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1.适体工作股票的制备<ol><li>遵循制造商的指示和（从基准18 5-的Cy5- CCTG GGGGAGTATTGCGGAGGAAGG-3，序列）溶于水的寡核苷酸，达到100μM的最终浓度。 </li><li>通过稀释寡库存至200nM用结合缓冲液制备的适体的工作溶液（20毫摩尔Tris，pH值7.6; 300毫摩尔NaCl; 5mM的MgCl 2的0.01％吐温20）。 </li><li>孵育2分钟，混合物在90℃，让样品立即冷却在冰上，并在室温下使用的样本。 </li></ol> 2.配体稀释系列的研制<ol><li>对于各配体（三磷酸腺苷（ATP），腺苷二磷酸（ADP），腺苷一磷酸（AMP），腺嘌呤，鸟苷三磷酸（GTP），三磷酸胞苷（CTP），脱氧腺苷三磷酸（的dATP），和S-腺苷甲硫氨酸（SAM） ;每个10毫米股票），准备一个16级串行diluti关于在200μl微反应管。 注:5分钟在14000 XG可能有助于除去聚集配股票的离心。低量，推荐使用低结合反应管，以避免分子吸附到所述管的壁。 </li><li>开始的比估计的亲和力高至少50倍的最大浓度和50％在各稀释步骤减少配体的浓度。 注:在控制软件实施的集中查找工具结合模拟数据，并寻找合适的浓度范围稀释系列帮助。 </li><li>填写20μl，以管1.配股票（10毫米）的加10μl适体结合缓冲液到微反应管2至16。 </li><li>转移10微升管1的向管2及通过上下吹打几次适当地混合。转移10微升到下管，并重复此稀释剩余的管子。 </li><li>丢弃10微升过剩从上管。避免以下buffer稀释效应。在管1和管2-16缓冲区必须相同。 </li></ol> 3.最终反应预混液的制备<ol><li>制备具有20微升体积的各个结合反应（10微升适体工作溶液+10微升的各配位体稀释的），以最小化移液误差。只有4微升的体积是苏夫网络cient到FI LL毛细管。 </li><li>添加10微升200nM的适体工作溶液到10微升各配体稀释，并通过上下抽吸数次适当地混合。 </li><li>通过浸渍毛细血管进入样品孵育在室温和音响LL样品为标准毛细管5分钟的样品。潜伏期较长时间可能对一些互动是必要的;然而，5分钟是足够的大多数。触摸仅在侧面的毛细管，而不是在中间的部分，其中该光学测量将采取。 </li><li>放置到毛细血管第Ë毛细盘和启动MST设备。 </li></ol> 4.启动MST设备注:设备提供了两个预安装的软件包，在&#39;控制"软件的实验条件和技术设置&#39;分析"，为产生的数据解释软件。 <ol><li>将毛细管盘成MST设备之前，开始控制软件，并调整通过选择&#39;&#39;"温度控制下拉菜单中的"启用手动温度控制"的总体要求的温度。调整温度至25℃以这种方式。 注:MST仪器就能温度控制在22至45℃。 </li><li>等待温度达到预期的水平，然后将毛细管盘成MST设备。 </li><li>设置LED通道到&#39;红色"对的Cy5染料和调节LED的功率以获得一个FL uorescenc在一个标准的传感器MST设备300到1000荧光单位E信号。 25％的LED功率在该研究中使用。 注:6000至18000荧光单元被推荐用于MST具有高灵敏度的传感器。 </li></ol> 5.毛细管扫描<ol><li>开展毛细管扫描选择了"控制"软件毛细血管位置，点击检查样品的不同质量等方面"开始扫描帽" 出发前的MST测量。 </li><li>检查是否有荧光增强/淬火坚持效果（U形或扁平峰）在软件毛细管扫描。 注:更多关于检测和荧光的处理和贴效果的详细信息可以在讨论中被发现。 </li></ol> 6. MST测量注:在开始测量MST之前，确保排除粘附作用，增强/淬火效果，或移液误差，并确保在毛细管扫描表明FL荧光信号是萨夫音响充分的。有关详细信息，请参阅讨论。 <ol><li>分配从稀释系列中的&#39;控制"的软件的各毛细管位置的配位体浓度考虑混合的适体和配体的稀释步骤。（1:1）。 </li><li>进入配体毛细管＃1的最高浓度（5毫米），选择正确的稀释型（在这里，1:1），点击最高浓度，使用拖动功能来自动分配的毛细血管其余浓度＃2 16。的最低浓度是152.6纳米。 </li><li>输入在控制软件的各个部分中的FL uorescent适体（这里，100纳米）的浓度。 </li><li>使用默认设置，其检测所述FL荧光持续5秒，记录台MST为30秒，并且激光的t灭活后记录再5秒的荧光<html> O监控分子的反扩散。 </li><li>调整激光功率，以20％的在控制软件的各部分。 注意:为了得到最好的信噪比，并避免非特异性的效果，推荐的20-40％的激光功率。在特定情况下，可能需要更高的激光功率，以便获得未结合的和结合的分子的良好分离。 </li><li>保存实验中选择目标文件夹后按&#39;开始MST测量"按钮开始测量MST。 注:.ntp文件将在目标文件夹中生成。使用这种设置，一个测量持续10-15分钟。 </li><li>重复实验过程至少两次以便更准确地确定EC 50值。 注意:为了测试技术再现性，相同的毛细管可以扫描多次（技术性重复）。 </li></ol> 7. MST数据分析核苷酸"&gt;注:分析软件能够在FL Y数据的分析测定时的分析软件绘制相对于配体浓度37 MST时间迹线和在规范化FL荧光（F 范数 ）的变化。 <ol><li>启动MST分析软件（MO.Affinity分析）和加载从目标文件夹中.ntp文件。选择"MST"作为在数据选择菜单中的分析类型。 注:在配体依赖性荧光效应的情况下，最初的荧光可以被选择用于分析。 </li><li>通过拖放和下降，或者按相应的实验运行下面的"+"按钮，各自的技术或生物的run（S）添加到一个新的分析。 </li><li>按各自的实验运行下面的信息按钮以获得关于该实验中，MST痕迹，毛细管扫描，毛细管形状，初始荧光，和漂白率的属性信息。 注意:这些原始数据，可以人因此在分析的后续步骤进行检查。 </li><li>目视检查MST痕迹的聚集和沉淀的效果，可见的颠簸和尖峰。 注:有关检测和聚集效应处理的详细信息，请阅读讨论。 </li><li>目视检查毛细管扫描和毛细管状覆盖吸附效果，扁平形或U形的山峰可见。目视检查毛细管扫描和荧光效应的初始荧光。目视检查漂白效果的漂白率。 </li><li>切换到剂量反应模式，按下相应的按钮改变分析设置为"专家"模式。选择"T-JUMP"作为MST评估策略。 </li><li>选择曲线拟合的"山"的模式。结合参数将自动进行计算。通过选择在"比较结果"菜单的各类型正常化的正常化的数据。导出数据无论是作为一个.xls或.pdf格式。 注:绑定图表下表总结了计算的结合参数。 </li></ol>

