我们感谢朱莉·霍顿博士批判性阅读的手稿和拉金德拉·普拉萨德博士有用的建议和讨论。这项研究最初发表在杂志“生物化学佐佐A 等部分。人，“DNA序列背景效应的糖基化酶活性的人8 -羟基鸟嘌呤DNA糖基化酶。”J Biol Chem的 287，36702-36710（2012）2。这项工作是支持的全部或部分，由美国国立卫生研究院资助计划Z01-ES050158，NIEHS院内研究计划。

1。酶和DNA底物的制备<ol><li>过作为GST融合蛋白在大肠杆菌中的表达OGG1 大肠杆菌 ，利用GST标签进行净化，然后取出GST标签裂解HRV-3C蛋白酶（ 图1）6。 </li><li>购买化学合成的5&#39;-6-羧基（6-FAM）标记的寡核苷酸包含一个8 oxoG残留和互补未标记的寡核苷酸链。的34聚体寡核苷酸的5&#39;-末端的17位中包含一个8-oxoG。这些寡核苷酸，通过聚丙烯酰胺凝胶电泳纯化。 </li><li>准备含有8-oxoG混合5&#39;-6-FAM标记的寡核苷酸，其互补链在退火缓冲液（1的10mM的Tris-HCl，pH值7.5，50 mM氯化钠，以摩尔比为1:1.2的双链DNA基板1mM EDTA）中，在1.5毫升微量离心管中。试管置于一个浮在沸水中5分钟。离开管到位，然后让水慢慢冷却到r的OOM温度（约需2小时）。 </li><li>执行下的实验样品的制备和低的或最小的光照条件下，以尽量减少光漂白的荧光标记。 </li></ol> 2。测量稳态的时间进程和活跃的网站滴定OGG1 2.1。样品制备和稳态时间过程<ol><li>准备另外DNA底物和OGG1解决方案，在反应缓冲液（50mM的HEPES，pH值为7.5，20mM的氯化钾，0.5mM的EDTA，0.1％牛血清白蛋白）在1.5ml微量离心管中，在冰上。 DNA的浓度为400纳米和OGG1的表观浓度为30，60，90，或120纳米，由Bradford蛋白测定法确定。放置在一个热块集的反应管在37℃下预孵育的酶和DNA底物溶液，分别在37℃，持续1分钟。 </li><li>通过移液的OGG1和DNA底物溶液等体积混合，开始反应。在混合这些解决方案之后：蒸发散1:1（体积/体积），终浓度是200 nM的DNA和15，30，45，或60 nM的OGG1分别。的时间间隔取出等分试样（10微升），并用1微升的1M NaOH中混合淬灭反应。作为控制的时间过程，10μl的反应混合物中不添加酶混合，用1微升的1M NaOH中。 </li><li>反应样品放置在90℃的加热块为5分钟，以裂解所得产物AP现场。加热后，加入1μl1 M盐酸中每个样品。 </li><li>加入等体积（12微升）的凝胶加样缓冲液（95％甲酰胺，20mM的EDTA，0.02％溴酚蓝，0.02％二甲苯青），每个反应样品中，然后将混合物在95℃下在一个热块2分钟，然后立即将管置于冰上。 </li><li>将样品（5微升）到15％的变性聚丙烯酰胺凝胶，含有8M尿素，89 mM的Tris-盐酸，pH为8.8，89 mM的硼酸，2 mM的EDTA。基片和切割的产品是完全分离后运行凝胶。 </li></ol> 2.2。凝胶成像<ol><li>扫描成像器，可以检测出荧光标记的DNA，和可视化的底物和产物的条带的凝胶。 </li><li>量化频带成像后的凝胶。请注意，可能会观察到一些背景裂解后用NaOH（ 图2A），衬底本身的治疗。从测得的各反应产物的量中减去当前背景。 </li></ol> 2.3。数据分析<ol><li>绘制形成的产物的量，在每个反应时间（t）（ 图2B）。分析使用公式1来确定的突发（A 0，y轴截距）的幅度和斜率的线性稳态阶段（V SS）的原始数据。 </li></ol><img fo:src="/files/ftp_upload/50695/50695eq1highres.jpg" src="/files/ftp_upload/50695/50695eq1.jpg" /><ol start="2"><li>情节y轴截距相对于总蛋白浓度（ 即表观酶浓度; 图2C），以提供一定程度的酶的活性级分。使用零截距的线性拟合与提供的校正因子，用于确定分数的活性酶。 </li><li>绘制的稳态速率与活性酶的浓度（ 图2D），提供产品的解离速率常数（ 即拟合直线的斜率）。 </li></ol> 3。测量前稳态时间课程在图2中所示，产物的形成过程中色同步信号相位的测量通过手动搅拌和淬火速度太快。因此，快速冷却流量仪表可以提供一个功能强大的方法来衡量快速反应，发生在毫秒时间尺度（ 图3）5。该仪器采用计算机控制驱动电机迅速混合ND解渴指定的反应时间后的反应。