Name: use of alu element containing minigenes to analyze circular rnas
Uploaded: 2020-03-10T12:00:00
Description: Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

这项工作得到了国防部国防部授予AZ180075的支持。斯特凡·斯塔姆感谢杰奎琳·努南捐赠。安娜·帕卢钦得到了德国学术交流项目DAAD的支持，贾斯汀·韦尔登是肯塔基大学马克斯·斯特克勒奖的获得者。

1. 结构设计
<ol><li>使用UCSC基因组浏览器24来识别循环RNA形成所需的重复元素，并将其纳入结构。重要的是，扩增的引素需要超出重复元素。</li>
	<li>将圆形RNA序列（补充图1是测试序列）粘贴到<a href="https://genome.ucsc.edu/cgi-bin/hgBlat?command=start" target="_blank">https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</a>并选择正确的有机体。提交序列并转到浏览器视图，缩小 1.5 倍或根据需?...

<ol>
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一般来说，圆形RNA是低丰1，这使得研究其功能和形成复杂化。与线性RNA13类似，使用报告器微基因可以识别调节圆形RNA形成的cis和跨作用因子。因此，这种方法产生假设，可以使用内源基因进一步测试。
最关键的步骤是记者基因的设计。DNA片段的酶组装（"吉布森克隆"27）有助于这种设计，因为它允许构建独立于限制位点?...

循环 RNA 圆形RNA（环RNA）是同价关闭的单滞留RNA，在大多数生物体中表达。它们通过将下游5'拼接站点连接到上游3'拼接站点生成，这个过程称为反拼接（图1A）1。在mRNA前序列，显示基础互补短为30-40nt，使回拼接位点在适当的对齐循环RNA形成2。在人类中，Alu元素1，约占基因组3的11%，由于自身互补4、5，在mRNA前形成广泛的双链RNA结构，从而促进环RNA1的形成。
目前，已经描述了环环境的三大功能。一些环RNA结合微RNA（miRNA），并通过封存行为像miRNA海绵6。循环RNA已涉及转录和后转录调节，通过竞争与线性拼接7或修改转录因子活动8。最后，循环RNA包含短的开放阅读框架和原理证明研究表明，他们可以翻译9，10。然而，大多数环苯A的功能仍然是神秘的。大多数循环RNA已经使用下一代测序方法11检测到。使用有针对性的RT-PCR方法对单个基因进行详细分析后发现，仍有大量圆形RNA有待发现。
使用记者基因分析mRNA前处理 从DNA报告器中提取的mRNA分析构造转染成细胞是研究替代预mRNA拼接的成熟方法，可应用于圆形RNA。一般来说，替代外子、其周围突长和构成外子被放大并克隆成真核表达向量。通常，缩内缩器会缩短。这些结构被转染成真核细胞，通常由RT-PCR13，14进行分析。该方法已被广泛用于绘制监管拼接站点和转效因子在联合转染实验13，15，16，17，18。此外，蛋白质表达微型基因的产生允许筛选改变替代拼接19，20的物质。
该方法已应用于圆形RNA。目前，至少有12个微型基因骨干在文献中进行了描述，并被汇总在表1中。除了基于tRNA的表达系统21，22，他们都依赖于聚合酶II启动子。在这里，我们描述了一种生成人类报告器微基因的方法，以确定循环RNA生成所涉及的cis和跨作用因子。图1显示了使用已发布报告基因23序列的方法的概述。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(PEI) Hydrochloride</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Builder tool</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Dark Reader Transilluminator.</td>
			<td>Clare Chemical Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic DNA assembly kit</td>
			<td>NEB</td>
			<td>E2621S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel and PCR cleanup kit</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyco Blue</td>
			<td>Thermo Fisher</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 cloning site</td>
			<td>Polycloning site</td>
			<td></td>
			<td><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf" target="_blank">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf</a></td>
		</tr>
		<tr>
			<td>Polymerase 1</td>
			<td>NEB</td>
			<td>M0491L</td>
			<td>Q5 DNA polymerase</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Biorad</td>
			<td>1725310</td>
			<td>Long range polymerase (NEB), iproof (BioRad)</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Qiagen</td>
			<td>206402</td>
			<td>Qiagen long range polymerase kit</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>12183025</td>
			<td>Ambion by Life Technologies</td>
		</tr>
		<tr>
			<td>RNAse R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td>Epicenter/Lucigen</td>
		</tr>
		<tr>
			<td>Stable competent cells</td>
			<td>NEB</td>
			<td>C3040H</td>
			<td>NEB stable cells</td>
		</tr>
		<tr>
			<td>Standard cloning bacteria</td>
			<td>NEB</td>
			<td>C2988J</td>
			<td>NEB5-alpha competent</td>
		</tr>
		<tr>
			<td>Web tool to design primers</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Web-based temperature calculations</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://tmcalculator.neb.com/#!/main" target="_blank">https://tmcalculator.neb.com/#!/main</a></td>
		</tr>
	</tbody>
</table>

