这项工作得到了帕多瓦大学制药和药理学系（PRIDJ-BIRD2019）的支持。

1.核酸及化学探针制备
<ol><li>核酸 注：名为“TBA”的寡核苷酸是 15-mer DNA 序列 5'-GGT-TGG-TGT-GGT-TGG-TGG-3'，由荧光基团 5-羧基荧光素 （FAM） 在 3'-末端标记，以实现凝胶上的可视化。未标记的寡核苷酸“cTBA”是其DNA互补序列5'-CCA-ACC-ACA-CCA-ACC-3'。采用TBA和cTBA获得三种不同的结构，如 表1所示。<ol><li>高压灭菌器吸头和 0.5 mL 试管，以获得无菌一次性用品并避免污染。</li>
		<li>制备将每种寡核苷酸溶解在超纯水中至终浓度为100μM的储备溶液。 使用制造商提供的260nm处的消光系数，使用紫外可见（UV-Vis）分光光度计确定确切的寡核苷酸浓度。 注：消光系数：TBA 和 cTBA 分别使用 164,300 M-1 cm-1 和 138,600 M-1 cm-1。</li>
		<li>将TBA和cTBA储备溶液储存在-20°C（在这些条件下储存数月）。</li>
	</ol></li>
	<li>化合物 B-CeP 1 注：化合物 B-CeP 1 的合成如前所述6.<ol><li>制备~10mM的B-CeP 1储备溶液。使用位于通风橱中的分析天平称取~1mg冻干化合物，并将其溶解在100%二甲基亚砜（DMSO）中。</li>
		<li>根据实际使用的化合物和DMSO（d = 1.1 g / cm3）的量计算确切的化合物浓度。 注意：始终戴手套处理化合物（冻干和溶解在DMSO中时）13,14。</li>
	</ol></li>
</ol>表1：该协议中使用的寡核苷酸结构。<a href="https://www.jove.com/files/ftp_upload/65373/65373_Table%201%20(5).xlsx" target="_blank">请按此下载此表格。</a>
2. 核酸构建体的折叠
<ol><li>缓冲液的制备<ol><li>制备BPE缓冲溶液（二磷酸 - 乙二胺四乙酸[EDTA]，5x：2mM NaH 2 PO 4,6mMNa 2 HPO 4,1mM Na2EDTA，pH7.4）和500mM KCl的超纯水溶液。通过0.22μm孔径过滤器过滤溶液。 注意：为获得最佳效果，请使用新鲜制备的溶液。BPE缓冲液可在4°C下储存长达15天。</li>
	</ol></li>
	<li>通过热复性程序折叠 G4-TBA、ssTBA 和 dsTBA 样品 注意：钾阳离子是折叠 G4 结构 （G4-TBA） 所必需的。不要在对照ssTBA和dsTBA的折叠溶液中添加钾阳离子。<ol><li>在1x BPE和100mM KCl中制备40μL的4μMG4-TBA溶液。 通过将管加热至95°C5分钟并使寡核苷酸G4-TBA溶液变性，并将其缓慢冷却至室温（RT），使TBA折叠成G-四链体。</li>
		<li>在1x BPE中制备40μL的4μMsTBA溶液。执行步骤 2.2.1 中提到的热复性程序，以单链形式折叠 TBA。</li>
		<li>通过在1x BPE中混合等摩尔量的TBA和cTBA，制备40μL的4μMdsTBA溶液。执行上述热复性程序（步骤2.2.1），使TBA退火到其互补序列cTBA并形成TBA的双链形式。 注：考虑到每个样品需要 5 μL 的 4 μM 溶液，每个折叠溶液的最终体积基于探测反应的样品数量。准备少量过量的每种溶液，以避免移液错误。</li>
	</ol></li>
</ol>3. 探测反应
注意：探测反应必须在热复性程序后立即进行。
<ol><li>当G4-TBA，ssTBA和dsTBA的折叠溶液冷却至室温时，进行短离心（室温下7,000× g ，持续5-8秒）并开始探测反应。</li>
	<li>准备 21 个空的 0.5 mL 高压灭菌管。如 表2所示，将它们组织在三组，每组七个管中，用于实验室样品。 注意：每个列集对应三种不同的 TBA 折叠条件 G4-TBA、ssTBA 和 dsTBA。每行对应三个不同的孵育时间。色谱柱内的每个细胞对应于最终的B-CeP 1探针浓度（表2）。确保清楚地标记试管。</li>
	<li>在每个试管中加入 3 μL 超纯水。</li>
	<li>在第一组的每个管中加入5μL折叠的G4-TBA（步骤3.2）。在第二组的每个管中加入 5 μL 折叠的 ssTBA。在第三组的每个试管中加入 5 μL 折叠的 dsTBA。</li>
	<li>在超纯水中将 B-CeP 1 储备溶液稀释至 250 μM 和 25 μM。 注意：B-CeP 1化学探针的稀释液必须是新鲜制备的，并立即与DNA底物反应，以避免与水发生竞争性反应。</li>
	<li>向样品中加入 2 μL 适当的 B-CeP 1 稀释液（25 μM 和 250 μM）。在三个对照样品（C）中用超纯水代替化合物，以便在没有化合物的情况下分析不同折叠的TBA。将所有样品在37°C孵育。</li>
	<li>孵育1小时，4小时和15小时后，通过将管置于-20°C直至下一步来停止反应。 注意：样品可以在这些条件下储存几天。</li>