例如，使用一个王技RQF-3快速淬火流量仪表来衡量最初的爆发阶段和后续产品形成催化OGG1稳态阶段。快速淬火流量仪表可从几个厂家。 3.1。样品的制备单独准备在1.5ml管2-1中所描述的DNA底物和OGG1解决方案。的DNA的浓度和活性OGG1是400纳米和80纳米。这将导致在最终浓度为200 nM DNA和40 nM的后：混合1:1（体积/体积）的活​​性OGG1。 3.2。的快速淬火流量仪表的制备<ol><li>将循环水浴快速淬火温度控制流量仪表（37°C）。 </li><li>根据制造商的指示，调整仪器参数，并选择适当的反应循环所需的时间点蒸发散。反应循环可变长度提供备用的反应时间。 </li><li>准备反应缓冲液和NaOH淬火解决方案鲁尔锁10毫升的一次性注射器，这些注射器连接到驱动器端口，并加载驱动油藏反应缓冲液（注射器B）和142毫米氢氧化钠（注射器Q）（注射器负载负荷位置阀门）要去除气泡的注射器，工作的解决方案，来回几次。 </li><li>下步进电机，直到它接触的注射器（注射器负载阀和样品负载阀在FIRE位置）的顶部。 </li><li>冲洗样品定量，反应的循环，并用水和甲醇的线出口。干燥潮红领域完全（阀门FLUSH位置）。 </li></ol> 3.3。预稳态时间过程<ol><li>具有上限的1.5毫升管使用16号针头的顶部的一个孔。号线出口连接管收集淬火反应。 </li><li>设置所需的reaction个时间（秒）使用键盘。步进电机将备份到调整循环量，从而保持恒定淬火体积。位置通过加入缓冲驱动端口连接到一次性注射器，注射器的柱塞B对步进电机平台。 </li><li>填写1毫升鲁尔锁一次性注射器DNA底物和OGG1解决方案，分别为（取样阀，负载位置）。将DNA底物的注射器和OGG1解决方案的每个样品负载端口，然后填写DNA底物的循环和OGG1解决方案的样品，分别为（样品负载负载位置的阀门）。 </li><li>将所有注射器负载阀和样品负载阀的火位置。由键盘冲程（ 例如，“G”或“启动”键按下）开始反应。 DNA底物和OGG1溶液（各18微升）立即混合在反应循环。 </li><li>等待，直到自动骤冷该反应所需的反应时间，通过混合36微升的reaction 86微升的142毫米NaOH的混合物。终止反应后，将样品从线出口排出。 </li><li>设置注射器负载阀的负荷状况及样品负载阀冲洗位置。冲洗样品定量，反应的循环，并用水和甲醇的线出口，并干燥3.2中所描述的。重复上述步骤，每个时间点。 </li><li>执行未加酶的对照实验，以确定背景校正，可以手动完成，或与快速骤冷的文书。要进行快速淬火仪器控制，填写DNA与DNA底物的样品环，但保持样品环OGG1空。设置了反应时间，进行混合并淬火3.3.4-3.3.6中所描述的。 </li><li>循环和样品反应循环与2 M 2 M氢氧化钠，盐酸，水和甲醇洗涤，然后干燥洗涤后的线。步进电机的设置到起始位置后，切换阀门注射器负载的负载的定位n和洗涤水驱油藏。 </li><li>治疗与热骤冷的反应样品，然后15％变性聚丙烯酰胺凝胶电泳，如2.1中描述的独立的底物和产物的DNA。 </li><li> 2.2中所描述的可视化和量化的频带上的凝胶。 </li></ol> 3.4。数据分析适合的时间过程的产品形成具有上升指数和线性的条件（公式2）提供的一级反应速率常数（k“ 观测值”），脉冲串的振幅（A 0）的方程，通过非线性回归分析以线性速率（V SS）。 <img fo:src="/files/ftp_upload/50695/50695eq2highres.jpg" src="/files/ftp_upload/50695/50695eq2.jpg" /> 4。单周转时间课程<ol><li>在单独的1.5 ml微量离心管中制备DNA底物和OGG1 2-1中所描述的。浓度DNA和活性OGG1蒸发散分别为100纳米和500纳米，这产生最终浓度为50nM的DNA混合后，1:1（体积/体积）和250 nM的OGG1。 </li><li>准备快速淬火流量仪表，并准备反应样本中所描述的3.2和3.3。 </li><li>治疗用热和骤冷的反应样品使其受到15％变性聚丙烯酰胺凝胶电泳分离底物和产物，如2.1中所述。 </li><li> 2.2中所描述的可视化和量化的频带上的凝胶。 </li><li>适合的时间过程的产品形成一个单一的指数来确定，如在式（3）给出的一级速率常数（k 观测值 ）。 </li></ol><img fo:src="/files/ftp_upload/50695/50695eq3highres.jpg" src="/files/ftp_upload/50695/50695eq3.jpg" />

