后进行 40 个循环，每个循环包含 95 °C 加热 30 秒，55 °C 退火 30 秒，60 °C 延伸 30 秒。 最后进行 60-95 °C 的熔解曲线分析。
Normalize data to the CL4 reference control.
将数据归一化到 CL4 参考控制。
This experiment demonstrates that the siRNA conjugation to the aptamer does not abrogate aptamer binding and internalization properties.
该实验表明，siRNA 与适配体的结合不会削弱其结合和内化特性。
3.6 AsiC Functional Activity Analyses by RT-qPCR 通过 RT-qPCR 分析 AsiC 的功能活性
To demonstrate the aptamer ability to deliver a functional siRNA, analyze the levels of the siRNA target by RT-qPCR and immunoblotting (see Subheading 3.7) upon cell construct treatment.
为了证明适配体能够递送功能性 siRNA，需要通过 RT-qPCR 和免疫印迹 (见下文 3.7 节) 分析细胞接受构建体处理后 siRNA 靶标的水平。
For RT-qPCR, recover RNAs from transfected or treated cells in 1 ml of TRiZol and extract according to manufacturer’s instructions.
对于 RT-qPCR，使用 1 ml TRIzol 从转染或处理过的细胞中提取 RNA，并按照制造商的说明进行操作。
Analyze the levels of the siRNA target by performing total RNA (1 μg) retrotranscription using a cDNA synthesis kit and subsequent amplification with SYBR Green Supermix and specific primers. Perform the amplification of a reference gene (i.e., actin, GAPDH) in parallel, and use the ΔΔCt method for relative quantization of gene expression.
分析 siRNA 靶标的水平，使用 cDNA 合成试剂盒对 1 μg 总 RNA 进行逆转录，然后使用 SYBR Green Supermix 和特异性引物进行扩增，从而分析 siRNA 靶标的水平。 同时扩增参考基因 (例如肌动蛋白、GAPDH)，并使用 ΔΔCt 方法进行基因表达的相对定量。
3.7 AsiC Functional Activity Analyses by Immunoblotting
For immunoblotting , prepare cell protein extracts from transfected or treated cells by washing cells in ice-cold PBS and lysing in lysis buffer. Determine protein concentration by using the Bradford assay and bovine serum albumin as standard.
对于免疫印迹法，需要用冰冷的 PBS 洗涤转染或处理过的细胞，然后用裂解缓冲液裂解细胞，获得细胞蛋白提取物。 使用 Bradford 法并以牛血清白蛋白为标准来测定蛋白质浓度。
Run samples on SDS-polyacrylamide gels and transfer into PVDF membranes.
将样品加入 SDS-聚丙烯酰胺凝胶电泳，并转印到 PVDF 膜上。
Probe filters with primary antibodies against the siRNA target (STAT3 in the reported example in Fig. 3). Use antibodies against a reference protein (anti-actin in the reported example) to confirm equal loading. Visualize signals with peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system.
用针对 siRNA 靶标 (本例中为 STAT3，见图 3) 的一抗探针孵育滤膜。 同时加入针对参考蛋白 (本例中为抗肌动蛋白) 的抗体，以确保样品上样量一致。使用增强型化学发光系统和过氧化物酶标记的二抗可视化信号。
An example of the results obtained with extracts from U87MG cells (PDGFRβ receptor-positive cells) treated with a chimera containing the anti-PDGFRβ internalizing aptamer conjugated to STAT3 siRNA by stick-end annealing (indicated as Gint4.T-STAT3) is shown in Fig. 3. As expected, the AsiC treatment results in a significant reduction of STAT3 levels as compared to cells left untreated or treated with a control aptamer or construct containing an unrelated aptamer. Notably, the extent of reduction is comparable to that obtained upon siSTAT3 duplex transfection.
图 3 显示了从 U87MG 细胞（PDGFRβ受体阳性细胞）提取的样品的结果示例，这些细胞经过使用包含 anti-PDGFRβ内化的 aptamer 和 STAT3 siRNA 的构建体（用 stick-end 退火法连接，表示为 Gint4.T-STAT3）处理。正如预期的那样，与未处理细胞或用控制适配体或包含无关适配体的构建体处理的细胞相比，AsiC 处理显著降低了 STAT3 蛋白水平。值得注意的是，这种降低程度与 siSTAT3 双链转染获得的降低程度相当。
Fig. 3 Functional analyses by immunoblotting. Cell extract from U87MG (PDGFRβ positive) cells left untreated (−) or treated with control construct (CtrlApt linked to siSTAT3), Gint4.T, control aptamer (CtrlApt), or Gint4.T-STAT3, or transfected with STAT3 siRNA duplex, were analyzed by immunoblotting with STAT3 or actin (used as loading control) antibodiesU87MG 细胞 (PDGFRβ 受体阳性) 的细胞提取物在未处理 (-)、用连接有 siSTAT3 的控制构建体 (CtrlApt linked to siSTAT3)、Gint4.T、控制适体 (CtrlApt) 或 Gint4.T-STAT3 处理或转染 siSTAT3 siRNA 双链后，通过免疫印迹与 STAT3 或肌动蛋白 (作为上样控制) 抗体进行分析。
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We recommend ordering HPLC-purification grade lyophilized RNAs. The RNAs are dissolved in sterile RNAse-free water before use.