	<li>在真空离心机中干燥样品。 注意：干燥的样品可以在-20°C下储存数周，然后再进行PAGE分析。</li>
</ol>表 2：探测反应的样品（结构、探针浓度和孵育时间）。 每个色谱柱组对应于三种不同的 TBA 折叠条件（G4-TBA、ssTBA 和 dsTBA）。每行对应于三个不同的孵育时间（1,4,15小时）。色谱柱内的每个细胞对应于最终的B-CeP 1探针浓度（5或50μM）。每组的对照（C）对应于在没有化合物的情况下孵育较长时间（15小时）的不同折叠TBA的样品。 <a href="https://www.jove.com/files/ftp_upload/65373/65373_Table%202%20(3).xlsx" target="_blank">请按此下载此表格。</a>
4.高分辨率PAGE
<ol><li>变性聚丙烯酰胺溶液的制备 注：提前准备 500 mL 20% 变性聚丙烯酰胺凝胶溶液。每个实验将使用大约 80 mL 的该溶液。使用琥珀色玻璃瓶或用铝箔盖住玻璃瓶，将溶液储存在室温下。 注意：聚丙烯酰胺具有神经毒性。在凝胶制备和倾倒的所有步骤中，戴上手套和实验室外套。将聚合丙烯酰胺丢弃在适当的盒子中，以处理受污染的材料。<ol><li>在 1 L 烧杯中称取 210 g 尿素。加入 250 mL 40% 丙烯酰胺/双丙烯酰胺 （19/1） 溶液和 50 mL 10x TBE（890 mM Tris-HCl、890 mM 硼酸盐、20 mM EDTA，pH 8）。</li>
		<li>将烧杯放在搅拌板上，用搅拌棒混合溶液。搅拌时用铝箔盖住烧杯，防止飞溅和污染。</li>
		<li>混合溶液，直到尿素完全溶解，溶液澄清。 注意：此步骤可能需要数小时。为了促进尿素的溶解，加入一小份等分试样的水，不要超过所需的最终体积。</li>
		<li>拆下搅拌棒。将溶液倒入圆柱体中，加水至精确的最终体积为 500 mL。</li>
	</ol></li>
	<li>凝胶装置的设置<ol><li>用70%乙醇清洗两块板（一块有缺口，一块无缺口），晾干，然后用二甲基二氯硅烷溶液处理板。 注意：硅烷化可以跳过，尽管当凝胶三明治被拆开时，它有助于从其中一个板中释放凝胶。 注意： 戴手套处理硅烷化溶液，并在通风橱中用该溶液进行板处理。</li>
		<li>将 0.4 毫米垫片沿较长板的长边放置，将短板放在另一块板的顶部，并将两块板对齐底部。</li>
		<li>沿着除顶部以外的所有边缘放置多层纸胶带。</li>
		<li>为避免在灌注过程中发生泄漏，请在凝胶底部添加一层额外的胶带。</li>
		<li>按照供应商的说明，用干净的夹子夹住玻璃夹层的侧面（不同的供应商使用的设备、夹层夹具和垫圈略有不同）。</li>
	</ol></li>
	<li>倒入凝胶 注意：在室温（25°C）下倒入凝胶，因为聚丙烯酰胺聚合对温度敏感。<ol><li>在烧杯中，立即使用前倒入 80 mL 先前制备的变性聚丙烯酰胺溶液（步骤 4.1）、450 μL 10% m/V 过硫酸铵 （APS） 溶液和 45 μL 四甲基乙二胺 （TEMED）。</li>
		<li>混合溶液并使用 50 mL 注射器将其快速倒入玻璃板之间。在玻璃板之间引入具有所需孔数的梳子，避免气泡。如有必要，加入凝胶溶液以完全填充三明治。放置四个夹子amps 在梳子上向下压，使孔均匀分布。</li>
		<li>让凝胶聚合至少45分钟。</li>
	</ol></li>
	<li>电泳凝胶<ol><li>聚合后，去除所有夹具和纸带层。慢慢取下梳子，用蒸馏水彻底冲洗孔。</li>
		<li>按照特定供应商的说明将凝胶夹心正确放置在垂直凝胶电泳仪中。</li>
		<li>在去离子水中制备TBE电泳缓冲液（1x：89mM Tris-HCl，89mM硼酸盐，2mM EDTA，pH 8），并用缓冲液填充顶部和底部储液槽。</li>
		<li>通过在50W下进行凝胶电泳的预运行至少30分钟来预热板。</li>
		<li>在超纯水中制备变性凝胶上样缓冲液（DGLB：1M Tris-HCl，80%甲酰胺，50%甘油，0.05%溴酚蓝）。 注：GLB有助于跟踪寡核苷酸样品进入凝胶系统的过程，并允许将样品加载到凝胶孔中。变性剂甲酰胺的存在使DNA物种能够根据大小分离，即使在非变性PAGE中也是如此。</li>
		<li>将干燥的样品（来自步骤3.8的样品）重悬于5μL的DGLB中。</li>
		<li>在加载样品之前，使用小注射器和上缓冲室中的TBE缓冲液清洁孔，以从孔中除去尿素。 注意： 重复此步骤数次以准确清洁孔，从而避免难以解释的条带。</li>
		<li>将样品加载到干净的孔中，并记下加载顺序。</li>
		<li>在50W下运行凝胶电泳2小时，或至少直到溴酚蓝染料在凝胶下运行2/3。</li>
	</ol></li>
	<li>凝胶成像<ol><li>电泳后，关闭电源，取下玻璃夹层，清洁眼镜。</li>
		<li>通过使用凝胶成像仪扫描来检测FAM标记的寡核苷酸条带的荧光。</li>
	</ol></li>
</ol>

