Martina Tuttolomondo and Henrik J. Ditzel
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Small interfering RNAs (siRNAs) are RNA molecules with promising therapeutic potential as a result of their selective mRNA cleavage. However, despite recent progress, low stability in the bloodstream is an impediment to successful administration in vivo. Thus, the availability of flexible and rapid methods for studying siRNA stability and vehicles is crucial for future novel siRNA-based therapeutics. Herein, we report a fast Förster resonance energy transfer (FRET) method based on agarose gel electrophoresis to evaluate the stability of siRNA in serum as well as siRNA interaction with serum proteins and enzymes.
小干扰 RNA (siRNA) 因其选择性切割 mRNA 而具有潜在的治疗前景。然而，尽管取得了巨大进展，siRNA 在血液中的稳定性较低，阻碍了其在体内成功发挥作用。因此，开发用于研究 siRNA 稳定性和载体的灵活快速的方法对于未来新型 siRNA 疗法至关重要。在这项研究中，我们报告了一种基于琼脂糖凝胶电泳的快速 Förster 共振能量转移 (FRET) 方法，用于评估 siRNA 在血清中的稳定性以及 siRNA 与血清蛋白和酶的相互作用。
Small interfering RNAs (siRNAs) are 20–25 base pairs, double-stranded, noncoding RNA molecules with promising therapeutic potential as a result of their selective mRNA cleavage. siRNA is currently commonly employed in vitro to investigate protein expression. Theoretically, siRNA can be employed in vivo to treat several diseases with much higher selectivity and potency compared to small molecule drugs. However, its administration in vivo is challenging and thus has been the focus of intense recent investigation. Major limiting factors for the clinical use of siRNA are instability of the naked siRNA in the blood due to RNase degradation and removal by immune cells, short half-life due to renal clearance, and low cell and tissue penetration. The siRNA can be chemically modified and associated with a carrier to prolong its stability in the bloodstream and allow efficient cell internalization and tissue permeation.
siRNA（小干扰 RNA）是一种长度为 20-25 个碱基对的双链非编码 RNA 分子，因其选择性切割 mRNA 而具有巨大的治疗潜力。目前，siRNA 常用于体外研究蛋白质表达。理论上，siRNA 可以用于体内治疗多种疾病，与小分子药物相比，具有更高的特异性和效力。然而，siRNA 的体内应用面临挑战，因此近年来备受关注。siRNA 临床应用的主要障碍包括：
● 裸露的 siRNA 在血液中不稳定，容易被 RNase 降解和免疫细胞清除；
● 由于肾脏清除作用，siRNA 的半衰期短；
● 细胞和组织穿透性差。
为了解决这些问题，我们可以通过化学修饰 siRNA 并将其与载体结合来延长其在血液中的稳定性，并实现有效的细胞内化和组织渗透。
Tracking the integrity of siRNA in serum is crucial to the design of stable siRNA delivery systems for clinical use. Radiolabeling to evaluate siRNA stability is very sensitive, but expensive, health hazardous, and highly impactful on the environment. Due to these factors, this method is usually reserved for in vivo experiments. For initial estimation of siRNA stability in vitro, most methods are based on the application of fluorescent tags or UV stains (e.g., ethidium bromide) or on the use of siRNA molecules labeled with a single fluorophores. These methods rely on a readout based on a single emission color. Thus, complete degradation of a relatively large amount of nucleic acid is needed to detect either decreased fluorescence or a shifted siRNA band in the agarose gel. The latter methods may lead to incorrect conclusions since they are not sufficiently sensitive to detect partially degraded siRNA. Labeling the siRNA with two distinct fluorescent moieties overcomes this limitation. If the fluorescent tags are chosen as Förster resonance energy transfer (FRET) pairs, a fluorophore, and a quenching fluorophore placed on opposing strands of the oligonucleotide, increases the fluorescence emission intensity in the wavelength of the quenched fluorophore upon cleavage. While the fluorescence intensity from the quenching dye is proportional to the amount of cleaved siRNA, this would be unchanged after cleavage and proportional to the total amount of siRNA, allowing normalization of the degraded siRNA vs. total siRNA in the sample. FRET-siRNA can be also administered to cells to evaluate the stability of internalized siRNA and can be employed in plate-based in vitro assays or agarose gel electrophoresis. Although the plate format is suitable when investigating degradation by a single enzyme, it does not allow delineation of interactions with serum components. We recently reported an effective agarose gel electrophoresis method to track siRNA integrity in serum by single-molecule FRET-labeling of siRNA with simultaneous evaluation of interaction with serum components.
