Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

下面的协议描述了用于执行基于荧光的RNA编辑分析的过程。该测定法可以在一个单一的PCR管，96孔或384孔取决于实验的范围板来进行。随后的荧光信号可以读合适的实时PCR检测系统。这里在测定中的384 - 孔板的上下文中被描述。 1，培养T。布氏细胞<ol><li>准备一个生长培养基为T。布氏 procyclic形式的细胞。 1 L培养基: <ol><li>溶解25.4克SDM-79粉末在800毫升miliQ水。 </li><li>添加2克的NaHCO 3和pH值至7.3，10 M氢氧化钠。 </li><li>加超纯水至900毫升，过滤消毒的最终体积。 </li><li>添加胎牛血清（FBS），青霉素 - 链霉素溶液和氯高铁血红素（2.5毫克/毫升）至终浓度为10％（体积/体积），分别为100单位/毫升和7.5毫克/升。 </li></ol></li><li>成长300毫升T. &lt;中EM&gt;布氏1.7A野生型（procyclic形式）细胞在28℃，在70转摇到1.5×10 7细胞/ ml的密度。注:这应该会产生3毫升活跃editosome与〜0.5毫克总蛋白，足够600编辑反应。 </li><li>离心6000×g离心10分钟收集细胞，在4℃下</li><li>用50ml冷PBSG缓冲液（10mM的磷酸氢二钠，10mM的的NaH 2 PO 4，145 mM氯化钠，以及6毫米的葡萄糖）洗涤沉淀，并再次离心收集到的细胞通过离心分离以10,000 xg在10分钟4°C。 </li></ol>原油线粒体2。隔离注意:所有的步骤应在冰上或4℃进行保存editosome活性。 <ol><li>重悬在30毫升的DTE缓冲液（1mM的Tris-HCl pH为8.0和1mM EDTA）中收获的细胞。用40毫升无菌Dounce匀浆（预冷）由抚摸着向上和向下的至少10倍于冰上，破坏细胞膜。 </LI&gt; <li>立即4.3毫升60％蔗糖（w / v的即 1.75 M）添加到匀浆至0.25米离心机的终浓度在15800×g下在4℃下10分钟，以优先减低线粒体。 </li><li>重悬线粒体沉淀4.6毫升STM缓冲液（20mM的Tris-HCl pH值8.0，250mM的蔗糖和2mM的MgCl 2）。添加13.8微升的0.1M的CaCl 2和4微升不含RNA酶的DNA酶I至终浓度分别为0.3毫米和9 U /毫升。为1小时，在冰上孵育混合物。 </li><li>添加4.6的STE缓冲液（20mM的Tris-盐酸pH值8.0，250mM的蔗糖和2mM EDTA）中在4℃以灭活DNA酶I。离心机在15800×g离心10分钟</li><li>重悬沉淀中加入400μl的裂解缓冲液（10mM的Tris-HCl（pH7.2）中的10mM的MgCl 2，100mM的氯化钾，1微克/毫升胃蛋白酶抑制素，1mM的DTT和1×完全不含EDTA的蛋白酶抑制剂），并转移到离心管。 </li><li>加10％的Triton X-100至1％的终浓度第二温育裂解物15分钟，在4℃下在管式旋转器。 </li><li>离心两次在17000×g离心15分钟，在4℃下清除线粒体裂解液;每次保留清除上清液。 </li></ol> 3。Editosome净化<ol><li>倒入10ml的10％-30％（V / V）线性梯度甘油（ 表1）中使用2个HHE梯度缓冲液（40mM的HEPES pH值为7.9，20mM的镁（醋酸）2，100mM的氯化钾超速离心管中，并2 mM EDTA）中，并通过下面的说明书的梯度壶。 </li><li>小心地从甘油梯度的顶部取出500微升的溶液，轻轻地加载500微升的清除线粒体裂解液。旋在178,000 xg离心6小时，在4℃下用超离心。 </li><li>收集500μl的级分连续地从顶部到梯度的在4℃下然后将其卡冻结使用液氮和储存在-80℃直至使用的馏分。 </li></ol><ol><li>退火含有互补序列的T7启动子序列（ 表2）与T7启动子的寡核苷酸（5&#39;-TAATACGACTCACTATAGGG-3&#39;）中，加热至90℃保持3分钟以1:1的摩尔比和冷却在RT相应DNA模板为至少10分钟。 </li><li>使用体外转录试剂盒由以下的说明书转录的RNA。 </li><li>停止转录反应通过加入7M尿素染料（7M尿素，0.05％二甲苯Cynol，和0.05％溴酚蓝）等体积。在消毒9％变性聚​​丙烯酰胺凝胶（9％丙烯酰胺，7M尿素，1×TBE）过滤器运行。 </li><li>使用紫外线（UV）遮蔽用短波紫外线灯进行定位和切除相应的RNA。将切下的凝胶块在一个离心管中，并加入400微升的凝胶洗脱缓冲液（20mM的Tris-HCl pH值7.5，250mM的醋酸钠，1mM EDTA和0.25％SDS）中。洗脱过夜在RT上一个管肩。 </li><li> PRECI通过加入1ml冰冷的100％乙醇并进行培养或在-80℃下30分钟或-20℃过夜pitate洗脱RNA。 </li><li>离心机以16,000×g离心30分钟，在4℃，以沉淀下来的核糖核酸。 </li><li>用1毫升75％乙醇洗涤沉淀。离心机以16,000×g离心20分钟，在4℃下</li><li>适当地重悬RNA沉淀在无RNA酶的水以达到所需的浓度，如表2所示。 </li></ol> 5，基于荧光的RNA编辑含量<ol><li>为一个单一的反应，结合1皮摩尔（1微升）preA6Rbz和2.5皮摩尔（1微升）gA6Rbz的（1:2.5摩尔比）在微量离心管中，孵育在70℃维持3分钟，并让其在室温下静置在至少10分钟。 </li><li>同时使用表3中制备主混合物，而不preA6Rbz和gA6Rbz，对于含有1x HHE缓冲液（25mM HEPES pH值为7.9，10mM的镁（醋酸）2，50mM的氯化钾和10mM EDTA），1 mM的编辑反应ATP，5mM的氯化钙2，圆酵母RNA，0.1％的Triton X-100和纯化的editosome 16纳克/微升。 </li><li>添加退火preA6Rbz和gA6Rbz完成主结构。 </li><li>免除18微升主混合物（ 表3）的成包含2微升不含RNA酶的水（井用无化合物）或2微升200μM的化学化合物的井，并按照图5包括对照样品中的板。 </li><li>密封板与板密封件和旋转盘下来，以消除任何气泡。孵化在28℃保温4小时。 </li><li>添加25皮摩尔gA6Rbz竞争者（2微升）到每个孔中。放置一个新密封剂，向下旋转的板，并把它实时PCR仪上。程序如下实验: 第1步:85℃5分钟;第2步:24℃，10分钟;步骤3:停止。 </li><li>加FRET底物的15皮摩尔（1微升）到每个孔中，以23微升的最终体积。用新鲜的封口机封板。快速旋转盘，并把它背在实时PCR仪上。 </li><li>设定新的实验步骤如下: 第1步:37℃1分钟;第2步:阅读;第3步:进入步骤1，40倍;步骤4:停止。 </li><li>安装板通过选择所有需要阅读和选择FAM过滤器的井。输入量为23微升并开始运行。 </li><li>计算从每个孔/样品获得通过绘制的斜坡上的条形图进行分析，以获得动力学测量值的斜率。注:动能读提高了样品和背景之间的信号噪声比;作为背景样品将有一个斜率接近于零。结束点读具有较高的背景噪音。 </li></ol>

