Martina Tuttolomondo and Henrik J. Ditzel
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3.3 Degradation Kinetics of FRET-siRNA in Serum FRET-siRNA 在血清中的降解动力学
Prepare a 2% agarose gel in 1× TAE buffer with an 8-wells comb.
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。
Set the thermomixer at 37 °C.
将热混合器设置到 37 °C。
Prepare 10 samples FRET ds-siRNA in PBS from 10 μM solution according to Table 3.
按照表 3 从 10 μM 溶液中制备 10 个 FRET 双链 siRNA 样品于 PBS 中。
Prepare a 0.2% solution of serum of interest.
配制 0.2%浓度的目标血清溶液。
Treat with 1 μl of 0.2% serum of interest at different time intervals for each sample and incubate at 37 °C according to Table 3).
在不同时间间隔内，用 1 μl 的 0.2%血清溶液处理每个样品，并在 37 °C 孵育（详见表 3）。
Perform electrophoresis, imaging, and analysis as described in Subheading 3.1 (see Fig. 4a).
按照 3.1 节中描述的方法进行电泳、成像和分析（详见图 4a）。
Plot the relative ratiometric FRET efficiency against the time (see Fig. 4b).
绘制相对比率 FRET 效率与时间的关系图（详见图 4b）。
Table 3 Preparation of serum-treated FRET-siRNA for kinetic analysis
1. Prepare 8 FRET-siRNA solutions | 2. Add 1 μl of 0.2% serum solution 3. Incubate at 37 °C for different time intervals | |||
|---|---|---|---|---|
siRNA solution | Volume of serum (μl) | |||
# | Incubation time | Volume of 10 μM siRNA (μl) | Volume of PBS (μl) | |
1 | – | 2.5 | 7.5 | – |
2 | 1 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 39 min |
3 | 2 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 38 min |
4 | 5 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 35 min |
5 | 10 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 30 min |
6 | 20 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 20 min |
7 | 30 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after 10 min |
8 | 40 | 2.5 | 6.5 | Add 1 μl of 0.2% serum after immediately |
Fig. 4 (a) Agarose gel electrophoresis of FRET-siRNA degraded by human serum in time. Green and magenta represent FAM and TAMRA emission intensities, respectively. Channels are shown both as separate images and merged.人类血清随时间降解 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。图像分别显示为独立通道和合并通道。 (b) Plot showing the relative ratiometric FRET efficiency of FRET-siRNA degraded in time obtained by ImageJ analysis of image shown in a. Data are shown as averages of three biological replicates. Bars are standard errors of the mean. The curve is two-phase decay exponential regression. (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)通过 ImageJ 分析a中图像获得的 FRET-siRNA 随时间降解的相对比率 FRET 效率图。数据为三个生物重复实验的平均值，误差条表示平均值的标准误差。曲线为双相衰减指数回归。（经许可转载自“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）
3.4 Evaluation of FRET-siRNA Stabilizing Effect of siRNA Carriers in Serum 评估 siRNA 载体在血清中对 FRET-siRNA 的稳定作用
Prepare a 2% agarose gel in 1× TAE buffer with a 4-wells comb.
用 1× TAE 缓冲液配置一个 2%的琼脂糖凝胶，并使用一个 4 孔梳子
Set the thermomixer at 37 °C.
将热混合器设置到 37°C
Prepare two samples of FRET ds-siRNA in PBS from 10 μM solution at a final concentration 1.5 μM. One sample will be untreated free siRNA and the other serum-treated free siRNA.
从 10 μM 溶液中制备两个最终浓度为 1.5 μM 的 FRET 双链 siRNA 样品于 PBS 中。一个样品为未处理的游离 siRNA，另一个为血清处理的游离 siRNA
Prepare two samples of FRET ds-siRNA encapsulated with the tested carrier according to standard lab protocol. One sample will be untreated FRET-siRNA+carrier and the other serum-treated FRET-siRNA+carrier.
按照标准实验室协议，制备两个包裹测试载体的 FRET 双链 siRNA 样品。一个样品为未处理的 FRET-siRNA+载体，另一个为血清处理的 FRET-siRNA+载体
Add serum of interest to the samples to be treated at a chosen final percentage. Add the same volume of PBS to the untreated samples.
在需要处理的样品中加入所需血清至选定的最终浓度。在未处理的样品中加入相同体积的 PBS
Incubate all samples at 37 °C for a desired time (see Note 10).
在 37°C 孵育所有样品至所需时间（详见注释10）
Release the FRET-siRNA from the carrier by adding a releasing agent. For peptides and Lipofectamine 2000, dextran sulfate sodium is suggested to a final concentration of 10 mg/ml.
通过加入释放剂从载体中释放 FRET-siRNA。对于肽和 Lipofectamine 2000，建议使用最终浓度为 10 mg/ml 的葡聚糖硫酸钠
Perform electrophoresis, imaging, and analysis as described in Subheading 3.1 (see Fig. 5aand Note 11).
按照 3.1 节中描述的方法进行电泳、成像和分析（详见图 5a 和注释11）
For each sample, normalize the relative ratiometric FRET efficiency value to the value from the corresponding untreated sample.
对每个样品，将相对比率 FRET 效率值归一化到相应未处理样品的值
Graph the normalized relative ratiometric FRET efficiency to compare the stabilizing effect of the carrier to the free FRET-siRNA degradation (see Fig. 5b).
