Name: continuous fluorescence-based endonuclease-coupled dna methylation assay to screen for dna methyltransferase inhibitors
Uploaded: 2022-08-05T13:00:00
Description: Watch this Scientific Journal Video about Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors at JoVE.com

作者感谢巴克内尔大学和化学系对这项工作的支持。

1. 为屏幕准备检测解决方案
注意：可以调整该测定中使用的底物浓度。对于缺乏RFTS的DNMT1，实验确定的发夹DNA底物和SAM的Km 值分别为1-2 nM和2μM，分别为26，40。
<ol><li>准备 600 μL 在冰上的微量离心管中测试的每个测定条件。 注意：所需的测定溶液总量取决于正在进行的重复次数。对于该协议，每个测定条件...

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为了鉴定和表征DNA甲基转移酶的抑制剂，必须测量酶的活性。存在几种检查DNA甲基转移酶活性的方法。通常使用放射性监测活动;SAM标记甲基的转移可以定量为29，30，31，32，33，34。利用甲基敏感核酸内切酶的基于凝胶的测定也已被描述为<sup class="xr...

DNA甲基化是调节基因表达和染色质结构的重要表观遗传标记。甲基化主要发生在 CpG 二核苷酸中——胞嘧啶，然后是鸟苷;将甲基加入到胞嘧啶的5位。正确的DNA甲基化模式，从而正确的基因表达，对于适当的细胞发育和功能是必需的。许多疾病状态与正常甲基化模式的改变有关1，2，3。例如，癌症的发生和进展以及DNA甲基化模式的改变之间存在联系。通常，癌细胞表现出较低的甲基胞嘧啶总体水平，这会导致基因组不稳定。同时，存在于基因组中的甲基胞嘧啶集中在肿瘤抑制基因的启动子区域，这导致这些重要蛋白质的基因沉默。值得注意的是，表观遗传变化是动态和可逆的，这与与肿瘤发生相关的DNA突变不同。这使得参与表观遗传基因调控的蛋白质成为有趣的药物靶点2，4。
DNA甲基转移酶（DNMT）是负责产生和维持DNA甲基化模式的蛋白质。三种催化活性同工酶DNMT1，DNMT3a和DNMT3b存在于人类中。在发育和分化过程中， 从头 甲基转移酶DNMT3a和DNMT3b建立甲基化模式。两种酶都可以结合催化无活性的DNMT3L蛋白，形成活性增加的复合物1，5。细胞分裂后，子细胞含有半甲基化的DNA——仅在双链的一条链中含有甲基胞嘧啶的DNA——因为新合成的DNA没有甲基化标记。DNMT1的主要功能是甲基化这种半甲基化的DNA，从而重建完整的甲基化模式1，5。
DNMT活动与癌症之间的联系已经确立。DNMT1的过表达，无论是通过转录还是翻译后机制，都是几种常见致癌途径的结果6，7，8，9。使用亚态性等位基因降低DNMT1活性的遗传方法导致Apc（Min）小鼠10的肿瘤形成减少。敲低DNMT1的反义寡核苷酸抑制细胞培养和小鼠肿瘤模型中的肿瘤形成11，12。因此，抑制DNMT1活性似乎是一种有前途的癌症治疗方法。然而，DNMT3同工酶所扮演的角色并不是那么简单。DNMT3a 突变见于急性髓系白血病13 和骨髓增生异常综合征14。已鉴定的突变中至少有一个已被证明会降低酶15的DNA甲基化活性。然而，DNMT3b在乳腺癌16和结直肠癌17中过表达。由于各种DNMT同工酶在致癌作用中发挥不同的作用，因此鉴定同工酶特异性抑制剂至关重要。这些化合物不仅可用于治疗药物的开发，而且同工酶特异性抑制剂也将成为剖析每种DNMT同工酶在癌症病因学中的作用的宝贵工具。
文献中报道了几种DNMT抑制剂。已知的DNMT抑制剂可分为两类：核苷和非核苷。核苷抑制剂通常是胞苷类似物。这些化合物被掺入DNA中并共价捕获DNMT。 5-氮杂胞苷和5-氮杂-2'-脱氧胞苷已被批准用于治疗骨髓增生异常综合征和急性髓系白血病4，18。这些化合物的高毒性、低生物利用度和化学不稳定性存在问题。正在进行的工作正在研究下一代核苷抑制剂的功效;SGI-110来源于5-氮杂-2'-脱氧胞苷，就是一个例子19，20。核苷抑制剂不是同工酶特异性的，会使遇到的任何DNMT同工酶失活。因此，用核苷去甲基化剂处理会导致所有DNMT同工酶4，18的耗竭。非核苷抑制剂不需要掺入DNA中即可发挥其抑制作用。相反，这些分子直接与DNMT结合，引入了同工酶特异性抑制的可能性。迄今为止，已经发现了几种非核苷抑制剂，包括SGI-102721，肼屈嗪22，普鲁卡因酰胺23，RG108及其衍生物24，以及天然产物（−）-表没食子儿茶素3-没食子酸酯（EGCG）25和乳酸A26，27。迄今为止发现的大多数非核苷抑制剂不是同工酶选择性的，或者对一种DNMT同工酶表现出弱的偏好。此外，这些分子的效力需要提高，特别是在细胞4，18中。因此，需要发现或开发更有效的同工酶选择性DNMT抑制剂。
发现新的DNMT小分子抑制剂的一个障碍是传统上用于检查DNMT活性的费力测定28。检测通常是不连续的，有多个步骤。DNMT的酶活性仍然常规使用放射性S-腺苷甲硫氨酸（SAM）29，30，31，32，33，34进行测定。DNA甲基化的非放射性测定也已经开发出来。例如，利用甲基敏感限制性核酸内切酶和电泳分离消化产物的测定已经描述35，36。这些类型的不连续、多步骤测定不容易用于药物发现。自 2000 年代中期以来，已经开发了几种具有更高通量的 DNA 甲基化测定28。闪烁邻近测定用于筛选DNMT1抑制剂37。另一种利用甲基敏感限制性核酸内切酶的测定用于筛选DNMT3a抑制剂25，38。虽然这两种检测方法都比传统的DNA甲基化测定具有更高的通量，但这些测定需要多个步骤，并且不允许实时观察甲基化活性。最近，已经描述了一种连续的动力学测定，该测定将甲基化反应的一种产物S-腺苷同型半胱氨酸（SAH）的形成与NADPH氧化39相关的340nm处的光谱变化耦合。该测定利用三种偶联酶产生光谱信号。
