Name: determining the thermodynamic and kinetic association of a dna aptamer and tetracycline using isothermal titration calorimetry
Uploaded: 2022-08-23T13:45:00
Description: Watch this Scientific Journal Video about Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry at JoVE.com

这项研究得到了Aptagen LLC的研发基金的支持。

注意： 图2 显示了ITC实验的主要步骤，用于确定DNA适配体和四环素的热力学和动力学关联。
1. 样品制备
注意：ITC样品需要在相同的缓冲液中制备适配体和配体，以避免混合样品池和注射器的不同缓冲液引起的热量释放。这通常是通过将所有材料透析到同一缓冲液中来实现的。缓冲液使用改编自3 kDa分子量截止（MWC）浓缩?...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+aptamers+specific+for+antibiotics+tetracyclines.">Single-stranded DNA aptamers specific for antibiotics tetracyclines.</a> Bioorganic and Medicinal Chemistry. 16 (15), 7245-7253 (2008).</li><li>Kim, Y. J., Kim, Y. S., Niazi, J. H., Gu, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+aptasensor+for+tetracycline+detection.">Electrochemical aptasensor for tetracycline detection.</a> Bioprocess and Biosystems Engineering. 33 (1), 31-37 (2010).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+indirect+competitive+assay-based+aptasensor+for+highly+sensitive+detection+of+tetracycline+residue+in+honey.">Development of an indirect competitive assay-based aptasensor for highly sensitive detection of tetracycline residue in honey.</a> Biosensors &amp; Bioelectronics. 57, 192-198 (2014).</li><li>Kim, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+colorimetric+aptasensor+using+gold+nanoparticle+for+a+highly+sensitive+and+specific+detection+of+oxytetracycline.">A novel colorimetric aptasensor using gold nanoparticle for a highly sensitive and specific detection of oxytetracycline.</a> Biosensors &amp; Bioelectronics. 26 (4), 1644-1649 (2010).</li><li>Thoa, T. T., Minagawa, N., Aigaki, T., Ito, Y., Uzawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+photosensitisation+processes+by+an+RNA+aptamer.">Regulation of photosensitisation processes by an RNA aptamer.</a> Scientific Reports. 7, 43272 (2017).</li><li>Horowitz, E. D., Lilavivat, S., Holladay, B. W., Germann, M. W., Hud, N. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt-mediated+two-site+ligand+binding+by+the+cocaine-binding+aptamer.">Salt-mediated two-site ligand binding by the cocaine-binding aptamer.</a> Nucleic Acids Research. 45 (3), 1041-1048 (2017).</li><li>Turnbull, W. B., Daranas, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+value+of+c:+Can+low+affinity+systems+be+studied+by+isothermal+titration+calorimetry.">On the value of c: Can low affinity systems be studied by isothermal titration calorimetry.</a> Journal of the American Chemical Society. 125 (48), 14859-14866 (2003).</li><li>Van Ness, J., Van Ness, L. K., Galas, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+reactions+for+the+amplification+of+oligonucleotides.">Isothermal reactions for the amplification of oligonucleotides.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (8), 4504-4509 (2003).</li></ol>

这里介绍的方法根据TA Instruments的说明进行了修改，足以确定我们中心许多选定的核酸适配体和靶标的结合亲和力和热力学。该过程的关键步骤包括交换缓冲液以获得与配体匹配的靶标，使用适当的参数运行样品，以及找到合适的结合拟合模型来分析数据。连续记录热释放需要消除所有噪声热量，例如缓冲液不匹配、细胞和注射器脏污以及样品内部气泡。在缓冲液交换步骤中，最好使用透析膜或?...