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The authors have nothing to disclose.

一种新型的高通量筛选方法来确定对锥虫的RNA编辑复杂的抑制剂已提交，为药物发现的新工具来对抗引起的锥虫病。基于FRET的核酶法已被广泛地用于不同的用途20-22;然而，我们动用的体外监测RNA编辑活动19 FRET为基础的核酶分析的能力。该测定可能适用于其他类型的RNA编辑在真核生物，如哺乳动物23的核编码的RNA的核苷酸替换编辑。 该测定中的?...

RNA编辑，转录后修饰的mRNA的过程中，首先被锥虫1发现。从那时起，大量的工作已经在后面学习中的RNA编辑布氏锥虫 2,3机制进行的。在一系列的酶反应中，editosome，约20蛋白核心复合物，产生成熟的mRNA线粒体的能量产生的氧化磷酸化系统的多个组件。催化事件的顺序是信使核糖核酸，尿苷酸（U）增加或删除，并结扎，所决定的指导的RNA（gRNAs）4。 除了 ​​核心editosome复杂的蛋白质，一些辅助因子也已确定5-7。这些蛋白质大多见于分组独立的复合物。然而，蛋白质装配在芯editosome复杂的顺序和核心复合物与该配件的交互模式配合物尚未确定。针对RNA编辑的过程中锥虫可提供化学解剖，援助在研究editosome复杂的装配和功能。此外，在几个editosome蛋白质功能的研究表明在不同人生阶段的必要性，指出其潜在的药物靶点8-12。因此，editosome的发现抑制剂也可作为对锥虫的先导化合物。这是及时的，因为目前市面上所引致的锥虫病的药物是有毒的，低效和昂贵13,14。 一个高效，便捷的体外试验要探索的化学宇宙，阻止RNA编辑特异性抑制剂。三个实验已经开发并用于监视editosome活动:（a）全轮体外 RNA编辑分析15（二）预切割体外 RNA编辑实验16,17，一ND（三）锤头状核酶（HHR）为基础的检测18。前两个实验依靠编辑的产品（ATP酶6基因）与放射性的帮助下直接观察。该HHR基测定法使用了被建模的行为作为经编辑的核酶的ATP酶6 mRNA的修改版本。功能核酶则特异性切割放射性标记的RNA底物，作为一名记者。最近，MOSHIRI 等人开发了一种"混合，并测量"HHR-基于体外报告基因分析来监测RNA编辑其中的放射性标记的RNA底物被替换为荧光共振能量转移（FRET）底物19。该测定法的原理的优点是:（a）它是一种快速和方便的混合和测定法的测量类型，为生产活性核酶和底物裂解的同时出现在同一试管中低体积（ 即 20微升），（二）它避免了使用放射性标记的物质，（C）的灵敏度是一个通过荧光仪在微滴定板格式，以及（d）一个高信噪比fforded。使用这种测定法，对纯化的editosome已知的RNA编辑连接酶抑制剂的作用被证实19。这个实验验证了分析的快速鉴定RNA编辑酶抑制剂，主要针对来自T.整个editosomes 布氏 。 图1是一个详细的一步一步的示意性的基于荧光的体外 RNA编辑测定。这个协议可以被用于监测在体外 RNA编辑或很容易地适应用于筛选各种比例的化合物库。