我们开发了一种基于荧光的核酸内切酶偶联DNA甲基化测定，该测定利用了单一的市售偶联酶，可以实时生成数据（图1）。含有三个甲基胞嘧啶的发夹寡核苷酸用作底物。底物DNA在5'端包含一个荧光团，在3'端包含一个淬灭剂。半甲基化CpG位点的甲基化产生核酸内切酶Gla I的切割位点 - 完全甲基化的GCGC。产物寡核苷酸的Gla I裂解从淬灭剂中释放荧光团并实时产生荧光。该测定可用于检查DNMT任何亚型的活性;然而，DNMT1观察到更高的活性，因为该同工酶优先甲基化半甲基化的DNA1，5。如果从DNMT1中删除自动抑制性复制病灶靶向序列（RFTS）结构域，则可以观察到更强大的活性。该结构域位于N末端调控区，与催化位点结合并阻止DNA结合。去除前 ~600 个氨基酸会导致截短酶比全长酶更活跃（kcat/Km 增加 ~640 倍）40。这种酶的活化形式，称为缺乏RFTS的DNMT1（氨基酸621-1616），由于其增加的催化能力，可以更容易地鉴定抑制剂。本文提出了一种在测定中利用缺乏RFTS的DNMT1来筛选潜在小分子抑制剂的方案。使用核酸内切酶偶联连续测定，在存在和不存在几个小分子的情况下确定初始速度。在两种浓度下检查每种潜在抑制剂，以寻找浓度依赖性DNMT1抑制。计算在每种情况下在存在小分子的情况下观察到的活性百分比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62949/62949fig01.jpg"> 图1：DNA甲基化测定。 使用半甲基化的发夹DNA，5'端有荧光团，3'端有淬灭剂作为底物。DNMT1催化甲基从 S-腺苷甲硫氨酸转移到非甲基化的CpG位点，产生 S-腺苷同型半胱氨酸和完全甲基化的DNA。DNA产物含有核酸内切酶Gla I的切割位点，该位点可切割完全甲基化的GCGC位点。产物DNA的切割从3'淬灭剂中释放5'荧光团，产生荧光。缩写：Fl = 荧光团;Q =淬火;DNMT1 = DNA甲基转移酶1;SAM = S-腺苷蛋氨酸;SAH = S-腺苷同型半胱氨酸。 <a href="https://www.jove.com/files/ftp_upload/62949/62949fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:791px;" width="791">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">96-well Half Area Black Flat Bottom Polystyrene Not Treated Microplate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:119px;">3694</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">96-Well Polystyrene Conical Bottom Plates</td>
			<td style="width:100px;">ThermoFisher</td>
			<td style="width:119px;">249570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Bovine Serum Albumin</td>
			<td style="width:100px;">NEB</td>
			<td style="width:119px;">B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 1</td>
			<td style="width:100px;">ChemBridge</td>
			<td>5812086</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 2</td>
			<td style="width:100px;">ChemBridge</td>
			<td>6722175</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 3</td>
			<td style="width:100px;">ChemBridge</td>
			<td>5249376</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dithiothreitol</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Gla I</td>
			<td style="width:100px;">SibEnzyme</td>
			<td style="width:119px;">E494</td>
			<td>methyl-sensitive endonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glycerol</td>
			<td style="width:100px;">RPI</td>
			<td style="width:119px;">G22025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Magnesium Chloride</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">M0250</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:251px;">Oligonucleotide (5&#39;-FAM-CCTATGCGmCATCAGTTTTCTGATGmCGmCATAGG-3&#39;-Iowa Black Quencher)</td>
			<td style="width:100px;">IDT</td>
			<td style="width:119px;">custom synthesized</td>
			<td>internally quenched hairpin DNA (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium Glutamate</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">G1501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">S-adenosylmethionine</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">A4377</td>
			<td>methyl-donating co-factor (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tris Base</td>
			<td style="width:100px;">RPI</td>
			<td style="width:119px;">T60040</td>
			<td></td>
		</tr>
	</tbody>
</table>