使用化学作图鉴定 DNA 模板中的 G-四链体

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作者没有要披露的利益冲突。

G-四链体是核酸二级结构，通常折叠在富含鸟嘌呤的DNA序列中，并且由于它们与遗传控制和疾病有关，因此是重要的研究目标。B-CePs 的化学定位是表征 DNA G4s 的有用方案，可用于鉴定在生理盐条件下参与 G-四分体形成的鸟嘌呤碱基。
该协议中使用的化学探针是B-CeP 1（图2A），其通过与鸟嘌呤核碱基的 N7特异性反应，可以区分参与G-四链体四链体形成的鸟嘌呤和不涉及这种排列的鸟嘌呤。事实上，G-四分体中鸟嘌呤之间的Hoogsteen氢相互作用（图1）损害了与探针的反应。探测反应产物通过高分辨率PAGE（图2B）进行解析，这是一种实验室可访问的程序，用于检测DNA序列中的烷基化加合物和切割位点。此外，使用用荧光团标记的DNA序列允许用户在安全条件下进行实验，避免放射性标记的核酸和使用插层剂的常规染色步骤。
B-CeP 1探针在固体和非水储备溶液中非常稳定，在进行探测反应时只会出现很少的问题。为了避免方案中的缺点，避免在探测溶液中存在不需要的亲核试剂非常重要，以避免竞争反应，这些反应可能会降低可用于与DNA形成加合物的活性探针的滴度。由于大多数常用的缓冲液都含有Tris碱，我们提醒读者，其亲核伯胺可以与探针亲电中心反应，从而抵消DNA底物10的鸟嘌呤的反应性，如图 3 所示的B-CeP 1。这是在磷酸盐缓冲液中进行探测反应的基本原理，如方案中详述的那样。为避免与水7发生反应，我们还建议B-CeP 1的所有稀释液均新鲜制备，并立即与核酸底物反应。
这里描述的 B-CePs 化学图谱是硫酸二甲酯 （DMS） 足迹方案的便捷替代方案，该协议代表了 G4s16 初始表征的主流检测方法。这两种方案给出的信息基本相同，即 G4 结构的形成和 G4 中涉及的鸟嘌呤的鉴定，而不管 G4 构象如何。然而，使用B-CePs进行化学图谱在DMS足迹方面具有关键优势，即协议的简单性。在探测反应之后，这里描述的方案不需要额外的步骤，而DMS足迹在与探针反应后需要随后通过热哌啶裂解，这意味着额外的样品纯化和缓冲液交换16。B-CePs方案中步骤数的减少意味着使用较少量的初始DNA底物。最后，本文描述的B-CePs的化学图谱，以TBA序列作为概念验证，将导致适用于任何其他潜在G-四链体形成序列的新实验。

