Name: determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers
Uploaded: 2022-08-09T14:30:00
Description: Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

这项工作得到了MCH的以下资助：NSF-BSF 1815508和NIH R01 GM124589。MRM得到了NIH T32 GM122740培训资助的部分支持。

1. 体外 动力学实验
<ol><li>通过PCR制备DNA模板<ol><li>设置PCR反应：要制备PCR反应，请将以下试剂合并到薄壁PCR管中： 33 μL 双蒸水 （ddH2O） 10 μL 5x 缓冲液，用于高保真 DNA 聚合酶 5 μL 脱氧核糖核苷三磷酸 （dNTP） 各 2 mM 0.5 μL 40 μM 正向引物 0.5 μL 40 μM 反向引物 0.5 μL （10-100 ng） DNA 模板（仅适用于菠菜2 PCR;西兰花底漆重叠） 0.5 μL 高保真 DNA 聚合酶（最?...

<ol>
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对于体外动力学实验，可以修改相同的通用方案以测量含有配体结合域和荧光团结合结构域的基于RNA的荧光生物传感器的体外动力学8。在这种情况下，RNA应在注射配体后进行测量之前与荧光团一起孵育，以获得配体反应动力学。如果在重复之间观察到高变异性，则可以通过在测量前检查每个样品是否允许在96孔板中平衡相同的时间来排除故障。每个样品或重复样品?...

荧光反应是产生荧光信号的化学反应。荧光RNA适配体通常通过结合小分子染料来提高其荧光量子产率来执行此功能（图1A）1。已经开发了不同的荧光RNA适配体系统，由特定的RNA适配体序列和相应的染料配体1组成。荧光RNA适配体已作为荧光标签附加到RNA转录本中，可实现mRNA和非编码RNA2，3，4的活细胞成像。它们也被放置在启动子序列之后作为基因表达的荧光报告基因，类似于使用绿色荧光蛋白（GFP）作为报告基因，只是报告功能在RNA水平5，6。最后，荧光RNA适配体已被掺入基于RNA的荧光生物传感器中，其设计用于响应特定的小分子触发荧光反应。基于RNA的荧光生物传感器已被开发用于各种非荧光代谢物和信号分子的活细胞成像7，8，9，10，11。
人们越来越关注荧光RNA适配体的开发，以可视化RNA定位，基因表达和小分子信号的动态变化。对于这些应用中的每一个，都需要获得实时测量，但测量的准确性取决于荧光反应的动力学是否快于采样频率。在这里，我们描述了使用配备样品进样器的酶标仪确定荧光RNA适配体Spinach2 12和Broccoli13的体外动力学的方法，并使用流式细胞仪确定在大肠杆菌中表达的Spinach2的细胞开启动力学的方法。之所以选择这两种RNA适配体，是因为它们已被应用于研究RNA定位2，3，4，它们已被用于报告5，6和生物传感器7，8，9，10，11，并且相应的染料配体（DFHBI或DFHBI-1T）已上市。表14，13，14给出了文献中确定的它们的体外特性的摘要，这为方案开发提供了信息（例如，使用的波长和染料浓度）。这些结果表明，受RNA适配体影响的荧光反应是快速的，不应妨碍所需细胞生物学应用的准确测量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP160500</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis equipment</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1A-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha D-(+)-lactose monohydrate</td>
			<td>Thermo Fischer Scientific</td>
			<td>18-600-440</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber 1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fischer Scientific</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate ((NH4)2SO4)</td>
			<td>Sigma-Aldrich</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow cytometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24861</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune 1x Focusing Fluid</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24904</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Shutdown Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24975</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Performance Tracking Beads</td>
			<td>Thermo Fischer Scientific</td>
			<td>4449754</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Wash Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>&#160;J24974</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>C3416</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorine Bleach</td>
			<td>Amazon</td>
			<td>B07J6FJR8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar 96-well plate</td>
			<td>Daigger Scientific</td>
			<td>EF86610A</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Tubes, 12 mm x 75 mm, 5 mL with attached dual position cap</td>
			<td>Globe Scientific</td>
			<td>05-402-31</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI</td>
			<td>Sigma-Aldrich</td>
			<td>SML1627</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI-1T</td>
			<td>Sigma-Aldrich</td>
			<td>SML2697</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose (anhydrous)</td>
			<td>Acros Organics</td>
			<td>AC410955000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA loading dye</td>
			<td>New England Biolabs</td>
			<td>B7025S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes (2.0 mL)</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs: dATP, dCTP, dGTP, dTTP</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, pH 8.0</td>
			<td>Gibco, Life Technologies</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter-tip micropipettor tips</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM12635, AM12648, AM12655, AM12665</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>FlowJo v10 Software</td>
		</tr>
		<tr>
			<td>Fluorescent plate reader with heating control</td>
			<td>VWR</td>
			<td>10014-924</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electrophoresis power supply</td>
			<td>Thermo Fischer Scientific</td>
			<td>EC3000XL2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen AM95010</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM95010</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Analysis software from Academic Group License&#160;</td>
		</tr>
		<tr>
			<td>Heat block&#160;</td>
			<td>Thomas Scientific</td>
			<td>1159Z11</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Inorganic pyrophosphatase</td>
			<td>Sigma-Aldrich</td>
			<td>I1643-500UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Molecular Weight DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N3233L</td>
			<td>Supplied with free vial of Gel Loading Dye, Purple (6x), no SDS (NEB #B7025).</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Fisher Scientific</td>
			<td>MFCD00011110</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge with temperature control</td>
			<td>Marshall Scientific</td>
			<td>EP-5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettors</td>
			<td>Gilson</td>
			<td>FA10001M, FA10003M, FA10005M, FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor tips</td>
			<td>Sigma-Aldrich</td>
			<td>Z369004, AXYT200CR, AXYT1000CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore water filter with BioPak unit</td>
			<td>Sigma-Aldrich</td>
			<td>CDUFBI001, ZRQSVR3WW</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow micropipettor pipette tips</td>
			<td>DOT Scientific</td>
			<td>RN005R-LRS</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP39920</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR clean-up kit</td>
			<td>Qiagen</td>
			<td>28181</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers and templates</td>
			<td>Integrated DNA technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler for thin-walled PCR tubes</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler for 0.5 mL tubes</td>
			<td>Techne</td>
			<td>5PRIME/C</td>
			<td></td>
		</tr>
		<tr>
			<td>pET31b-T7-Spinach2 Plasmid</td>
			<td>Addgene</td>
			<td>Plasmid #79783</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase&#160;</td>
			<td>New England Biolabs</td>
			<td>M0530L</td>
			<td>Purchase of Phusion High-Fideldity Enzyme is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, and MgCl2 and DMSO solutions.</td>
		</tr>
		<tr>
			<td>Polyacrylamide gel electrophoresis gel comb, C.B.S. Scientific</td>
			<td>C.B.S. Scientific</td>
			<td>VGC-1508</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide gel electrophoresis equipment</td>
			<td>C.B.S. Scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Genesee Scientific</td>
			<td>38-101</td>
			<td></td>
		</tr>
		<tr>
			<td>rNTPs: ATP, CTP, GTP, UTP</td>
			<td>New England Biolabs</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Short wave UV light source</td>
			<td>Thermo Fischer Scientific</td>
			<td>11758221</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic, anhydrous</td>
			<td>Thermo Fischer Scientific</td>
			<td>S375-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>SoftMax Pro 6.5.1 (platereader software) obtained through Academic Group License</td>
		</tr>
		<tr>
			<td>Sterile filter units</td>
			<td>Thermo Fischer Scientific</td>
			<td>09-741-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>Thermo Fischer Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer for agarose gel electrophoresis</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9869</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>TRIS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone (granulated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea-PAGE Gel system&#160;</td>
			<td>National Diagnostics</td>
			<td>EC-833</td>
			<td></td>
		</tr>
		<tr>
			<td>UV fluorescent TLC plate</td>
			<td>Sigma-Aldrich</td>
			<td>1.05789.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Vis spectrophotometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>ND-8000-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Thermo Fischer Scientific</td>
			<td>2215415</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Sigma-Aldrich</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract (Granulated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP9727-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers

荧光RNA适配体已被应用于活细胞中，以标记和可视化RNA，报告基因表达，并激活荧光生物传感器，以检测代谢物和信号分子的水平。为了研究每个系统中的动态变化，需要获得实时测量，但测量的准确性取决于荧光反应的动力学比采样频率快。在这里，我们描述了分别使用配备样品进样器和流式细胞仪的酶标仪来确定荧光RNA适配体的体外和细胞开启动力学的方法。我们表明，Spinach2和西兰花适配体荧光活化的体外动力学可以建模为两相反合反应，并且具有不同的快速相速率常数0.56 s-1和0.35 s-1。此外，我们表明，大肠杆菌中Spinach2荧光激活的细胞动力学仍然足够快，可以在分钟时间尺度上实现准确的采样频率。这些分析荧光活化动力学的方法适用于已开发的其他荧光RNA适配体。

体外 动力学 作为合成寡核苷酸购买的DNA模板和引物的序列如 表2所示， 试剂配方如 补充文件1所示。PCR扩增用于扩大带有T7启动子的DNA模板的量，这是随后的 体外 转录（IVT）反应所必需的。此外，PCR扩增可用于同一反应中的两个目的：通过引物延伸生成全长西兰花DNA模板，以及放大DNA模板。
在IVT反应合成RNA?...

Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

该协议提供了两种确定荧光RNA适配体菠菜2和西兰花动力学的方法。第一种方法描述了如何使用酶标仪 在体外 测量荧光适配体动力学，而第二种方法详细介绍了通过流式细胞术测量细胞中荧光适配体动力学。

荧光RNA适配体 外和细胞 开启动力学的测定

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

研究体外和细胞荧光适配体动力学使用户能够评估适配体染料相互作用是否足够快，可以实时研究感兴趣的过程。所介绍的技术允许在细胞内部和外部进行定量动力学测量，根据适配体的应用，其中任何一种都可能相关。所介绍的方法可应用于任何荧光RNA系统，包括替代适配体和基于RNA的小分子生物传感器。首先，从热循环仪设置RNA复性程序。通过选择创建新程序创建程序“添加新阶段”并添加新步骤“多次添加特定于温度的步骤，然后按保存”根据程序调整其他设置。要使菠菜二和西兰花RNA变性，请在0.5毫升薄壁PCR管中的双蒸水中制备每种RNA的两微摩尔茎。然后加入等体积的两倍复性缓冲液，制成一微摩尔RNA溶液。将试管放入热循环仪中。打开保存的复性程序并按运行“接下来，设置酶标仪进样器程序。在荧光酶标仪上，选择温度“并将其设置为37摄氏度。在开始动力学之前，请确保温度已平衡到此值。打开酶标仪软件。选择设置“然后采集视图”要输入动力学测量程序，请选择循环“对于每个孔。接下来，将基线设置设置为 60 个基线读数，并将智能注射设置设置为 10 微升注射。将荧光读数激发波长设置为 448 纳米，将发射波长设置为 506 纳米，将墨盒设置为单声道。将总读取时间设置为 10 分钟，读取间隔为 0.5 秒。将 PMT 和光学元件设置为每次读取六次闪光。然后输入循环到下一个井。要准备酶标器进样器，请在读板器上选择进样器“，并根据指示提供废物收集板。然后选择“洗涤”并按照酶标仪上的说明用一毫升体积的双蒸水，75%乙醇和蒸馏水清洁注射管。接下来选择吸气“允许注射器喷射多余的液体。然后选择prime“用两个260微升的100微摩尔D F H B灌注进样器 I.To 进行一次动力学实验，将80微升结合缓冲液预混液加入到96孔透明底板的一个孔中。然后加入10微升变性RNA。让板和进样器中的 D F H B I 溶液平衡至 37 摄氏度 15 分钟。接下来，在读取区域下的软件设置中选择要分析的孔。然后在主页选项卡下，选择运行“以执行前面描述的动力学程序。重复此过程，直到所有实验完成。运行后，如前所述，清洗进样器以从注射管中取出剩余的D F H B I溶液。要设置实验文件，请打开流式细胞仪和计算机。在实验资源管理器选项卡中创建一个新文件，方法是右键单击流式细胞仪“用户名。然后选择“新建实验”在下拉窗口中，当计算机屏幕上弹出一个新窗口时，选择实验类型为试管“然后选择确定”在新实验文件中右键单击组文件夹并选择添加新样品管“标记每个特定时间点的样品管并通过右键单击样品进行复制”并选择重命名“在下拉菜单中。接下来，通过在培养管中的AI培养基中加入1.5毫升1 X P B S和3微升诱导细胞来制备稀释的细胞溶液。在添加染料之前，测量细胞的背景荧光，以观察折叠随时间推移而打开。加入染料后，将含有P B S细胞的培养管放入样品管升降器中，并将升降器抬高到样品注射针上。然后在收集面板选项卡中选择适当的样品文件并单击记录“运行完成后，用手用培养管降低样品管升降机。启动冲洗步骤，冲洗流体系统，并最大限度地减少每个生物重复样品之间的残留。要移动到以下样品，请单击记录图标下样品管名称附近的右箭头图标，选择下一个示例文件。要用染料测量细胞的荧光，将1.4微升浓缩染料原液加入一个带有细胞的X P B S中，得到终浓度为50微摩尔D F H B I ONE T.固定培养管盖并倒置三至五次以使溶液均匀混合。接下来，取下盖子并将培养管放入样品管升降器中。将支架抬高到样品进样针头后，单击正确样品文件下的记录“图标。然后，通过按开始开始定时器“荧光适配体菠菜二和西兰花的体外动力学，显示用于结合D F H B I染料的两相缔合动力学。对于这两种核酸适配体，在较长的测量时间内，两相关联比单相结合的动力学数据拟合得更好。最佳拟合曲线决定了快速和慢速关联的速率常数和半衰期值。菠菜二处于结合能力状态，显示出比西兰花更快的开启。两种核酸适配体的第二相动力学相似，可能对应于一个共同的限速步骤。核酸适配体动力学曲线由相似程度的快速结合RNA组成。对于表达菠菜两个T RNA的细胞，平均荧光强度或M F I在零时间点立即增加。此外，细胞荧光在五分钟内达到其最大平衡M F I值。相比之下，对照细胞显示出低背景荧光，并且在D M S O添加的情况下M F I值没有变化。 所提出的方法使研究人员能够确定如何更快地在荧光适配体中制造基于RNA的传感器。

Research

JoVE Journal

Biochemistry

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

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科研

生物化学

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

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Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

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In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

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体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

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利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

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Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

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体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

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Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

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荧光RNA适配体外和细胞开启动力学的测定