<table><tbody><tr><td>SDM-79 Medium</td><td>Gibco by Life Technologies</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>12483-020</td><td>heat inactivation at 55 &amp;#176;C for 1 hr</td></tr><tr><td>Hemin, minimum 80%</td><td>Sigma</td><td>H5533-10G</td><td></td></tr><tr><td>Penicillin-Streptomycin Sollution</td><td>Fisher Scientific</td><td>MT-30-002-CI</td><td></td></tr><tr><td>Dnase 1 recombinant, Rnase Free&amp;nbsp;</td><td>Roche</td><td>4716728001</td><td></td></tr><tr><td>T7 RiboMax Express Large&amp;nbsp; scale RNA&amp;nbsp; production system</td><td>Promega</td><td>P1320</td><td></td></tr><tr><td>Kimble Kontes Dounce Tissue Grinders&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>K885300-0040&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Gradient Master, ver 5.25&amp;nbsp;</td><td>Biocomp</td><td>107-201M</td><td></td></tr><tr><td>Ultra Clear Tube, 13.2 ml</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Optima L-100XP&amp;nbsp; Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>392052</td><td></td></tr><tr><td>SW 41 Ti ROTOR</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>MicroSeal &#039;B&#039; Seal, Seals</td><td>Biorad</td><td>MSB1001</td><td></td></tr><tr><td>CFX 384 Touch Real-Time PCR Detection System</td><td>Biorad</td><td>185-5484</td><td></td></tr><tr><td>Acryl/Bis solution (19:1), 40% (w/v)</td><td>Bio Basic</td><td>A0006-500ML</td><td></td></tr><tr><td>Urea, Molecular biology grade, 1 kg</td><td>Life Technologies</td><td>AM9902</td><td></td></tr></tbody></table>

rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

实质性的进展已经取得了确定的线粒体RNA编辑在锥虫的机制。同样，相当大的进展已经取得了识别editosome络合物的催化RNA编辑的组件。但是，目前还不清楚这些蛋白质是如何协同工作。从对editosome高通量筛选获得的化学化合物可以阻止或影响在编辑周期的一个或多个步骤。因此，新化合物的鉴定将产生有价值的分子探针解剖editosome功能和组装。在以前的研究中， 在体外测定法的编辑进行了使用放射性标记的RNA。这些测定法是耗时，低效和不适合于高通量的目的。在这里，均匀的荧光为基础的"混合和计量"锤头状核酶体外记者分析监测RNA编辑，呈现。仅作为RNA编辑的结果锤头状核酶荧光共振能量转移（FRET）寡基板发生裂解。这反过来导致荧光团与猝灭剂，从而产生一个信号分离。与此相反，当editosome功能受到抑制，荧光信号会被淬灭。这是一个高度敏感和简单的分析，应该是普遍适用于体外 RNA编辑或化学物质，可以抑制editosome功能的高通量筛选监控。

以证明需要设立一个大型屏幕必要的步骤， 图2-5是与测定的质量代表对照实验。这些都是必不可少的对照实验为一致的测定过筛选的几天或不同的屏幕之间的对比。 评估荧光信号噪声比 以确保在一个大型安装的荧光素标记的寡核糖核苷酸底物的稳定性和质量，Z&#39;因子，其定义为在测定背景和最大信号之间的差用的是活性核酶...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

rna-catalyst-as-reporter-for-screening-drugs-against-rna-editing

Research

JoVE Journal

Biology

8.4K 次观看.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

视频: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

生物化学

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA甲基转移酶活性分析的无抗体测定

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

CRISPR/Cas9 使用dpy-10联合 CRISPR 筛选标记和组装的核糖核蛋白复合物编辑C. elegans rbm-3.2基因。

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

免疫与感染

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

一种快速检测RNA编辑的无序列方法

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...