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K., Mitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+DNA+glycosylase+activity+of+OGG1+by+NEIL1:+functional+collaboration+between+two+human+DNA+glycosylases.">Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases.</a> Biochemistry. 43, 11596-11604 (2004).</li><li>Sidorenko, V. S., Nevinsky, G. A., Zharkov, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+interaction+between+human+8-oxoguanine-DNA+glycosylase+and+AP+endonuclease.">Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease.</a> DNA Repair. 6, 317-328 (2007).</li></ol>

作者什么都没有透露。

动力学这里描述的方法，概括的方法来定义基本的动力学常数。如果一个时间过程的产品形成了第一种酶营业额迅速发生双相，然后经化学的一个步骤是在随后的催化失误限速。 OGG1的情况下，可以使用高浓度的酶与限制（S &lt;E)或高(S&gt;É）DNA的浓度测量的第一个营业额。在第一种情况下，该反应是有限的“单一营业额，并提供一定程度的酶的活性位点的催化速率。在后者的情况下，被评估多种?...

一个有氧环境加快基因组不稳定。甲的主要promutagenic DNA病变导致的氧化应激是7,8 - 二氢-8 - 羟基鸟嘌呤（8-oxoG）。这是由于8-oxoG的暧昧编码潜力。 8 oxoG发起碱基切除修复负责人8 - 羟基鸟嘌呤DNA糖基化酶（OGG1）。 OGG1切除8 oxoG基地，导致一个的嘌呤网站（AP站点）产品DNA糖基化酶活性。在某些情况下，一个弱OGG1裂解酶活性可以切割的AP站点。 一般动力学特性的DNA糖基化酶发现，他们表现出双相时间课程。在经过最初的快相产物的形成（ 即脉冲串），观察到的线性稳态阶段1-3。这种行为是表示化学（ 即构象变化或产品释放）过程中的时间过程的线性部分的限速的步骤，而钻针圣相，通常被称为“过渡阶段，在第一个周期的反应产物的形成，可在酶的活性位点相对应。当产品发布速率限制在稳态阶段，活动的测量提供了定性测量产品DNA结合的亲和力，但没有提供有关的酶的活性部位（ 即化学）事件的动力学信息。因此，需要的方法来分离和测量指数的预稳态脉冲串相位探测事件的过程中的第一酶在酶的活性位点4的营业额。 有三种标准的动力学方法OGG1催化行为的特征，（1）稳定状态，（2）预稳定的状态，和（3）单成交。这些方法彼此不同的酶的浓度在反应混合物中的每一种方法中使用的酶底物比。在一个典型的稳态的方法，有时也被称为多周转动力学，使用低浓度的酶按照产物的形成。当底物浓度大大超过酶的浓度，以便多个酶失误不显着影响底物浓度。在这种情况下，时间场应该是线性的，它往往是难以辨别是否发生突发过程中的第一个营业额，由于在这种方法中使用的酶浓度低;注意，脉冲串的幅度是酶的浓度相当于。这是可以克服的，通过使用较高的酶浓度和外推线性时间当然零时间检测是否很快就发生了第一种酶营业额。纵坐标（y轴）上的截距应该是酶的浓度成比例，并提供了一​​个衡量积极从事与底物的酶。虽然这种方法原则上可以提供证据的存在突发阶段，DIFferent方法需要测量的动力学突发阶段。在许多情况下，的突发阶段是太快了人工搅拌淬火技​​术测量。在这种情况下，预稳态和单营业额动能（ 即瞬态动能）的方法往往需要快速混合和猝灭反应5按照早的时间点的仪器。在一个稳定状态前的方法，使用高浓度的酶，以便在第一个营业额，形成显着数量的产品。 ，由于多个失误其次观察催化循环（ 即突发如下的线性相位），底物浓度是大于酶浓度（[酶] &lt;[基板]）。隔离事件的酶的活性部位无催化单车，单营业额的条件下被使用。在这种情况下，底物是饱和的酶（E &gt;&gt; S），以使所有的基片将参加我在单笔成交金额，通常会表现出一个单一的指数的时间过程。 如上所述酶表现出一个突发阶段，产品发布（K掉 ）往往限制的稳态阶段的时间过程的速率。产品放行的速度（V SS，浓/时间）可确定从斜坡的线性稳态阶段。