Name: kinetic screening of nuclease activity using nucleic acid probes
Uploaded: 2019-11-01T12:30:00
Description: Watch this Scientific Journal Video about Kinetic Screening of Nuclease Activity using Nucleic Acid Probes at JoVE.com

作者要感谢路易莎·埃尔南德斯（林克平大学）对手稿的认真修订和宝贵建议。这项工作得到了克努特和爱丽丝·沃伦贝格基金会以及瑞典政府战略研究区在林雪平大学高级功能材料材料战略研究区的支持（学院授予SFO-Mat-LiU No.2009-00971）。

1. 橄榄核苷酸库的设计与准备
<ol><li>图书馆设计<ol>
<li>根据以下标准设计一个8至12mer长的淬火荧光寡核苷酸探针库，以避免二次结构形成，所有探头的长度相等。<ol>
<li>包括至少一个DNA和一个RNA随机序列，其中包含腺苷（A）、瓜宁（G）、细胞氨酸（C）和胸腺（T）/尿（U）（表1中的DNA和RNA探针的组合）。 注：表1中描述的DNA和RNA序列作为对未知核酸酶活性（DNase或RNase...

<ol>
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核酸酶活性的改变与各种各样的疾病表型有关，包括不同类型的癌症和细菌感染。这些改变被建议为一个条件14的致病剂，而在其他情况下，它们是有害生理事件20或致病剂16，26的结果。毫不奇怪，使用核酸酶和核酸酶活性作为诊断生物标志物的尝试被描述为15，22，23，26，具有相当大的前景。</...

核酸酶是一类酶，能够分解形成核酸分子骨干结构的磷酸键。核酸酶的多样化使得它们的分类相当困难。然而，有一些常用的标准用于描述核酸酶，如基质偏好（脱氧核糖核酸（DNA）或核糖核酸（RNA）），裂解位点（内糖酶或外糖酶），或金属组依赖，等等1。核酸酶是高度保守的催化酶，在原核生物和真核生物中具有基本作用，并已作为基因编辑工具2使用并继续使用。他们也是DNA维护和复制的基本参与者，帮助保持基因组稳定和参与校对过程3。例如，在细菌中，核酸酶被确定为重要的毒治因素，能够通过降低宿主免疫系统4、5、6、7的功效来促进细菌生存。，8.在哺乳动物中，核酸酶被建议与凋亡9、线粒体生物生成和维持10和抗菌和抗病毒先天免疫反应的中介有关11。毫不奇怪，核酸酶活性的改变，无论是增强还是缺乏，都与广泛的人类疾病有关。这些疾病范围从多种癌症12，13到心脏肥大10或自身免疫性疾病14。因此，核酸酶已成为人类异常群体的生物标志物，成为有趣的候选物。事实上，核酸酶已经显示出其作为检测特定细菌制剂（如金黄色葡萄球菌或大肠杆菌15）引起的感染的成功诊断工具的潜力。 16.在许多癌症类型中，葡萄球菌类核酸酶领域含微糖蛋白1（SND1）核糖酸酶的表达表明预后不佳17。在胰腺癌患者中，升高的核糖核酸I（RNase I）血清水平已报告18，并提议与癌细胞表型19相关。在缺血性心脏疾病中，如心肌梗死或不稳定的心绞痛，脱氧核糖核酸I（DNase I）血清水平已被证明是有效的诊断标记20，21。
据推测，在健康和疾病状态中，核酸酶活性的全球蓝图可能有所不同。事实上，最近的报告已经利用核酸酶活性的差异来区分健康表型22或以特定物种15、23的方式识别致病性细菌感染。这些发现为利用核酸酶作为疾病的生物标志物开辟了一条新途径。因此，有必要开发一种全面的筛选方法，能够系统地查明核酸酶活动中与疾病相关的差异，这可能对开发新的诊断工具至关重要。
在此，我们介绍并描述了一种新的体外筛选方法（图1），以识别敏感和特定的探针，这种探针能够区分健康不健康中的核酸酶活性，或特定于某类细胞或细菌的活动。利用核酸的模块化，我们设计了一个初始的淬火荧光寡核苷酸探针库，该库由一套全面的不同序列和化学成分组成，都是库设计的重要参数。这些寡核苷酸探针的两侧分别有荧光团（氟辛石、FAM）和5'和3'端的环流（潮汐淬火2，TQ2）（表1）。通过使用这种基于荧光共振能量转移 （FRET） 的荧光测定来测量酶降解动力学，我们能够识别具有识别核酸酶活性差异模式的候选探针与健康或疾病状态相关。我们设计了一个迭代过程，其中基于最佳候选探测器创建新库，从而可以在后续筛选步骤中识别更具体的候选探测器。此外，这种方法利用核酸酶的催化性质来增加灵敏度。这是通过利用报告器探针的可激活性以及核酸酶持续处理基质分子的能力来实现的，这两者都代表了替代抗体或基于小分子的筛选方法的关键优势。
这种方法提供了一个高度模块化，灵活和易于实施筛选工具，用于识别能够区分健康状态和疾病状态的特定核酸探针，并为开发新的可适应未来临床应用的诊断工具。因此，这种方法用于识别从沙门氏菌（以下简称沙门氏菌）衍生的核酸酶活性，以特定鉴定这种细菌。在下面的协议中，我们报告一种使用动力学分析筛选细菌核酸酶活性的方法。