除了典型的 Watson-Crick 双螺旋结构外，核酸还可以采用各种二级结构，例如替代的 G-四链体 （G4） 形式，因为它们富含鸟嘌呤的序列。G4 结构基于平面四聚体的形成，称为 G-四聚体，其中四个鸟嘌呤通过 Hoogsteen 氢键相互作用。G-四分体通过在鸟嘌呤核心中心配位的一价阳离子堆叠并进一步稳定（图1）1。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65373/65373fig01.jpg"> 图 1：G 四链体结构的示意图。 （A） G-四联体的示意图。平面阵列通过Hoogsteen碱基配对和中心阳离子（M +）稳定。<a href="https://www.jove.com/files/ftp_upload/65373/65373fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
具有至少两个连续鸟嘌呤核苷酸的四次或更多次运行的序列是潜在的 G 四链体形成序列 （PQS），它们可以折叠成 G 四链体结构。PQS 位于许多不同的细胞环境中，例如端粒、基因启动子、核糖体 DNA 和重组位点，并参与许多生物过程的调节2。因此，目前主要通过计算工具进行的人类基因组中 G4 的鉴定和实验验证是一个生物学相关问题3。为了支持计算预测或检测不可预测的 G4 结构，本文展示了一种基于化学图谱的可识别方法，用于识别 DNA 模板中 G4 的形成，从而能够精确识别形成 G-四体结构的鸟嘌呤。
报道的化学定位分析利用了双-3-氯哌啶 （B-CePs） 在 G4 结构形成后与鸟嘌呤的不同反应性。由于 B-CeP 与亲核试剂 4,5,6,7,8,9 的高反应性，因此是核酸烷基化剂，能够非常有效地与鸟嘌呤核苷酸10 的 N 7 位置反应。烷基化之后是单链和双链 DNA 构建体中的脱嘌呤和链切割。相反，参与 G4 排列中 G-四分体形成的鸟嘌呤不受 B-CeP 烷基化的影响，因为鸟嘌呤的 N7 位置与 Hoogsteen 氢键有关。B-CePs 的这种特异性反应性不仅可以检测 G4 结构，还可以鉴定形成四联体的鸟嘌呤，因为与单链和双链 DNA 中的鸟嘌呤相比，它们可以从相对保护中推断出免受烷基化的影响。
本文报道了使用 B-CeP 1（图 2A）作为表征凝血酶结合适配体 （TBA） 的探针的化学定位方案，TBA 是一种 15 聚体 DNA，能够在钾阳离子存在下呈现 G4 排列11,12。将TBA（G4-TBA）的G4排列直接与两个对照进行比较，即单链形式的TBA（ssTBA）和TBA退火到其互补序列以形成双链结构（dsTBA）（表1）。探测反应产物通过在烷基化鸟嘌呤上定位单个烷基化加合物和 DNA 链切割，在单核苷酸水平上通过高分辨率聚丙烯酰胺凝胶电泳 （PAGE） 进行解析。通过将TBA寡核苷酸与3'端的荧光基团偶联，可以在凝胶上实现可视化（表1）。该协议显示了如何将TBA折叠在其不同的构象（G4和对照）中，以及如何使用B-CeP后跟PAGE进行探测反应。