CE和TS是2bind有限公司，提供生物物理分析服务的员工。此视频文章出版费用由2bind有限公司支付。

质量控制: 非特异性粘着/样品材料的表面，以及聚集效应的吸附，对亲和数据的质量有巨大影响。但是，只有少数国家的最先进的技术提供准确，快速的选择，监督和避免这些影响。台MST提供了检测和帮助克服这些问题，从而允许技术设置的逐步优化集成质量控制。上粘附和荧光效果的重要信息可以从毛细管扫描和毛细管形状（步骤5.2和7.5）被提取，而聚集/沉淀的效果（步骤7....

分子之间的相互作用是自然的基础。因此，科学家基础和应用研究的众多领域尝试了解不同种类的分子相互作用的基本原则。 MicroScale程序热泳（MST），科学家可以在溶液中进行快速，精确，成本效益和质量控制的分子间相互作用的特征，与缓冲区的自由选择。目前已经有超过1000出版物使用MST，2016年独自一人，描述不同类型的分析，包括图书馆放映，结合事件验证，竞争分析，和实验与多个合作伙伴的结合1-8。在一般情况下，台MST允许古典结合参数，如任何类型的分子相互作用的结合亲和力（1pM至毫米），化学计量，和热力学，研究。 MST的一大优点是研究独立的互动合作伙伴的大小结合事件的能力。即使CHAL目标小核酸适体之间有挑战性相互作用（15-30个核苷酸）和诸如小分子，药物，抗生素，或代谢产物可以定量。 当前国家的最先进的技术来表征适体靶相互作用或者是实验室强烈和高度复杂的或无法量化适配子的小分子的相互作用9,10。表面等离子体共振（SPR）为基础的试验11,12和真正的无标记热量的方法，如温滴定量热（ITC）13-15度洗脱16，平衡渗透17,18，在线探测19，凝胶-移位测定，stopped-溢流FL荧光光谱20,21，荧光各向异性（FA）22,23，单分子FL荧光成像24,25，和生物层干涉测量（BLI）26也无论是不精确或适体-小分子不相容互动。其他principa这些方法升问题是灵敏度低，高样品消耗，固定化，在表面上的质量传递的限制，和/或缓冲的限制。只有少数这些技术提供了聚集和吸附作用的综合控制。 台MST表示一个强大的工具为科学家克服这种限制，研究其它靶的适体和小分子27-29之间的相互作用，以及如蛋白质30-33。该技术依赖于分子通过温度梯度的运动。这种定向运动，被称为"热泳，"取决于大小，电荷，并且该分子34,35的水化外壳。配体分子的结合将直接改变这些参数中的至少一个，从而产生改变热泳迁移率。小尺寸的配体可能没有从绑定尺寸的变化对束缚态方面相当大的影响，但可以拥有博士<html>上水合壳和/或电荷amatic影响。在分子与结合配偶体相互作用后的热泳运动的变化使得基本结合参数2,7,34,36,37的量化。 如在图1A中所描绘的MST装置由红外激光聚焦到使用相同的光学元件作为用于荧光检测的玻璃毛细管内的试样。而激光建立温度梯度（2-6℃的ΔT）可监控的蛋白质经由色氨酸6或荧光标记的相互作用配偶3,8的固有FL荧光的热泳的运动。在空间，ΔT，所产生的温度差会导致在升高的温度下的区域耗尽或分子的积累，这可通过索瑞量化系数音响cient（S T）的 : G"/&gt; C 热表示在被加热区域的浓度，和c 冷是在初始冷区域中的浓度。 如图1B所示 ，一个典型的MST试验结果在MST运动轮廓（时间跟踪），由不同的相位，这可以通过它们各自的时间尺度来分离的。初始荧光在第一5秒中不存在温度梯度的测量来定义的精确开始荧光和检查漂白或p​​hotoenhancement。温度跳跃（T-跳转）表示相，其中热泳动前的荧光变化。荧光这个初始下降取决于uorophore量子产率FL的热依赖性变化。热泳相位如下，其中达到荧光减少（或增加），由于分子直至稳态分布的热泳运动。如在图1B中所示的激光被关闭后，可以观察到的反向TJump和FL uorescent分子伴随背面扩散。为了获得基本的绑定参数，相互作用伙伴不同的摩尔比进行了分析和比较。通常情况下，16个不同的比例进行了研究一个MST实验，而光可见分子被保持恒定，并且与未标记的配体的增加量供给。两个结合配偶之间的相互作用诱导的热泳的变化，并因此在规范化FL荧光，女范数 ，这是因为以下计算: <img alt="方程" src="/files/ftp_upload/55070/55070eq2.jpg" /> ˚F 热和F 冷代表在MST痕迹德科幻奈德时间点的平均荧光FL强度。结合亲和力（K D或EC 50值）可以通过CURV计算易连接（ 图1C）。 总体来看，MST是一个强大的工具来研究任何种类的分子间的相互作用。这份手稿提供了一个协议来表征小分子三磷酸腺苷（ATP 0.5 kDa的）之间的相互作用充满挑战和25-nt的单链DNA短适体DH25.42（7.9 kDa的）。在手稿的过程中，在ATP分子的适体的结合位点被映射下来到腺嘌呤基团的ATP。

<table><tbody><tr><td>Aptamer binding buffer</td><td></td><td></td><td>20 mM Tris pH 7.6; 300 mM NaCl; 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;; 0.01% Tween-20</td></tr><tr><td>Fluorescently labeled ATP aptamer</td><td>IDT, Leuven, Belgium</td><td></td><td>sequence: DH25.42 50-Cy5-CCTGGGGGAGT-&lt;br/&gt;ATTGCGGAGGAAGG-3</td></tr><tr><td>ATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2383</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>ADP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2754</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>AMP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2252</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Adenine</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A8626</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>SAM</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A7007</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>dATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>11934511001</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>CTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>C1506</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>GTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>G8877</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Monolith