跟踪血清中 siRNA 的完整性对于设计稳定的 siRNA 递送系统以供临床使用至关重要。放射性标记可以非常灵敏地评估 siRNA 稳定性，但这种方法昂贵、危害健康且对环境影响很大。因此，这种方法通常仅用于体内实验。对于体外初步评估 siRNA 稳定性，大多数方法基于以下几种手段：
● 使用荧光标记或紫外染料（例如溴化乙锭）
● 使用标记有单个荧光团分子的 siRNA 分子
这些方法的检测结果都依赖于单一的荧光发射颜色的读书。因此，需要相对大量的核酸完全降解才能检测到荧光减弱或琼脂糖凝胶电泳条带移动位置的变化。后一种方法由于灵敏度不足以检测部分降解的 siRNA，可能会导致错误的结论。用两种不同的荧光基团标记 siRNA 可以克服这一限制。如果将荧光团选择为 Förster 共振能量转移 (FRET) 对，则将荧光团和淬灭荧光团分别放置在寡核苷酸 的对立链上，则切断后受抑制的荧光团的波长处会增加荧光发射强度。虽然淬灭染料的荧光强度与切割的 siRNA 量成正比，但在切割后不会改变，并且与样品中 siRNA 的总量成正比，从而允许归一化降解的 siRNA 和总 siRNA。FRET-siRNA 还可以用于评估细胞内 siRNA 的稳定性，并可在平板式体外测定或琼脂糖凝胶电泳中使用。虽然平板格式适用于研究单个酶的降解，但它不能区分与血清成分的相互作用。我们最近报道了一种有效的琼脂糖凝胶电泳方法，通过单分子 FRET 标记 siRNA 并同时评估与血清成分的相互作用，来跟踪血清中 siRNA 的完整性。
The method relies on a double-stranded siRNA labeled at the 5′-end with the fluorophore carboxyfluorescein (FAM) and at the 3′-end with the quencher carboxytetramethylrhodamine (TAMRA) (Fig. 1a). The FRET pair TAMRA (Absmax = 557 nm; Emmax = 583 nm) and FAM (Absmax = 495 nm; Emmax = 520 nm) is based on the overlap between the absorption spectrum of TAMRA (acceptor) and the emission spectrum of FAM (donor) (see Fig. 1b). When the intact FRET-siRNA is excited in the wavelength of FAM absorption, the emission from FAM is weak due to TAMRA quenching. When degraded siRNA is excited in the wavelength of FAM absorption, the distance between FAM and TAMRA is more extended and the quenching effect of TAMRA decreases, leading to increased intensity of the green fluorescence. In contrast, the red fluorescence is similar for both intact and degraded siRNA.
该方法依赖于在 5′端标记有荧光团羧基荧光素（FAM）和在 3′端标记有猝灭剂羧基四甲基罗丹明（TAMRA）的双链 siRNA（图 1a）。FRET 对 TAMRA（吸收峰=557 nm；发射峰=583 nm）和 FAM（吸收峰=495 nm；发射峰=520 nm）基于 TAMRA（受体）的吸收光谱与 FAM（供体）的发射光谱之间的重叠（见图 1b）。当完整的 FRET-siRNA 在 FAM 吸收波长下被激发时，由于 TAMRA 的猝灭作用，FAM 的发射较弱。当降解的 siRNA 在 FAM 吸收波长下被激发时，FAM 和 TAMRA 之间的距离增加，TAMRA 的猝灭效果减弱，导致绿色荧光强度增加。相反，对于完整和降解的 siRNA，红色荧光的强度相似。
Fig. 1 (a) A graphic representation of the fluorescence emission shift resulting from FRET-siRNA degradation.FRET-siRNA 降解导致荧光发射变化的图示 (b) Overlap of FAM emission and TAMRA absorption spectra. (Data retrieved from Spectra Viewer is available at www.thermofisher.com/order/spectra-viewer. Reprinted with permission from “Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability” by Tuttolomondo M, Ditzel HJ, 2020. ACS Med Chem Lett 11(2):195–202. Copyright 2020 American Chemical Society)FAM 发射光谱与 TAMRA 吸收光谱的重叠。（数据来自 Thermo Fisher 公司的 Spectra Viewer，网址为：www.thermofisher.com/order/spectra-viewer。经许可转载自“Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability” by Tuttolomondo M, Ditzel HJ, 2020。ACS Med Chem Lett 11(2):195–202。版权所有 2020 年美国化学学会）
In this chapter, we will provide detailed protocols to exploit our recently proposed agarose gel electrophoresis of FRET-siRNA for the evaluation of the stability of siRNAs or siRNA-delivery systems. Initially, we describe the design of a FRET-siRNA molecule, and thereafter we provide protocols to employ the FRET-siRNA for the evaluation of siRNA stability in the presence of degrading enzymes (e.g., RNase A) and sera (e.g., fetal bovine, mouse, and human serum) as dose-response and kinetic assays. We also provide a detailed protocol to analyze the gel bands using FIJI ImageJ software.