<table><tbody><tr><td>Acrylamide/bis-acrylamide solution 40%</td><td>Applichem</td><td>A3658</td><td>R45-46-20/21-25-36/38-43-48/23/&lt;br/&gt; 24/25-62</td></tr><tr><td>Ammonium per-sulfate (APS)</td><td>Sigma Aldrich</td><td>A7460</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td></td><td></td></tr><tr><td>Autoclave</td><td>pbi international</td><td></td><td></td></tr><tr><td>Boric acid</td><td>Sigma Aldrich</td><td>B0252</td><td></td></tr><tr><td>Bromophenol blue Brilliant blue R</td><td>Sigma Aldrich</td><td>B0149</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dodecahydrate</td><td>Fluka</td><td>71649</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>276855</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>Integrated DNA Technologies</td><td>synthesis of custom sequences</td><td></td></tr><tr><td>EDTA disodium</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>Formamide</td><td>Fluka</td><td>40248</td><td>H351-360D-373</td></tr><tr><td>Gel imager</td><td>GE Healtcare</td><td>STORM B40</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Micro tubes 0.5 mL</td><td>Sarstedt</td><td>72.704</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Sequencing apparatus</td><td>Biometra</td><td>Model S2</td><td></td></tr><tr><td>Silanization solution I</td><td>Fluka</td><td>85126</td><td>H225, 314, 318, 336, 304, 400, 410</td></tr><tr><td>Sodium phosphate monobasic</td><td>Carlo Erba</td><td>480086</td><td></td></tr><tr><td>Speedvac concentrator</td><td>Thermo Scientific</td><td>Savant DNA 120</td><td></td></tr><tr><td>TEMED</td><td>Fluka</td><td>87689</td><td>R11-21/22-23-34</td></tr><tr><td>Tris-HCl</td><td>MERCK</td><td>1.08387.2500</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>51456</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Thermo Scientific</td><td>Nanodrop 1000</td><td></td></tr></tbody></table>

in vitro chemical mapping of g-quadruplex dna structures by bis-3-chloropiperidines

G-四链体 （G4s） 是生物学相关的非典型 DNA 结构，在基因表达和疾病中起重要作用，代表重要的治疗靶点。在潜在的 G 四链体形成序列 （PQS） 中对 DNA 进行 体外 表征需要可用的方法。B-CeP是一类烷化剂，已被证明是研究核酸高级结构的有用化学探针。本文描述了一种新的化学定位分析方法，利用 B-CePs 与鸟嘌呤 N7 的特异性反应性，然后在烷基化 Gs 处直接链裂解。 
也就是说，为了区分 G4 折叠和未折叠的 DNA 形式，我们使用 B-CeP 1 探测凝血酶结合适配体 （TBA），这是一种能够承担 G4 排列的 15 聚体 DNA。B-CeP 响应鸟嘌呤与 B-CeP 1 的反应产生的产物可以通过高分辨率聚丙烯酰胺凝胶电泳 （PAGE） 在单核苷酸水平上解析，方法是在烷基化鸟嘌呤上定位单个烷基化加合物和 DNA 链切割。使用 B-CePs 进行定位是一种简单而强大的工具，用于体 外 表征 G-四链体形成的 DNA 序列，能够精确定位参与 G-四链体形成的鸟嘌呤。