活性酶浓度（E）产品发布的速度是需要转换的内在速率常数K掉 = V SS / [E]。重要的是，活性酶的浓度一般低于所测得的蛋白浓度因杂质，处于非活动状态的酶，酶的非生产性基板结合，和所使用的方法，以确定蛋白浓度。活性酶的浓度可以由突发幅度当产品释放缓慢的。因此，推断一个稳定状态的时间当然要零时间提供了一个估计需要从观察到的稳态率计算K掉 （产品发布）的活性酶。 要测量的动力学的突发，预稳态的方法是在第一营业额前发生的线性稳态阶段，要遵循产品的形成。该突发动力学如下酶产品的中间体的形成。一旦反应过程是由酶与底物混合的酶产品的量迅速增加，直至反应达到稳定状态阶段。如果催化比产品的发布更迅速，脉冲串的幅度是等于积极参与的酶和所观察到的指数接近平衡（十一观测值 ）对应的底物向产物的化学转化率，假设反向速率化学是可以忽略不计。 在某些情况下催化CY保鲜干扰预稳态分析，如化学和产品发布率的幅度并不显着不同。在这种情况下，采用酶相对过剩防止绑定到一个单一的营业额酶的催化循环和限制基板基板。因此，第一化学反应步骤可以分离和精确地确定作为一级反应速率常数（k 观测值 ）。此速率常数为k应该是相似的OBS从上述预稳态的方法确定。 这里，我们描述如何将这些动力学的方法，可以用来分析OGG1糖基化酶活性的。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>5'-6-FAM labeled oligonucleotides containing a single 8-oxoG</td>
 <td>Eurofins MWG Operon</td>
 <td></td>
 <td>5'-P32 radiolabeled oligonucleotides can be used as well. Polyacrylamide gel purification grade is recommended. </td>
 </tr>
 <tr>
 <td>Unlabeled oligonucleotides (complementary strand)</td>
 <td>Eurofins MWG Operon</td>
 <td></td>
 <td>Polyacrylamide gel purification grade is recommended. </td>
 </tr>
 <tr>
 <td>1 ml BD Luer Lock disposable syringe</td>
 <td>BD Medical</td>
 <td>309628</td>
 <td>Lue Lock disposable syringe from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>10 ml BD Luer Lock disposable syringe</td>
 <td>BE Medical</td>
 <td>309604</td>
 <td>Luer Lock disposable syringe from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Circulating water bath</td>
 <td>Any vender</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RQF-3 Rapid Quench-Flow Instrument</td>
 <td>KinTek Corporation</td>
 <td></td>
 <td>Rapid Quench-Flow Instrument from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>Typhoon Phosphorimager 8600</td>
 <td>GE Healthcare Life Sciences</td>
 <td></td>
 <td>Imager from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>KaleidaGraph</td>
 <td>Synergy Software</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

steady-state, pre-steady-state, and single-turnover kinetic measurement for dna glycosylase activity