在本章中，我们将提供详细的实验方案，利用我们最近提出的 FRET-siRNA 琼脂糖凝胶电泳方法评估 siRNA 或 siRNA 递送系统的稳定性。首先，我们描述了 FRET-siRNA 分子的设计，然后提供了使用 FRET-siRNA 评估在降解酶（如 RNase A）和血清（如胎牛血清、小鼠血清和人血清）存在下 siRNA 稳定性的剂量反应和动力学实验方案。我们还提供了使用 FIJI ImageJ 软件分析凝胶条带的详细步骤。
10 μM Double-stranded FRET-labeled siRNA: Design the siRNA based on the desired target mRNA sequence using an available online tool (e.g., siDirect, http://sidirect2.rnai.jp/ ( http://sidirect2.rnai.jp/ )) to which suitable stabilizing chemical modifications are added. Label with FAM and TAMRA tags at 3′-end and 5′-end, respectively, on each strand of a double-stranded siRNA. In this way, the fluorescent intensity and FRET efficiency will be optimal, and a shift of fluorescence will be observed either in the case of siRNA cleavage or dissociation of two siRNA strands. Fluorescent-labeled oligonucleotides for the sense and antisense siRNA can be obtained from a synthesis service (e.g., GenScript) or produced in house (see Note 1). Resuspend the oligonucleotides at 20 μM in RNase-free water. To anneal the two siRNA strands, mix equal volumes of each strand solution, incubate at 65 °C for 5 min, and cool down to room temperature for 30 min.
10 μM 双链 FRET 标记的 siRNA：根据所需的目标 mRNA 序列，使用可用的在线工具（例如，siDirect，http://sidirect2.rnai.jp/）设计 siRNA，并添加适当的稳定化学修饰。在双链 siRNA 的 3′末端和 5′末端分别标记 FAM 和 TAMRA 标签，这样荧光强度和 FRET 效率会达到最佳，不论 siRNA 切割或是两条 siRNA 链解离，都能观察到荧光的变化。可以通过合成服务（如 GenScript）获得荧光标记的正义链和反义链寡核苷酸，或自行生产（参见注释 1）。将寡核苷酸溶解在 RNase-free 水中至 20 μM。为了退火两条 siRNA 链，将等体积的每条链溶液混合，在 65°C 下孵育 5 分钟，然后在室温下冷却 30 分钟。
PBS.
10 mg/ml (700 U/ml) RNase A.
A vial mixer with thermal control.
带有热控功能的涡旋混合器
High-resolution agarose for Molecular Biology, powder.
分子生物学用高分辨率琼脂糖粉
10× TAE buffer: 50 mM EDTA disodium salt, 2 M Tris base, 1 M glacial acetic acid. Add about 800 ml deionized water to a 1 l graduated cylinder or a glass beaker. Weigh 242 g Tris base and transfer to the cylinder. Add 18.61 g of disodium EDTA to the solution. Add 57.1 g Acetic Acid to the solution. Add deionized water to 900 ml. Mix and adjust pH with NaOH. Add deionized water to 1000 ml. Store at room temperature. Dilute to 1× prior to use.
10× TAE 缓冲液：50 mM EDTA 二钠盐，2 M Tris 碱，1 M 冰乙酸。将约 800 ml 去离子水加入到 1 升量筒或玻璃烧杯中。称量 242 g Tris 碱并转移到量筒中。加入 18.61 g 二钠 EDTA。加入 57.1 g 冰乙酸。加入去离子水至 900 ml。混合并用 NaOH 调节 pH 值。加入去离子水至 1000 ml。室温保存。使用前稀释至 1×
Sample loading solution: 30% glycerol in ultrapure water (see Note 2).
样品加载溶液：30%甘油在超纯水中（参见注释 2）
Horizontal electrophoresis apparatus.
水平电泳装置
Serum of interest: fetal bovine, mouse, or human serum.