use of alu element containing minigenes to analyze circular rnas

除了线性 mRNA 之外，许多真核基因产生圆形 RNA。大多数圆形RNA是通过将5'拼接位点与上游3'拼接位点在mRNA前结合而生成的，这个过程称为回拼接。这种循环可能得益于mRNA前的二级结构，这些结构使拼接点非常接近。在人类基因中，Alu元素被认为能促进这些二次RNA结构，因为Alu元素非常丰富，并且在mRNA前以相反方向存在时，表现出彼此的基础互补性。在这里，我们描述了大型Alu元素的生成和分析，该元素包含构成圆形RNA的记者基因。通过优化克隆协议，可以生成插入长度高达20kb的分子基因。他们在联合转染实验中的分析可以识别监管因素。因此，该方法可以识别与循环RNA形成有关的RNA序列和细胞成分。

报告基因允许确定影响循环RNA形成的调节因子。然而，这些报告基因很大，并且含有经常使DNA构造不稳定等重复性元素。由于其大尺寸，通常需要删除部分的内科，这是通过放大基因组片包含外子和较小的侧翼在电子部分。这些DNA片段是酶组装的，允许结构不受限制的酶。
从微管相关蛋白tau （MAPT） 生成的圆形RNA示例显示了微基因方法分析圆形RNA的应用。本例中使用的tau 9_12...

Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

我们克隆和分析生成圆形RNA的记者基因。这些报告基因大于构造，用于分析线性拼接并包含Alu元素。为了研究圆形RNA，构造被转染到细胞中，在去除线性RNA后使用RT-PCR分析产生的RNA。

