Name: rna catalyst as a reporter for screening drugs against rna editing in trypanosomes
Uploaded: 2014-07-22T13:00:00.000+00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

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

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

科研

遗传学

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 ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

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 ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

生物化学

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

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

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

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) ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

功能金报告系统测量特定分枝杆菌误调速率

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

免疫与感染

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 ...

生物工程

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...