NT.115&amp;nbsp;</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-G008</td><td>Blue/Red Channel&lt;br/&gt; MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector</td></tr><tr><td>Monolith NT.115 capillaries Standard</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-K002</td><td></td></tr><tr><td>Eppendorf PCR tubes</td><td>Eppendorf, Germany</td><td>30124537</td><td></td></tr><tr><td>Monolith control software. 2.1.33, pre-installed on the device</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>MO.affinity analysis v2.1.1</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>Kaleidagraph 4.5.2</td><td>Synergy Software</td><td></td><td></td></tr></tbody></table>

mapping the binding site of an aptamer on atp using microscale thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

在这项研究中，MST施加以表征DH25.42的DNA适体18的结合位点上的ATP。相对于其他的研究表征的ATP或ATP的模拟与一个或更多个荧光团38-40随机标记的蛋白质的小分子的相互作用，本研究包括的7.9 kDa的单链DNA适体与一种Cy5的分子标记的版本上的5&#39;端。不同的ATP衍生物和相关分子，都在不同的位置从ATP的不同的，分别用于映射的ATP分子的结合位点。稀释系列（在...

Watch this Scientific Journal Video about Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis at JoVE.com

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

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

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 ...

mapping-binding-site-an-aptamer-on-atp-using-microscale

Research

JoVE Journal

Biochemistry

10.5K 次观看.  2bind GmbH. MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

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

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

科研

化学

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

生物化学

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

癌症研究

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

生物学

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

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 application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

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 ...

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

基于适配体的靶标检测通过 3 级 G-四链体等温指数扩增反应进行

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

生物工程

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Source: Mittermaier, A. K., et al. <a href="https://app.jove.com/v/55959">Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions.</a> J. Vis. Exp. (2017)
This video demonstrates differential scanning calorimetry &#8212; a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic analysis.

差示扫描量热法研究 DNA 适配体-不稳定性配体相互作用

Source: Mittermaier, A. K., et al. Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions. ...