目标血清：胎牛血清、小鼠血清或人血清。
A siRNA stabilizing agent (see Note 3).
siRNA 稳定剂（参见注释 3）
A siRNA releasing agent that can release the siRNA from the tested carrier. For peptides and Lipofectamine 2000, we suggest 50 mg/ml dextran sulfate sodium.
siRNA 释放剂：能够从测试载体中释放 siRNA。对于肽和 Lipofectamine 2000，建议使用 50 mg/ml 葡聚糖硫酸钠。
Gel imager with the possibility of setting the excitation at 465 nm and the emission detection at 600 nm for TAMRA and 520 nm for FAM (see Note 4)
凝胶成像仪：需能够设置激发光波长为 465 nm 和发射光检测波长为 600 nm（TAMRA）和 520 nm（FAM）（参见注释 4）
FIJI ImageJ (NIH) software (available for free of charge download at https://imagej.net/Fiji.
FIJI ImageJ（NIH）软件：可免费下载，网址：https://imagej.net/Fiji。
3.1 Validation of Designed FRET-siRNA by RNase A Digestion 通过 RNase A 消化实验验证 FRET-siRNA 的设计
Prepare a 2% agarose gel in 1× TAE buffer with an 8-wells comb (see Note 5).
用 1× TAE 缓冲液配置一个 2%的琼脂糖凝胶，并使用一个 8 孔梳子（详见注释 5）。
Set the thermomixer at 37 °C.
将热混合器设置到 37°C。
Prepare eight Eppendorf tubes containing 7.5 pmol (22 μg) FRET ds-siRNA in PBS: add 2.5 μl of 10 μM siRNA to 6.5 μl of PBS (see Note 6 and Table 1).
准备八个 Eppendorf 管，每个管中加入 7.5 pmol（22 μg）的 FRET 双链 siRNA 溶于 PBS 中：加入 2.5 μl 的 10 μM siRNA 到 6.5 μl 的 PBS 中（详见注释 6和表 1）。
Prepare serial dilutions of RNase A from the 10 mg/ml stock solution according to Table 1.
按照表 1 从 10 mg/ml 的 RNase A 储备溶液制备一系列稀释溶液。
Add 1 μl of corresponding RNase A solution according to Table 1.
根据表 1 向每个管中加入 1 μl 相应浓度的 RNase A 溶液。
Incubate the samples at 37 °C for 2 min with shaking.
在 37°C 下摇动孵育样品 2 分钟。
Add 2 μl of sample loading solution to each sample and load them on the agarose gel according to the sample number.
向每个样品中加入 2 μl 的样品加载缓冲液，并将它们按样品编号加载到琼脂糖凝胶中。
Perform the electrophoresis in 1× TAE buffer at 120 V for about 10 min.
在 1× TAE 缓冲液中以 120 V 电泳约 10 分钟。
Image the gel using a fluorescence gel imager. Excitation should be close to 465 nm for both fluorophores. Detection should be close to 520 nm for FAM and close to 600 nm for TAMRA. The suggested exposure time is 15 s (see Note 7).
用荧光凝胶成像仪拍摄凝胶图像。激发波长应接近 465 nm，用于两种荧光团。检测波长应分别接近 520 nm 和 600 nm，用于 FAM 和 TAMRA。建议曝光时间为 15 秒（详见注释 7）。
Table 1 Preparation of RNase-treated FRET-siRNA
1. Prepare 8 FRET-siRNA solutions | 2. Prepare 7 serial dilutions of RNase A from 10 mg/ml solution 3. Add 1 μl of corresponding RNAse A dilution 4. Incubate for 2 min at 37 °C | ||||
|---|---|---|---|---|---|
9 μl siRNA solution | RNase serial dilutions | ||||
# | RNase A amount (μg) | Volume of 10 μM siRNA (μl) | Volume of PBS (μl) | Volume of RNase A (μl) | Volume of PBS (μl) |
1 | – | 2.5 | 7.5 | – | – |
8 | 1 | 2.5 | 6.5 | 1μl of 10 mg/ml stock solution | 9 |
7 | 0.5 | 2.5 | 6.5 | 5 μl of 1 mg/ml solution | 5 |
6 | 0.25 | 2.5 | 6.5 | 5 μl of 0.5 mg/ml solution | 5 |
5 | 0.1 | 2.5 | 6.5 | 1 μl of 1 mg/ml solution | 9 |
4 | 0.05 | 2.5 | 6.5 | 5 μl of 0.1 mg/ml solution | 5 |
3 | 0.025 | 2.5 | 6.5 | 5 μl of 0.05 mg/ml solution | 5 |
2 | 0.01 | 2.5 | 6.5 | 1 μl of 0.1 mg/ml solution | 9 |
3.1.1 Evaluating Band Fluorescence Intensity by FIJI ImageJ Gel Analyzer Tool 使用 FIJI ImageJ 凝胶分析工具评估条带荧光强度
Import the images to FIJI ImageJ and assign the red channel to TAMRA and the green channel to FAM.