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

该协议是重要的，因为它允许用户生成一个基因组表达系统，可以经历替代拼接，在这种情况下，形成循环RNA，可以测试其功能和疾病关联。该技术的主要优点是，在研究圆形RNA形成和功能的替代拼接时，它将为其他人在分子生物学和克隆中使用这一协议铺平道路。我给第一次尝试这种技术的人的建议是优化PCR条件。运行梯度或执行接触PCR与不同的退火温度和延长时间。通常，超过 6 KB 的较长片段将延长每个周期 1 KB，需要测试不同的退火温度以获得最佳放大。此方法的演示至关重要，因为它将为用户提供更好的可视化表示过程中比较困难的方面，尤其是底像的生成。要开始此过程，请加载 UCSC 基因组浏览器，并用它来识别循环 RNA 形成所需的重复元素，并将其合并到构造中。重要的是，用于放大的底剂需要位于重复元素之外。将圆形RNA序列粘贴到人类BLAT搜索中，并选择合适的生物体。提交序列，转到浏览器视图，并缩小 1.5 计时器或酌情。接下来，将鼠标悬停在重复元素上，以在浮动窗口中标识其子类型。阿卢元素在正弦线中。如果获取不正确的图像，请使用窗口下的默认轨道按钮重置浏览器。首先去查看，在USS基因组浏览器的顶部行的DNA下载窗口中显示的DNA序列。在排序格式选项中，选择扩展大小写/颜色选项。选择默认大小写为较低，然后为 NCBI RefSeq 选择切换大小写。为重复掩应选择下划线和粗体以及"一元"的"形体"。单击"提交"。将有外文作为大写字母和内文作为小写字母。检查 exon/intron 边框。如果浏览器显示反向补码，请返回并选择反向补码框，直到显示正确的 exon/intron 边框。接下来，将具有正确方向的文件复制到文字处理文档中并突出显示 exons。选择要放大的片段，确保内子不会以重复区域开始或结束，因为这些区域中的底因不会放大特定序列。首先，使用 Web 工具设计克隆的底项。对于向量序列，将插入位点添加为最后一个核苷酸，然后添加片段。由于矢量编号不是从给定的插入站点开始，因此向量中插入的站点位于，下游部分位于上游序列的前面。如果其熔点相距超过 4 摄氏度且在放大中不起作用，请调整底向器。在这项研究中，克隆并分析了产生循环RNA的记者基因。优化的 PCR 产品在含有 1X 凝胶的绿色 1% agarose 凝胶上分离。单个频段表示将用于酶DNA组装的PCR产品。然后从凝胶中切出这些带子并进行纯化。纯化的 PCR 产品没有达到预期尺寸，使用凝胶绿色，因此产品也分离在 1%的 agarose 凝胶上，随后沾染溴化乙二苯胺，以确保产品尺寸正确。首先，设置一个酶DNA组装套件。以一比二的摩尔比例合并矢量和插入。接下来，添加10微升的DNA组装主混合物。在50度下孵育样品60分钟。在孵育过程中在冰上解冻称职的细胞。电池的体积应为 50 微升。接下来，使用总组装反应转换称职的细胞。将冷冻组装产品的两微升添加到主管单元中。轻轻轻拂管子四到五次。不要漩涡。将混合物放在冰上30分钟。热在42摄氏度下冲击混合物30秒。然后把它放回冰上两分钟。在此之后，向管中加入950微升室温S、C介质。在37摄氏度下孵育60分钟，在300 RPM下摇晃。在此孵育过程中，加热两个含有适当抗生素的选择板。孵育后，将反应管在10，000克离心30秒，将细胞进行颗粒化，并在一个选择板上取出25%的细胞，将75%的细胞板化。在37摄氏度下孵育这些板块过夜。在你开始转染之前，通过限制消化检查你的DNA。在这里，一个代表性的tau微基因，包含外子9至12被削减与限制酶指示，以排除主要重组。对于转染，首先以pH2浓度每毫升1毫克的速度将线性聚乙烯盐酸溶解在水中。使用氢氧化钠使 pH 值高达 7，并使用 0.22 微米过滤器将溶液无菌过滤。将溶液存放在摄氏四度，直到准备好使用。然后将细胞分成六孔板的井中，让它们在含有10%FBS的DMEM介质中过夜生长。第二天，在无菌管中加入一微克报告的基因，并加入200微升的无菌过滤150毫摩尔氯化钠。通过漩涡混合。接下来，将聚乙二烯胺溶液加入到这种混合物中，每一微克DNA，聚乙二烯胺的比例为三微升。短暂离心，收集管底的样品。在室温下孵育样品10分钟。然后直接将其添加到 HEK293 细胞中。用5%的二氧化碳在37摄氏度下孵育细胞过夜。第二天，使用RNA隔离试剂盒分离RT-PCR的RNA。从来自人类脑组织的两个样本中提取的cDNA被放大与圆形RNA底色环tau exon 1210反向和圆tau exon 1211前进。虽然与tau圆形RNA对应的预期波段被看到，但其他强频段是与人类基因组不匹配的神器。本实验在相同的PCR条件下重复，但逆转录只能用圆头1210反向底因进行。只有预期的波段通过测序被放大和验证。然后使用RNARSe R进行治疗，去除线性RNA。循环RNA在治疗后可检测，而线性RNA则不再提供可检测信号。RNA在转染后24小时被分离，并由RT-PCR进行分析。线性 tau mRNA 的放大显示了两个波段，由于 exon 10 的替代拼接。它们的比例与拼接因子的过表情变化有关。圆形1210 tau RNA的扩增显示了tau圆RNA表达对一些拼接因子，特别是Cdc2样激酶CLK2和SR蛋白9G8的表达的依赖性。尝试此过程时，要记住的最重要的事情是构造微基因时底向器的设计和位置。底像不应位于重复元素中，需要根据被放大的片段在不同条件下进行优化。按照此过程，可以执行的其他方法将测试循环 RNA 的功能。用户可以测试蛋白质的转化或封存，以确定用户感兴趣的特定圆形RNA的功能。这项技术为利用微基因系统中的基因组片段铺平了道路，这种片段可以过度表达循环RNA，允许用户测试和识别其功能，并在某些方面与疾病关联。这项技术的含义延伸到特定疾病的潜在疗法和诊断，例如与tau病理学相关的神经退行性疾病。我们发现，微管相关蛋白tau，称为 MAPT，产生圆形RNA，我们相信，这是有助于疾病病理学和这种方法使我们能够充分研究这些循环RNA，并确定其功能和与疾病的关系。这种方法可以深入了解循环RNA，它们的功能是什么，以及它们在某些疾病中可能发挥的作用。此方法可用于克隆其他微型基因，这些小基因可以跨物种表达循环RNA，以便深入了解其功能。PCR产品的扩增被证明是最困难的，从基因组DNA中扩增的长片段，以及片段中具有多个重复元素。

use-of-alu-element-containing-minigenes-to-analyze-circular-rnas

Research

JoVE Journal

Biochemistry

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

科研

生物化学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

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

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

使用微尺度热泳测量蛋白质-脂质相互作用

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

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 multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

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

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

即用型qPCR，用于检测 克氏锥虫 或其他致病微生物的DNA

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 ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...