图2显示了在折叠成三种不同结构的TBA寡核苷酸上进行化学定位测定的代表性结果，如方案中所述的B-CeP 1。TBA 的 G-四链体排列 （G4-TBA） 是通过在 BPE 中折叠寡核苷酸并在 K+ 阳离子存在下获得的，而同一 TBA 序列的单链形式 （ssTBA） 是在无钾的情况下折叠的。通过在BPE缓冲液中将TBA退火至其互补序列来制备双链构建体（dsTBA）。将终浓度为5或50μM的B-CeP 1与2μM的G4-TBA（图2B的左泳道）、ssTBA（图2B的中央泳道）和dsTBA（图2B的右泳道）样品反应1小时、4小时和15小时。其他对照是未与探针反应的 DNA 构建体。对三种不同的TBA褶皱进行圆二色性（CD）分析，证实了它们的正确结构（补充图S1）。dsTBA 的 CD 光谱实际上具有 ~260-280 nm 的正长波段和 ~245 nm 处的负波段15，与 B 形式的 DNA 螺旋一致。在不存在 K+ 离子的情况下，发现 ssTBA 结构较差，而在存在 K+ 离子的情况下，G4-TBA 折叠成反平行的 G4，其特征在于 260 nm 处的负带和 290 nm 处的正带15。
图2B中报告的高分辨率PAGE结果提供了B-CeP 1对G4-TBA、ssTBA和dsTBA的时间和浓度依赖性反应的全面视图，清楚地揭示了G4-TBA的烷基化模式与观察到的ssTBA和dsTBA构建体相比明显不同。与未处理的样品 （C） 相比，迁移迁移率较低的条带与 DNA 底物烷基化一致。在G4-TBA底物的情况下，根据仅存在一种可用于烷基化的鸟嘌呤（G8）（图2C中鸟嘌呤的编号），仅清楚地观察到一个烷基化加合物。相反，当底物是 ssTBA 或 dsTBA 时，可以区分多个加合物，所有鸟嘌呤都具有 N7 位置，可以自由探测。相反，迁移速度更快的条带归因于链切割的产物。在 G4-TBA 底物的情况下，参与 G-四链体四联体的鸟嘌呤（G1、G2、G5、G6、G10、G11、G14、G15）不受烷基化的影响，而 G8 作为烷基化的后续步骤受到裂解的影响。在 ssTBA 和 dsTBA 构建体中，所有鸟嘌呤都容易发生裂解，如检测到的众多条带所示，这些条带在较长的孵育时间内逐渐变得更加强烈。
该协议允许区分参与G-四链体四联体形成的鸟嘌呤和不涉及这种排列的鸟嘌呤，从而阐明G4-TBA结构（图2C）。
该实验的一个可能的缺点是反应环境中存在不需要的亲核试剂。例如，从反应缓冲液中的盐中提取的亲核试剂与DNA竞争与B-CePs的反应性，导致探针与底物的反应效率低下。图3显示了在含有亲核Tris碱基的Tris缓冲液中进行的探测反应的结果。将B-CeP 1（5和50μM）与2μM样品的G4-TBA（图3的左泳道），ssTBA（图3的中央泳道）和dsTBA（图3的右泳道）反应1小时，4小时，15小时和24小时。在这些条件下，探针对所有三种DNA底物的反应性明显降低。对于G4-TBA样品，我们只能观察到加合物的形成，而不能检测到链断裂。在ssTBA和dsTBA的情况下，只有在24小时后，DNA片段化才与图2B中孵育15小时后观察到的DNA片段相当。B-CeP与Tris的不良竞争反应性导致缺乏核酸结构信息。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65373/65373fig02.jpg"> 图 2：B-CeP 1 在 BPE 缓冲液中对 TBA G 四链体结构的化学定位。 （A） B-CeP 1 的化学结构。（B） BPE缓冲液中B-CeP 1和TBA探测反应的时间和浓度依赖性。TBA的等分试样在三种不同的条件下折叠，以获得G-四链体排列（G4-TBA）、单链寡核苷酸（ssTBA），TBA退火到其互补序列以形成双链构建体（dsTBA）。TBA寡核苷酸在3'端用荧光基团（FAM）标记，以实现凝胶系统的可视化。G4-TBA，ssTBA和dsTBA的等分试样（2μM）在BPE缓冲液中以5或50μM终浓度处理，并在37°C下孵育指定时间。通过高分辨率变性PAGE（20%聚丙烯酰胺[PAA]，7M尿素，1x TBE）分析探测反应的结果。“C”表示每个TBA构建体的对照样品。（C） G4-TBA的G-四链体排列和TBA序列中鸟嘌呤编号的动画。唯一不参与 G4 结构的鸟嘌呤，因此与 B-CeP 1 反应，以红色突出显示。绿色星号代表荧光基团FAM。 缩写：TBA = 凝血酶结合适配体;B-CeP 1 = 双-3-氯哌啶 1;BPE = 磷酸氢盐-EDTA;G = 鸟嘌呤;G4 = 鸟嘌呤四链体;ss = 单股;ds = 双链;FAM = 5-羧基荧光素;PAA = 聚丙烯酰胺;PAGE=聚丙烯酰胺凝胶电泳;TBE=三硼酸盐-EDTA。<a href="https://www.jove.com/files/ftp_upload/65373/65373fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65373/65373fig03.jpg"> 图 3：存在 Tris 缓冲液时化学分布效率低下。 Tris缓冲液中B-CeP 1和TBA探测反应的时间和浓度依赖性。TBA的等分试样在三种不同的条件下折叠，以获得G-四链体排列（G4-TBA）、单链寡核苷酸（ssTBA），TBA退火到其互补序列以形成双链构建体（dsTBA）。TBA寡核苷酸在3'端用荧光基团（FAM）标记，以实现凝胶系统的可视化。G4-TBA、ssTBA和dsTBA的等分试样（2μM）在10mM Tris-HCl缓冲液中以5或50μM终浓度处理，并在37°C下孵育指定时间。通过高分辨率变性PAGE（20%PAA，7M尿素，1x TBE）分析探测反应的结果。“C”表示每个TBA构建体的对照样品。在探测反应过程中用作缓冲液的Tris的亲核性导致信号降低和结构信息丢失。缩写：TBA = 凝血酶结合适配体;B-CeP 1 = 双-3-氯哌啶 1;BPE = 磷酸氢盐-EDTA;G = 鸟嘌呤;G4 = 鸟嘌呤四链体;ss = 单股;ds = 双链;FAM = 5-羧基荧光素;PAA = 聚丙烯酰胺;PAGE=聚丙烯酰胺凝胶电泳;TBE=三硼酸盐-EDTA。 <a href="https://www.jove.com/files/ftp_upload/65373/65373fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图S1：G4-TBA、ssTBA和dsTBA的CD谱图。在三种不同结构正确折叠后，在25°C下记录CD光谱。 dsTBA 的 CD 光谱以 ~260-280 nm 处的正长波段和 ~245 nm 处的负波段为特征16，与 DNA 螺旋的 B 型一致。与ssTBA相比，ssTBA在没有K+离子的情况下结构较差，G4-TBA在K+离子存在下折叠成反平行G4，其特征在于260nm处的负带和290nm处的正带15。缩写：TBA = 凝血酶结合适配体;B-CeP 1 = 双-3-氯哌啶 1;BPE = 磷酸氢盐-EDTA;G = 鸟嘌呤;G4 = 鸟嘌呤四链体;ss = 单股;DS = 双链。<a href="https://www.jove.com/files/ftp_upload/65373/CD.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping at JoVE.com