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		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Black bottom, non-treated 96 well plate</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:128px;">10000631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Cytation1</td>
			<td style="width:199px;">BioTek</td>
			<td style="width:128px;">CYT1FAV</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Eppendorf tubes</td>
			<td>Thermofisher</td>
			<td style="width:128px;">11926955</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Escherichia coli</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:128px;">25922</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Microbank cryogenic storage vial containing beads</td>
			<td style="width:199px;">Pro-Lab Diagnostics</td>
			<td style="width:128px;">22-286-155</td>
			<td></td>
		</tr>
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			<td height="38" style="height:39px;width:344px;">Nucleic acid probes</td>
			<td style="width:199px;">Biomers.net</td>
			<td style="width:128px;">#</td>
			<td></td>
		</tr>
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			<td height="38" style="height:39px;">Phosphate Buffer Saline containing MgCl2 and CaCl2</td>
			<td style="width:199px;">Gibco&#8482;</td>
			<td style="width:128px;">14040117</td>
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			<td height="38" style="height:39px;width:344px;">Salmonella enterica subs. Enterica</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:128px;">14028</td>
			<td></td>
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			<td height="38" style="height:39px;">Tris-EDTA</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:128px;">10647633</td>
			<td></td>
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			<td height="38" style="height:39px;width:344px;">Tryptone Soya Agar with defibrinated sheep blood</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:128px;">10362223</td>
			<td></td>
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			<td height="38" style="height:39px;width:344px;">Tryptic Soy Broth</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:128px;">22092</td>
			<td></td>
		</tr>
	</tbody>
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kinetic screening of nuclease activity using nucleic acid probes

核酸酶是一类酶，通过催化连接核糖的磷酸键的水解来分解核酸。核酸酶在原核生物和真核生物中表现出各种重要的生理作用，从保持基因组稳定到提供对病原体的保护。改变的核酸酶活性与几种病理疾病有关，包括细菌感染和癌症。为此，核酸酶活性显示出作为特定生物标志物被开发的巨大潜力。然而，基于此活动的可靠和可重复的筛选方法仍然非常可取。
本文介绍一种利用核酸探针作为基质进行核酸活性筛选的方法，其范围是区分病理条件和健康条件。此方法提供了以迭代方式设计新的探测库的可能性，其特异性也越来越高。因此，利用化学修饰的核酸的可用性，需要进行多轮筛选，以增强特性改进探针的设计。所拟提出的技术的巨大潜力在于其灵活性、高可重复性和用于筛查与疾病条件相关的核酸酶活性的多功能性。预计这项技术将开发有前途的诊断工具，在临床上具有巨大的潜力。

图 1显示了此方法的工作流程，该方法分为两个筛选轮。在第一轮筛选中，我们使用5个DNA探针（DNA、DNA聚A、DNA聚A、DNA聚子和DNA聚氨酯）和5个RNA探针（RNA、RNA聚A、RNA聚Dna聚氨酯和RNA聚氨酯）。本筛选轮的原始数据可在补充表 1 中找到。在第二轮中，通过用化学修饰的核苷（所有2'-Fluoro和所有2'-O甲基）替换RNA序列，或者结合RNA和纯化或化学修饰的紫杉醇（RNA Pyr-2...

Watch this Scientific Journal Video about Kinetic Screening of Nuclease Activity using Nucleic Acid Probes at JoVE.com

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

改变的核酸酶活性与不同的人类条件有关，这是其作为生物标志物的潜力的基础。本文提出的模块化且易于实施的筛选方法允许选择特定的核酸探针，以利用核酸酶活性作为疾病的生物标志物。