我们建议订购经过 HPLC 纯化 (HPLC chun hua) 的冻干 (dong gan) RNA。使用前 RNA 需要溶解于无菌的 RNase 酶阴性水中。
2′F-Py RNAs are used to increase nuclease resistance.
2′F-Py RNAs 用于增加核酸酶的抵抗力。
The carbon linker increases the distance of the stick sequence from the aptamer, reducing possible interferences with the correct aptamer folding.
碳链连接子 可以增加棒-桥序列与适配体 之间的距离，减少对适配体正确折叠的潜在干扰。
The sequence of the stick portion may vary based on the specific aptamer used. It is important that the stick portion: (a) does not alter the correct aptamer folding; (b) produces a duplex with the most favorable annealing in the construct.
棒-桥的序列可能因所使用的特定适配体而异。重要的是，棒-桥片段应该：（a）不改变适配体正确的折叠；（b）在构建体中形成具有最优退火的双链。
In addition to 2′-FPy, the stick portion contains 2′-O-methyl-purines to further increase the base pairing stability of the duplex.
除了 2′-FPy 外，棒状部分还含有 2′-O-甲基嘌呤，进一步增加了双链的碱基配对稳定性。
Alterative nonspecific competitors (e.g., polyinosinic acid) may be used.
可以使用替代性的非特异性竞争物（例如，多肌苷酸）。
The reference control is an unrelated RNA that is used as an internal control to normalize any experimental errors between the analyzed samples.
参考对照是一种不相关的 RNA，用作内部控制，以规范所分析样品之间的任何实验误差。
The same primers are used for the aptamer and the construct.
相同的引物用于适配体和构建物。
Commercially available pre-casted gels may be used.
可以使用市售的预制凝胶
Transfer buffer can be used up to three times, checking that the voltage is maintained at a constant rate.
转移缓冲液可重复使用最多三次，需检查电压是否保持在恒定速率。
An example is the online tool available at www.Dharmacon.com.
可用的在线工具示例是 www.Dharmacon.com 上的工具。
A prediction of bimolecular RNAs can be performed with common structure prediction software such as RNAStructure.
可以使用常见的结构预测软件（例如 RNAStructure）对双分子 RNA 进行预测。
The correct annealing can be monitored by the presence of a shifted band of migration as compared to the antisense stick and the sense strands on non-denaturing PAGE gel.
通过非变性 PAGE 凝胶上的移动带的出现与反义棒和正义链相比，可以监测正确退火的存在。
The correct efficacy of the generated siRNA duplex can be monitored by analyzing the levels of the siRNA target by RT-qPCR and immunoblot following cell transfection with the duplex.
通过细胞转染与双链后，可以通过 RT-qPCR 和免疫印迹法分析 siRNA 靶标的水平来监测所生成 siRNA 双链的正确功效
Once dissolved, we recommend the solution be aliquoted and stored at −20 °C.
溶解后，我们建议将溶液分成小份并储存在 -20 °C。
When constructs are stored at −20 °C, we recommended avoiding freezing-refreezing cycles.
当构建体储存在 -20 °C 时，我们建议避免反复冷冻-解冻 循环。
The gel is run until the sample dye front settles to the bottom.
电泳运行直到样品染料前端沉淀到底部。
Before treatment, constructs are warmed up to 37 °C, and the aptamers are subjected to the denaturation-renaturation steps (5 min 85 °C, 2 min snap-cooling on ice, warming up to 37 °C).
处理前，将构建体预热至 37 °C，并将适配体进行变性-复性步骤（85 °C 5 分钟，冰上快速冷却 2 分钟，升温至 37 °C）。
To ensure a correct aptamer folding, denaturation-renaturation steps must be performed at low aptamer concentration (no more than 20 μM).
为了确保适体正确折叠，必须在低适配体浓度（不超过 20 μM）下进行变性-复性步骤。
We recommend preparing a unique solution of TRIzol with the reference control for all experimental points.
我们建议为所有实验点准备含有参考对照的唯一 TRIzol 溶液。