作者聚焦：通过基于 Bis-3-氯哌啶的化学定位表征 DNA G-四链体 

双-3-氯哌啶 （B-CePs） 是有用的化学探针，用于在体外鉴定和表征 DNA 模板中的 G-四链 体结构。该协议详细介绍了用B-CeP进行探测反应和通过高分辨率聚丙烯酰胺凝胶电泳分离反应产物的过程。

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

G-四链体是核酸二级结构，通常在富含鸟嘌呤的 DNA 序列中形成。B-CePs 对 G4 的化学定位旨在对 G4 进行体外表征，并允许鉴定形成 G-四分体的特定鸟嘌呤。在潜在的G-四链体形成序列中识别和实验验证G4s是一个生物学相关问题，目前主要通过计算工具解决。为了支持这种预测并检测未预测的G4，B-CePs的化学图谱代表了一种在DNA序列中识别G4的可行方法。B-CePs 的化学图谱是广泛使用的硫酸二甲酯足迹方案的便捷替代方案，用于表征 G-四链体。B-CePs的主要优点是它减少了方案中的步骤数，并减少了DNA底物的初始量。本文描述了 B-CePs 的化学图谱，并使用 TBA 序列作为概念证明。该协议将导致适用于任何其他潜在的G-四链体形成序列的新实验，探索DNA和RNA结构。

in-vitro-chemical-mapping-g-quadruplex-dna-structures-bis-3

Nanyang Technological University

Research

JoVE Journal

Biochemistry

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

1.2K 次观看.  University of Padova. G-四链体是核酸二级结构，通常在富含鸟嘌呤的 DNA 序列中形成。B-CePs 对 G4 的化学定位旨在对 G4 进行体外表征，并允许鉴定形成 G-四分体的特定鸟嘌呤。在潜在的G-四链体形成序列中识别和实验验证G4s是一个生物学相关问题，目前主要通过计算工具解决。为了支持这种预测并检测未预测的G4，B-CePs的化学图谱代表了一种在DNA序列中识别G4的可行方法。B-CePs 的化学图谱是?...

视频: 作者聚焦：通过基于 Bis-3-氯哌啶的化学定位表征 DNA G-四链体 

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

科研

遗传学

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.

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 ...

生物化学

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

CD光谱学研究DNA-蛋白质相互作用

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

生物学

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.