| Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening
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Author:
Date:
2020-12-20
[Abstract] Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of covalent fragment binding. However, robust hit identification requires characterization of the kinetics of covalent binding that can be readily achieved using quantitative irreversible tethering. This screening platform uses a non-specific cysteine-reactive fluorogenic probe to monitor the rate of reaction between covalent fragments and cysteine containing biomolecules. Fragment libraries are simultaneously screened against the target protein and ...
[摘要] [摘要]与蛋白质反应形成共价键的小分子被广泛用作生物学工具和治疗剂。筛选针对蛋白质靶标的半胱氨酸反应性片段是鉴定共价探针开发的化学起点的有效方法。质谱通常用于鉴定共价片段结合的位点和程度。然而，强大的命中鉴定需要的共价结合的动力学表征是 使用定量不可逆的网络共享可以轻松实现。该筛选平台使用非特异性的半胱氨酸反应性荧光探针来监测共价片段与含半胱氨酸的生物分子之间的反应速率。同时针对目标蛋白和作为对照的谷胱甘肽筛选片段文库，以鉴定具有动力学选择性的命中片段，以对目标进行共价修饰。通过定量不可逆的束缚进行筛选，可以解释单个片段内在反应性的变化，从而实现可靠的命中鉴定和排名。
[背景]位点定向配体发现最早在2000报道，并利用表面暴露半胱氨酸残基共价陷阱二硫键连接的片段，其结合在相邻的口袋（Erlanson等人，2000 ）。从那时起，不可逆的半胱氨酸靶向抑制剂已广泛普及，现在已成为多种肿瘤学适应症的一线治疗方法，其中值得注意的例子是针对BTK和EGFR以及以前不可药物治疗的靶标，例如KRAS（G12C）。随着这些发展，基于共价片段的配体发现已成为设计靶标特异性共价抑制剂的有效途径（Resnick et al。，2019 ...
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| High Resolution Melting Temperature Analysis to Identify CRISPR/Cas9 Mutants from Arabidopsis
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Author:
Date:
2018-07-20
[Abstract] CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results in single-bp insertion or deletion. While single-bp insertions or deletions (indels) effectively destroy the function of protein-coding genes through frameshift, detection is difficult due to the small size shift. High-resolution melting temperature analysis allows quick detection, and it does not require any additional pipetting steps after the PCR amplification of the region of interest. In this protocol, we will describe the steps required for ...
[摘要] CRISPR / Cas9可以对许多植物物种进行定向诱变和基因组编辑。 内切核酸酶用于植物遗传学的方法之一是产生功能丧失突变体，其通常由通过非同源末端连接（NHEJ）途径的错误DNA修复引起。 大多数错误的修复事件导致单bp插入或缺失。 虽然单bp插入或缺失（插入缺失）通过移码有效地破坏蛋白质编码基因的功能，但由于小的移位，检测很困难。 高分辨率熔解温度分析允许快速检测，并且在PCR扩增感兴趣区域后不需要任何额外的移液步骤。 在该方案中，我们将描述分析潜在的纯合突变体所需的步骤。
【背景】CRISPR / Cas9核酸酶是一种核糖核蛋白，能够在特定的22个核苷酸序列上切割DNA双链。与其他核酸酶（例如锌指核酸酶和转录激活因子样效应核酸酶（TALEN））相比，CRISPR / Cas9系统的主要优点在于序列特异性由RNA赋予，并且不需要针对每种靶序列的单独蛋白质。这大大降低了成本，单个构造可以定位多达32个目标。由于这种低成本和高效率，CRISPR / Cas9系统现在广泛用于许多植物物种（Baltes和Voytas，2015; Belhaj et al。，2015）。
当CRISPR / Cas9诱导的双链DNA断裂被NHEJ途径错误修复时，修复的序列最常导致小插入缺失，其中一个bp插入缺失是最常见的（Ma et al。，2015; ...
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