核酸适配体是通过进化过程选择的ssDNA或RNA片段，与目标靶标1，2具有高结合亲和力和特异性，可作为高级识别元件或化学抗体3，4，5。因此，核酸适配体与其靶标的结合亲和力和特异性在适配体的选择和应用中起着至关重要的作用，等温滴定量热法（ITC）已被广泛用于这些表征目的。许多方法已被用于确定核酸适配体的亲和力，包括ITC、表面等离子体共振（SPR）、比色滴定、微量热泳（MST）和生物层干涉测量（BLI）。其中，ITC是确定两种分子在溶液相中的热力学和动力学结合的最新技术之一。该方法使用无标记分子进行连续滴定，并在每次滴定产生的结合事件6，7上记录随时间释放的热量。与其他方法不同，ITC可以提供结合亲和力，多个结合位点以及热力学和动力学结合（图1A）。根据这些初始参数，吉布斯自由能变化和熵变化使用以下关系确定：
ΔG = ΔH-TΔS
这意味着ITC提供了分子相互作用的完整热力学曲线，以阐明结合机制（图1B）。由于核酸适配体和靶标之间的尺寸大不相同，因此很难确定小分子与适配体的结合亲和力。同时，ITC可以在不标记和固定分子的情况下提供灵敏的测量，这提供了一种在测量过程中保持核酸适配体和靶标自然结构的方法。通过上述属性，ITC可用作表征适配体和小靶点之间结合的标准方法。
经过Gu组的选择，该适配体与不同的平台集成，包括基于电化学适配体的生物传感器、竞争性酶联适配体测定和微量滴定板，可实现四环素8，9，10的高通量检测。然而，其结合特性尚未得到充分阐明，无法选择合适的平台8;值得使用ITC表征适配体与四环素的结合。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5&#39;-CGTACGGAATTCG CTAGCCCCCCGGCAGGCCACGG 
			C TTGGGTTGGTCCCACTGCGCG 
			TGGATCCGAGCTCCAC GTG-3&#39;</td>
			<td>Integrated DNA Technologies, Inc</td>
			<td></td>
			<td>The sequence is adopted from Gu&#39;s research, which has not identified Kd using ITC (refer references 8 and 9)</td>
		</tr>
		<tr>
			<td>Affinity ITC Auto Low Volume (190 &#181;L) System Complete&#8211;Gold Cells</td>
			<td>TA Instruments</td>
			<td>61000.901</td>
			<td>Isothermal titration calorimetry system</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Avantor (VWR)</td>
			<td>E506-100ML</td>
			<td>Calcium chloride 1 M in aqueous solution, Biotechnology Grade, sterile</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>The Eppendorf 5417R is unsurpassed in safety, reliability and ease-of-use. Very easy to maintain with a brushless motor that spins up to 16,400 RPM with maximum RCF up to 25,000 x g.</td>
		</tr>
		<tr>
			<td>Complete Degassing Station (110/230V)</td>
			<td>TA Instruments</td>
			<td>6326</td>
			<td>This degasser provides a self-contained stirring platform, vacuum chamber, vacuum port, temperature control and electronic timer for proper sample preparation.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>TekNova</td>
			<td>E0375</td>
			<td>EDTA 500 mM, pH 7.5</td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>ND-ONE-W</td>
			<td>UV-Vis Spectrophotometer</td>
		</tr>
		<tr>
			<td>Nanosep, Nanosep MF and NAB Centrifugal Devices</td>
			<td>Pall Laboratory</td>
			<td>OD030C34</td>
			<td>3 kDa molecular weight cutoff concentrator</td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>IBI Scientific</td>
			<td>IB70165</td>
			<td>Buffer containing Sodium phosphate, Sodium chloride, Potassium phosphate, and Potassium chloride Ultra-Pure Grade Sterile filtered using 0.2 &#181;m filter. Autoclaved at 121 &#176;C for greater than 20 min.</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Large Cap Microcentrifuge Tubes</td>
			<td>labForce (a Thomas Scientific Brand)</td>
			<td>1149K01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline, Hydrochoride</td>
			<td>EMD Millipore Corperation</td>
			<td>CAS64-75-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the thermodynamic and kinetic association of a dna aptamer and tetracycline using isothermal titration calorimetry

确定适配体与其靶标之间的结合亲和力和行为是选择和使用适配体进行应用的最关键步骤。由于核酸适配体和小分子之间存在巨大差异，科学家们需要付出很多努力来表征它们的结合特性。等温滴定量热法（ITC）是实现此目的的有效方法。ITC不仅可以确定解离常数（Kd），还可以提供溶液相中两个分子之间相互作用的焓变化和结合化学计量。该方法使用无标记分子进行连续滴定，并在每次滴定产生的结合事件上记录随时间释放的热量，因此该过程可以灵敏地测量大分子与其小靶标之间的结合。本文介绍了使用小靶标四环素对选定的核酸适配体进行ITC测量的分步程序。这个例子证明了该技术的多功能性及其在其他应用中的潜力。

ITC提供了精确的解离常数（Kd），结合化学计量和双分子相互作用的热力学参数6。在该示例中，Kim等人选择的适配体9，11与四环素结合，结合亲和力为Kd 1 = 13 μM，Kd 2 = 53 nM。有趣的是，该结合是使用平衡过滤方法测定的，报告的Kd为63.3 nM，这与有利的结合位点（位点2）没有?...