人类8 - 羟基鸟嘌呤DNA糖基化酶（OGG1），切除从DNA诱变氧化性DNA损伤-8 - 氧代-7,8 - dihydroguanine（8-oxoG）。 OGG1动力学特性进行测量8 oxoG切除率和产品发布。当的OGG1浓度的低于底物DNA，产物的形成的时间过程是双相的，随后由一个线性稳态阶段产品形成的快速指数阶段（ 即脉冲串）。产物的形成的初始脉冲串对应的正确啮合在基片上的酶的浓度，和脉冲串的幅度取决于酶的浓度。一阶速率常数的突发对应的内在8 oxoG切除率和速度较慢的稳态测量产品的发行率（产品DNA解离速率常数，k 关闭 ）。在这里，我们描述了稳定状态，稳定状态，单营业额的方法来隔离和测量SPecific步骤在OGG1催化的循环。含病变的荧光标记的寡核苷酸和纯化OGG1是用来方便精确的动力学测量。由于酶浓度低，用来做稳态测量，人工搅拌反应试剂和淬火，可以进行确定的稳态率（K 关闭 ）。此外，外推在零时间，纵轴上的点的稳态速率表明的产物的形成过程中发生一个脉冲串的第一个营业额（ 即 y轴截距为正）。可以测量使用的是快速混合和猝灭技术，检查形成在短的时间间隔（&lt;1秒）之​​前的稳态阶段的产品的量和对应的速率为8的一级速率常数的指数的脉冲串相位oxoG切除术（化学）。化学步骤也可以使用一个单一的营业额的方法测量催化循环防止饱和底物DNA酶（E&gt; S）。这些方法可以测量的基本速率常数的影响的效率去除的DNA损伤。

稳态动力学分析是由使 ​​用200纳米的DNA底物和四种不同浓度明显OGG1（15，30，45和60纳米）确定通过Bradford蛋白检测2。时间课程适合产品形成一个线性方程来确定y轴截距，分别为2.2，11，15，和26纳米，分别相对于每个蛋白浓度（ 图2B）。的y截距，进一步绘制相对于每个实际的蛋白质浓度（ 图2C）。从图2C中的线的斜率，被确定为38％的活性酶的馏分?...

Watch this Scientific Journal Video about Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity at JoVE.com

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

时间8 - 羟基鸟嘌呤DNA糖苷酶糖基化酶活性课程双相参展产品的形成和爆发出的线性稳态阶段。利用淬火流技术中，突发和可以测量的稳态速率，分别对应到8 - 羟基鸟嘌呤和释放从产物DNA糖基化酶切除。

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

steady-state-pre-steady-state-single-turnover-kinetic-measurement-for

Research

JoVE Journal

Chemistry

19.2K 次观看.  NIEHS, National Institutes of Health. 时间8 - 羟基鸟嘌呤DNA糖苷酶糖基化酶活性课程双相参展产品的形成和爆发出的线性稳态阶段。利用淬火流技术中，突发和可以测量的稳态速率，分别对应到8 - 羟基鸟嘌呤和释放从产物DNA糖基化酶切除。

视频: Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&amp;#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

该DNA氧化标志物的高效液相色谱法测量，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷，在培养细胞和动物组织

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

科研

化学

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel &#945;-diimine ethylene polymerization catalyst. Initial polymerizations with the catalyst lead to optimized reaction conditions, including catalyst concentration, ethylene pressure and reaction time. Using gas-uptake data for these reactions, a procedure to calculate the initial rate of propagation (kp) is presented. Using the optimized conditions, the ability of the nickel &#945;-diimine polymerization catalyst to undergo chain transfer with diethylzinc (ZnEt2) during ethylene polymerization was investigated. A procedure to assess the ability of the catalyst to undergo chain transfer (from molecular weight and 13C NMR data), calculate the degree of chain transfer, and calculate chain transfer rates (ke) is presented.

A protocol for the high-throughput analysis of polymerization catalyst, chain transfer polymerizations, polyethylene characterization, and reaction kinetic analysis is presented.

乙烯聚合中使用并联电抗器的压力和链转移聚合的动力学分析

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

光子温度计的制作与测试

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

用于从单一DNA分子大规模平行蛋白质合成的Femtoliter滴体阵列

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.