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

该协议为筛选核酸酶活性作为疾病的生物标志物提供了一种强有力的替代方案，即使对于不太专门研究核酸探针的研究人员，也提供了易于实施的方法。该技术的主要优点是能够选择核酸探针，可以识别已知和未知的核酸酶活动，利用探针-核酸酶动态相互作用。这种方法的其他优点是灵活性、高可重复性和易用性。示范将由硕士生卡迪娅和来自我们实验室的博士后巴里斯进行。在设计寡核苷酸库时，至少包括一个DNA和一个RNA随机序列，其中包含腺氨酸、瓜氨酸、细胞氨酸和硫胺或乌拉西尔的组合。为了准备寡核苷酸探针，旋转低干寡核苷酸探针，以每微升浓度500皮摩尔稀释Tris-EDTA缓冲液中的每个探针，以防止核酸酶降解。对于固体介质上的细菌培养，将单个多孔玻璃珠从低温储存直接卷到含有 TSA 的培养皿上，并辅以除颤羊血，以划掉单个细菌菌落。然后将板在37摄氏度下放置24小时。对于液体介质中的细菌培养，将单个菌落从固体培养体转移到 50 毫升 TSB，在 37 摄氏度下孵育，每分钟 200 次旋转 24 小时。第二天，在新鲜的TSB中，以1比500的比例稀释培养物，并在37摄氏度下再孵育细菌24小时，并在摇动的培养箱中每分钟旋转200次。要建立核酸酶活性测定，首先将荧光计预热至37摄氏度，并小心地将96微升无菌TSB或上清液从沙门氏菌或大肠杆菌液体培养物添加到每个探针1个1.5毫升无核酸酶微离心管中。在每个管中加入四个微升探针工作溶液，并使用移液器彻底混合每个管内，直到实现均匀溶液，注意避免气泡。接下来，小心地将95微升的每个溶液装到靠近黑底壁的个别井壁上，不经过处理的96孔板，注意避免气泡。添加所有溶液后，盖住板并目视检查盖子上是否带有笔标记或灰尘，这些标记或灰尘可能会引入测量伪影。要设置核酸酶活性测量软件，请打开合适的采集软件程序。从"任务管理器"窗口中选择"现在阅读"，然后选择"新"以创建动力学测量协议。单击"设置温度"以选择 37 摄氏度，然后单击"确定"确认并保存设置。单击"开始动力学"。在弹出窗口中，在"运行时"输入框中选择两个小时，在间隔输入框中选择两分钟，然后单击"确定"以确认和保存设置。单击"阅读"。在弹出窗口中，选择荧光强度作为检测方法，选择端点动力学作为读取类型，选择滤波器作为光学类型。然后单击"确定"。在弹出窗口中，从"筛选集"中选择"绿色"，然后单击"确定"。在"过程"窗口中，选择"使用盖子"并单击"验证"。将出现一个弹出窗口，确认创建的协议有效。在"协议"菜单下，选择"过程"。在"过程"窗口中，定义要测量的井，并在"文件名称输入"框中输入实验的名称。然后将板加载到板读卡器中，注意板位于右方向，然后单击"读取新"按钮开始采集。对于数据分析，请在相应的分析软件中打开数据，并在一个窗口的板中选择一个测量孔。单击"选择井"并在"井选择对话框"窗口中包括所有测量的孔，然后单击"确定"。然后选择板一窗口中的数据以可视化制表结果，然后单击"快速导出"将数据导出到电子表格。在电子表格中，将数据列标记为适合每个样本和探头，并绘制相对荧光单位与数据生成动能图的时间。在这个具有代表性的实验中，在第一轮筛查之后，沙门氏菌培养超先剂报告了RNA探针比DNA探针的明显偏好。基于对RNA作为沙门氏菌核酸酶首选核酸类型的鉴定，设计了一个新的RNA库，用于第二轮筛选。相比之下，培养基对控中的E.Coli表明，降低RNA探针的能力非常有限。在使用化学修饰核苷酸进行第二轮筛选，旨在提高RNA探针的特异性后，可根据其化学修饰确定RNA苯丙胺2'O-甲基和RNA紫菜2'O-甲基分别表现出最佳的动力学行为，与RNA pyrimidine 2'氟和RNA紫福二氟相比，表现出最佳的动力学行为。这些结果表明，沙门氏菌具有重要的RNAse活性，具有差分基质化学偏好，可用于选择能够专门识别这种细菌的探针。此过程能够选择能够识别与疾病条件（如癌症或细菌感染）相关的核酸酶活动的探针，从而开发新的临床诊断工具。

kinetic-screening-of-nuclease-activity-using-nucleic-acid-probes

Research

JoVE Journal

Biochemistry

A Polyaniline-based Sensor of Nucleic Acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

核酸的聚苯胺传感器

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

科研

生物化学

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

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

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

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

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

使用硫二甘酸（TGA）估计植物生物质木质素含量

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

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 ...

使用化学作图鉴定 DNA 模板中的 G-四链体

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

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

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 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

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 ...