continuous fluorescence-based endonuclease-coupled dna methylation assay to screen for dna methyltransferase inhibitors

DNA甲基化是表观遗传基因调控的一种形式，对正常的细胞功能很重要。在细胞中，称为DNA甲基转移酶（DNMT）的蛋白质建立并维持DNA甲基化模式。正常DNA甲基化模式的变化与癌症的发展和进展有关，使DNMT成为潜在的癌症药物靶标。因此，鉴定和表征这些酶的新型小分子抑制剂非常重要。本文提出了一种可用于筛选DNA甲基转移酶抑制剂的方案。连续偶联动力学测定允许在存在和不存在潜在小分子抑制剂的情况下确定DNA甲基化的初始速度。该测定使用甲基敏感的核酸内切酶Gla I将半甲基化DNA底物的甲基化偶联到荧光生成。
这种连续测定允许实时监测酶活性。在微量滴定板中进行小体积测定可降低试剂成本。使用该测定方法，对DNMT1抑制剂进行了小型示例筛选，DNMT1是人类中最丰富的DNMT同工酶。高度取代的蒽醌天然产物乳酸A是一种有效的DNA竞争性DNMT1抑制剂。在这里，我们检查三种潜在的小分子抑制剂 - 蒽醌或具有一到三个取代基的蒽醌样分子 - 在两种浓度下描述测定方案。初始速度用于计算在每个分子存在下观察到的活性百分比。检查的三种化合物中的一种表现出DNMT1活性的浓度依赖性抑制，表明它是DNMT1的潜在抑制剂。

活动 DNMT1 是此分析的先决条件。缺乏RFTS的DNMT1在 大肠杆菌 中表达，并按照先前发表的程序纯化至均一性41。为了确保纯化的酶具有活性，使用不连续的核酸内切酶偶联测定来检查DNA甲基化活性36。该测定利用位于Sau3A1切割位点的32个碱基对双链DNA和单个半甲基化CpG。Sau3A1可以切割半甲基化的底物DNA;然而，当DNA完全甲基化时，切割被阻断。将缺乏RFTS的DNMT1...