Watch this Scientific Journal Video about Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry at JoVE.com

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

本协议描述了使用等温滴定量热法（ITC）来分析DNA适配体和四环素之间结合的关联和解离动力学，包括样品制备，运行标准品和样品，以及解释结果数据。

使用等温滴定量热法测定DNA适配体和四环素的热力学和动力学关联

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

该协议允许研究人员确定适配体对溶液中游离靶标的结合亲和力，同时提供对结合机制的见解。由于ITC测量由于结合引起的热量变化，因此不需要像SPR等技术那样标记或功能化适配体或功能化。新用户应密切注意所有绑定组件中的匹配缓冲区。此外，请注意，该技术具有很高的样品要求，对于较大的配体（如蛋白质）可能难以满足。首先，激活透析柱的膜，填充PBS缓冲液，在室温下平衡10分钟，并以5， 000倍G离心15分钟。取出缓冲液并将500微升适配体样品加载到色谱柱中。以5， 000倍G离心，重复四次以将原始缓冲液换成PBS。使用移液管收集透析的DNA适配体并将其转移到新的1.5毫升管中。收集最后的流通缓冲液以溶解四环素。确保注射器中实验的缓冲液与参比池中的缓冲液匹配。使用紫外可见光谱仪再次测定适配体浓度。使用最后的交换缓冲液将浓度调节至40微摩尔四环素和2微摩尔适配体。折叠DNA适配体，在90摄氏度下加热10分钟，在4摄氏度下冷却10分钟，然后回到室温20分钟。使用脱气站或真空泵对折叠的适配体和透析的四环素进行脱气25分钟，以消除溶解的气体。清理并确认机器的清洁度后，使用默认程序并按照制造商的说明使用包含 EDTA 和氯化钙的标准套件测试机器的准确性。设置运行参数。使用正在运行的程序计算器检查所需的卷。使用此运行参数，使用 ITC 在 ITC 注射器中使用 230 μL 40 μmol 四环素和在 ITC 样品池中使用 485 μL 两微摩尔适配体进行 ITC 测量。使用移液器将透析的四环素注射器板和折叠的适配体装入样品池中，避免气泡。通过单击软件上的“开始”按钮开始运行ITC仪器。双击打开数据分析软件，开始分析数据。打开保存的原始数据的路径以了解绑定的趋势。打开“建模”选项卡，并使用不同的绑定模型来查找最适合数据曲线的模型。然后，软件自动计算ITC热图和各种热力学参数，包括焓、熵、自由能、平衡结合常数和化学计量。收集根据数据和拟合模型信息确定的热力学参数。创建一个报告，包括ITC热图和各种热力学参数的图片。Kim等人选择的适配体与四环素结合，结合亲和力为解离常数1等于13微摩尔，解离常数2等于53纳摩尔。ITC的拟合模型和化学计量反映了适配体通过与序贯结合模型的2：1结合比与四环素结合。ITC测量确定的位点2的热力学参数表明，焓克服了相对显着的熵损失，驱动了强结合。焓驱动的与熵损失的结合与核酸构象变化有关，这已被报道为RNA与小分子之间的结合行为。核酸适配体和配体必须在同一缓冲液中。如果通过复叠开发，则应重新折叠适配体，并且必须对样品进行脱气。解决这些考虑因素将确保只有适体-配体相互作用对测量信号有贡献。由于适配体靶标的大小，适当的后续方法可能是微尺度热泳，以确认测得的溶液中游离结合亲和力。

Research

JoVE Journal

Biochemistry

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

科研

生物化学

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential Scanning Calorimetry ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

用限制性酶来测定染有菁染料的 DNA

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...