绘制归一化的相对比率 FRET 效率图，以比较载体对游离 FRET-siRNA 降解的稳定作用（详见图 5b）
Fig. 5 (a) Agarose gel electrophoresis of FRET-siRNA encapsulated with lipofectamine, TAT peptide, and SRCRP2-11R peptide and treated (+) or not (−) with human serum in PBS. Green and magenta represent FAM and TAMRA emission intensities, respectively. 包裹在脂质体、TAT 肽和 SRCRP2-11R 肽中的 FRET-siRNA 在 PBS 中经人血清处理（+）或未处理（−）的琼脂糖凝胶电泳图。绿色和品红色分别表示 FAM 和 TAMRA 的发射强度。(b) Graphs showing the FRET efficiency obtained by ImageJ analysis of images shown in a. Data are shown as averages of three biological replicates. Bars are standard deviations. Asterisks indicate the level of significance, based on the Student’s t-test图表显示了通过 ImageJ 分析a中图像获得的 FRET 效率。数据为三个生物重复实验的平均值，误差条表示标准偏差。星号表示基于 Student's t检验的显著性水平。 (*p = 0.0021–0.04332, * p* = 0.0002–0.0021, * p= 0.0001–0.0002, ** p < 0.0001)
As sense and antisense siRNA, we employed the following oligonucleotide sequences: FAM-5′-GGUCUAGCUACAGAGAAAU-3′-TAMRA (sense siRNA strand); FAM-5′-AUUUCUCUGUAGCUAGACC-3′-TAMRA (antisense siRNA strand). We suggest purifying the oligonucleotides by RNase-free HPLC.
作为正义链和反义链 siRNA，我们使用了以下寡核苷酸序列：FAM-5′-GGUCUAGCUACAGAGAAAU-3′-TAMRA（正义链 siRNA）；FAM-5′-AUUUCUCUGUAGCUAGACC-3′-TAMRA（反义链 siRNA）。我们建议通过无 RNase 的 HPLC 纯化这些寡核苷酸。
We suggest using a custom-made sample loading solution without dye since we observed that most dyes would run on top of the siRNA band, reducing the detection efficiency.
我们建议使用不含染料的定制样品加载液，因为我们发现大多数染料会在 siRNA 带的顶部运行，降低检测效率。
As stabilizing agents, we employed lipofectamine 2000 (Thermofisher), 0.5 mM TAT peptide (Genscript, GRKKRRQRRRPQ), and 1 mM SRCRP2-11R peptide (obtained by custom-made synthesis at 95% purity from Genscript, GRVRVLYRGSW).
作为稳定剂，我们使用了 Lipofectamine 2000（Thermofisher）、0.5 mM TAT 肽（Genscript, GRKKRRQRRRPQ）和 1 mM SRCRP2-11R 肽（由 Genscript 定制合成，纯度为 95%，GRVRVLYRGSW）。
We used Spectrum In Vivo Imaging System (IVIS, PerkinElmer).
我们使用了 Spectrum In Vivo 成像系统（IVIS, PerkinElmer）。
The gel should be prepared without addition of any nucleic acid stain (e.g., Ethidium Bromide, Gel Red). Indeed, the imaging of the siRNA on the gel will rely on the presence of the fluorophores. Please note that for small oligonucleotides, a high percentage agarose gel is usually suggested to obtain a good resolution of the ladder. In this experiment, the ladder is not needed and we recommend employing a low-resolution gel to reduce the agarose background.
制备凝胶时不应添加任何核酸染料（如溴化乙锭、Gel Red）。实际上，siRNA 在凝胶上的成像将依赖于荧光团的存在。请注意，对于小寡核苷酸，通常建议使用高浓度的琼脂糖凝胶以获得良好的梯形分辨率。在本实验中不需要梯形分辨率，我们建议使用低分辨率凝胶以减少琼脂糖背景。
Work in a RNase-free area and keep all tubes in ice. Please note that in Table 1samples are numbered after the loading order on the agarose gel.
在无 RNase 的区域工作，并将所有试管置于冰上。请注意，表 1 中的样品是按照在琼脂糖凝胶上的加载顺序编号的。
Exposure time should be optimized based on gel thickness and instrument features. We recommend testing different exposure times before saving a protocol in the machine.
曝光时间应根据凝胶厚度和仪器特性进行优化。我们建议在机器中保存协议之前测试不同的曝光时间。
To move the rectangular selection to the next lane, you can use the mouse or the arrows on the keyboard. Do not create a new rectangular selection as the selection must be exactly the same size for all lanes.
要将矩形选择移动到下一个泳道，可以使用鼠标或键盘上的箭头。不要创建新的矩形选择，因为选择必须对所有泳道完全相同的大小。
As visible in Fig. 2a, if the resolution of the gel is high, it is possible to observe a slight shift of the siRNA band in parallel with fluorescence color shift that is due to the siRNA degradation.
如图 2a 所示，如果凝胶的分辨率很高，可以观察到 siRNA 带的轻微移动，同时荧光颜色也会发生变化，这是由于 siRNA 降解所致。
We have encapsulated our FRET-siRNA with peptide SRCRP2-11R, peptide TAT, and lipofectamine 2000 (Thermofisher) in 1% human serum for 30 min.
我们在 1%人血清中将 FRET-siRNA 与肽 SRCRP2-11R、肽 TAT 和 Lipofectamine 2000（Thermofisher）包裹 30 分钟。
For this assay, we observed that using completely degraded FRET-siRNA as a positive control could result in an overexposed band from a strong fluorescence intensity of FAM, which could lead to an increased background in the close lanes. To avoid this, we suggest leaving one empty well between samples when loading the agarose gel.
在此实验中，我们观察到使用完全降解的 FRET-siRNA 作为阳性对照可能会导致 FAM 的强荧光强度产生过度曝光带，从而增加临近泳道的背景。为避免这种情况，我们建议在加载琼脂糖凝胶时在样品之间留一个空孔。