Watch this Scientific Journal Video about Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors at JoVE.com

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA甲基转移酶是潜在的癌症药物靶标。在这里，提出了一个协议来评估DNA甲基转移酶抑制的小分子。该测定利用核酸内切酶将DNA甲基化与荧光生成偶联，并允许实时监测酶活性。

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

像我们这样强大的酶标仪检测对于潜在甲基转移酶抑制剂的初步筛选非常有用。实时收集数据的能力是连续核酸内切酶偶联测定的一大优势。首先在冰上的微量离心管中分别制备含有20微摩尔和50微摩尔化合物的600微升测定条件。为此，向每个试管中加入双蒸水和5X甲基化缓冲液，以达到1X甲基化缓冲液的最终浓度。接下来，向每个样品中添加 3.15 微升每毫升 20 毫克牛血清蛋白或 BSA。然后，向每个样品中加入两微升 3.15 微摩尔发夹 DNA 底物和 1.33 微升 4.75 微摩尔 S-腺苷蛋氨酸或 SAM。向适当的样品中加入抑制剂，以达到21微摩尔或52.5微摩尔的终浓度和等量的二甲基亚砜（DMSO），然后通过以每分钟3， 000转的速度涡旋3秒来混合溶液。然后，将样品在1， 200倍G的台式小型离心机中旋转几秒钟，以确保溶液收集在管底部。将每种测定溶液的95微升等分到黑色半区96孔板中的六个连续孔中。如前所述，在微量离心管中制备75微升Gla I或DNMT1加Gla I酶溶液至一次性缓冲液的终浓度。接下来，向每种溶液中加入 1.2 微升每微升 Gla I 的 10 单位，以达到每微升 0.16 单位的最终浓度。向DNMT1加Gla I溶液中加入0.6微升5微摩尔缺乏RFTS的DNMT1，最终浓度为40纳摩尔。轻轻混合后，将样品在1， 200 G的台式小型离心机中旋转几秒钟，以确保溶液收集在管底部。然后，将12微升每种酶溶液等分到锥形底部96孔板中的六个孔中。对于DNA样品的甲基化测定，将酶标仪预热至37摄氏度。然后，将含有测定溶液的黑板插入酶标仪中。以425 CPM摇动板后，以485纳米的激发波长和528纳米的发射波长测量荧光。然后，将板在 37 摄氏度下孵育五分钟。从读板器中取出测定板，向每个孔中加入5微升酶溶液，然后通过上下移液混合溶液。将板插入读板器并摇动板，然后每53秒记录一次荧光，持续30分钟。获得每种条件的一式三份Gla I对照测定的平均荧光，并从在缺乏RFTS的DNMT1存在下获得的三次重复迹线中减去含有对照反应托盘的平均Gla I。然后，确定校正重复的平均值和标准误差。绘制平均校正反应迹线并将初始线性部分拟合到一条线上以确定初始速度。通过将在化合物存在下观察到的速度除以在含有对照反应的DMSO中观察到的速度并乘以100来确定活性百分比。不连续的核酸内切酶偶联测定的结果表明，完全甲基化的产物DNA被保护免受切割，证实了缺乏RFTS的DNMT1具有活性并且能够甲基化半甲基化底物DNA。在基于荧光的测定中，在没有Gla I的情况下，单独添加DNMT1或缓冲液不会影响背景荧光。随后将Gla I添加到所有测定中，仅在含有DNMT1的测定中产生荧光。将缺乏RFTS的DNMT1和Gla I同时添加到三种测定中，可产生稳定的荧光。单独添加Gla I不会产生荧光。为了筛选潜在的抑制剂，在DMSO，化合物一，化合物二或化合物三存在下，以20和50微摩尔浓度检查缺乏RFTS的DNMT1的DNA甲基化活性。在这种偶联测定中减少荧光产生的化合物必须进一步验证。确保化合物不抑制偶联酶是重要的下一步。

Research

JoVE Journal

Biochemistry

A Fluorescence-based Assay of Phospholipid Scramblase Activity

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

科研

生物化学

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

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

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

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

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

CD Spectroscopy to Study DNA-Protein Interactions

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 ...

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...