将图像导入 FIJI ImageJ，分配红色通道给 TAMRA，绿色通道给 FAM
Convert the image to 8-bit (go to Image, Type, 8-bit) to obtain a grayscale image of the gel.
将图像转换为 8 位灰度图（在菜单栏选择图像->类型->8 位）
Choose the Rectangular Selections tool from the ImageJ toolbar.
在 ImageJ 工具栏选择矩形选择工具
Draw a rectangle around the first lane.
在第一个泳道区域绘制矩形
Go to Analyze, Gels, Select First Lane to set the selection as “first lane.” A number 1 will appear over the first lane selection.
在菜单栏选择分析->凝胶->选择第一个泳道，将当前选择设为“第一个泳道”。第一个泳道上会出现数字 1
Move the rectangle selection to the next lane (see Note 8).
移动矩形选择工具到下一个泳道区域（详见注释 8）
Go to Analyze, Gels, Select Next Lane to set the selection as “second lane.” A number 2 will appear over the second lane selection.
在菜单栏选择分析->凝胶->选择下一个泳道，将当前选择设为“第二个泳道”。第二个泳道上会出现数字 2
Repeat steps 6 and 7 for the remaining lanes of the gel.
对剩余的泳道重复步骤 6和步骤 7
Go to Analyze, Gels, Plot Lanes to obtain a profile plot of each lane. A pick will correspond to each lane.
在菜单栏选择分析->凝胶->绘制泳道，生成每个泳道的轮廓图。每个泳道都会出现一个峰值
Choose the straight-line selection tool from the ImageJ toolbar.
在 ImageJ 工具栏选择直线选择工具
Draw a line across the base of each peak to enclose the peak and remove the background noise.
绘制一条线穿过每个峰值的底部，围住峰值并去除背景噪音
Use the wand tool from the toolbar to click inside each peak. The measurements of the pick areas will appear in the Results window. The results can be exported to a spreadsheet.
使用工具栏中的魔棒工具点击每个峰值内部。峰值面积的测量结果会显示在结果窗口中，并可以导出到电子表格
Repeat the procedure for each channel (FAM and TAMRA).
对每个通道（FAM 和 TAMRA）重复此过程
Create a merged image (see Note 9 and Fig. 2a) .
创建一个合并图像（详见注释 9和图 2a）
The amount of degraded siRNA is proportional to the FRET efficiency. Calculate the ladder as “relative ratiometric FRET efficiency,” or “relative proximity ratio,” EPR by using Eq. 1 as from Eyal Nir et al. :
降解的 siRNA 量与 FRET 效率成正比。使用 Eyal Nir 等人的方程 1 计算“相对比率 FRET 效率”或“相对接近比率”（EPR）
Factors on Eq. 1 have been defined in our previous work “IA
is the fluorescence intensity of the acceptor, I D is the fluorescence intensity of the donor, is the minimum fluorescence intensity of the acceptor, and I A 0 is the minimum fluorescence intensity of the donor”. I D 0 方程 1 中的参数在我们的前期工作中已定义：“
I A 是受体的荧光强度， I D 是供体的荧光强度， 是受体的最小荧光强度， I A 0 是供体的最小荧光强度”。 I D 0 Plot the relative ratiometric FRET efficiency against the RNase A amount (Fig. 2b).
绘制相对比率 FRET 效率与 RNase A 量的关系图（图 2b）
Fig. 2 (a) Agarose gel electrophoresis of FRET-siRNA degraded with RNase A. Green and magenta represents FAM and TAMRA emission intensities, respectively. Channels are shown both as separate images and merged.显示经过 RNase A 降解的 FRET-siRNA 的琼脂糖凝胶电泳图。绿色和品红色分别代表 FAM 和 TAMRA 的发射强度。通道分别显示为独立图像和合并图像。 (b) Graph showing the relative ratiometric FRET efficiency of FRET-siRNA obtained by ImageJ analysis of images shown in a. Data are shown as averages of three biological replicates. Bars are standard errors of the mean. (Adapted with permission from “Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability” by Tuttolomondo M, Ditzel HJ, 2020. ACS Med Chem Lett 11(2):195–202. Copyright 2020 American Chemical Society)图表显示了通过 ImageJ 分析a中图像获得的 FRET-siRNA 相对比率 FRET 效率。数据为三个生物重复的平均值，误差条表示平均值的标准误差。（经许可改编自“Tuttolomondo M, Ditzel HJ, 2020. 简单 FRET 电泳方法用于精确和动态评估血清 siRNA 稳定性”. ACS Med Chem Lett 11(2):195–202. 版权归 2020 年美国化学会所有）
