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Uridine 5'-triphosphate trisodium salt hydrate

Uridine 5′-triphosphate trisodium salt hydrate

Company: Sigma-Aldrich
Catalog#: U6625
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A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
Author:
Date:
2017-08-20
[Abstract]  Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, ... [摘要]  原代神经元是鉴定用于治疗神经变性疾病的治疗性寡核苷酸的理想细胞系统。然而,由于原代细胞的敏感性,使用经典方法转染小干扰RNA(siRNA)是费力的,并且通常显示低效率。寡核苷酸化学的最新进展使得稳定和疏水修饰的小干扰RNA(hsiRNA)的发展成为可能。这种新型的寡核苷酸治疗剂显示出非常有效的自我传递性质,并且在体外和体内支持有效和持久的效果。我们开发了高通量的体外测定法来鉴定和测试原代神经元培养物中的hsiRNA。为了简单,快速,准确地量化数百个hsiRNA的mRNA沉默,我们使用QuantiGene 2.0定量基因表达测定法。这种高通量,96孔板测定法可以直接从样品裂解液中定量mRNA水平。在这里,我们描述了一种制备96孔板格式的小鼠原代皮质神经元的短期培养物用于寡核苷酸治疗剂的高通量测试的方法。该方法支持在短短两周内测试hsiRNA文库和鉴定潜在的治疗方法。我们详细介绍了从初级神经元准备到数据分析的高通量测定工作流程的方法。该方法可以帮助鉴定用于治疗各种神经疾病的寡核苷酸治疗剂。
【背景】寡核苷酸治疗剂代表了通过沉默突变蛋白的表达,可以靶向任何遗传定义的病症的新一类药物。具体地,siRNA是负载于RNA诱导的沉默复合体(RISC)中的双链寡核苷酸,并且可以在mRNA翻译之前使mRNA沉默。然而,未修饰的siRNA是不稳定的,并且不能在没有阳离子脂质制剂的帮助下进入细胞,其可能对原代细胞如神经元有毒性。在本协议中,我们使用自我递送,疏水修饰的siRNA(hsiRNA)进行mRNA沉默。最近在寡核苷酸化学方面的进展使得这些稳定的hsiRNA的设计促进了细胞内化,有效进入RISC以及有力击倒靶基因(Byrne等,2013; ...

Measurement of Nucleotide Triphosphate Sugar Transferase Activity via Generation of Pyrophosphate
Author:
Date:
2015-04-20
[Abstract]  Nucleotide triphosphate (NTP) transferases (EC. 2. 7. 7. X) transfer a nucleoside monophosphate moiety from NTP to another substrate. NTP sugar transferases form a large member of the NTP transferase. There are many variations for the substrate combination of the NTP sugar transferases. It is important to measure the precise enzymatic activity of such NTP sugar transferases by a simple and efficient method. In our method, we measure pyrophosphate as a byproduct of nucleotide diphosphate (NDP)-sugar generation using the pyrophosphate assay kit. The kit reagents include two enzymes that convert pyrophosphate to phosphate, and then phosphorolyze chromogenic substrate to allow color development at 360 nm (see details below). Thus, the NDP-sugar formation can be simply traced as production of ... [摘要]  核苷三磷酸(NTP)转移酶(EC.2.7.7.X)将核苷单磷酸部分从NTP转移到另一底物。 NTP糖转移酶形成NTP转移酶的大成员。 NTP糖转移酶的底物组合有许多变化。重要的是通过简单有效的方法测量这种NTP糖转移酶的精确酶活性。在我们的方法中,我们使用焦磷酸测定试剂盒测量焦磷酸作为核苷酸二磷酸(NDP)糖产生的副产物。试剂盒试剂包括将焦磷酸盐转化为磷酸盐,然后磷酸化发色底物以允许在360nm显色的两种酶(见下文详述)。因此,NDP-糖形成可以简单地追溯为焦磷酸盐的产生,其通过在360nm处的吸光度监测。该方法对于测量包括NTP糖转移酶的各种产生焦磷酸的酶是可靠的和多用途的。

[原理和概述] NTP转移酶催化可逆反应如下:NTP +糖-1P - NDP-糖+ PPi
酶反应可以监测为无机焦磷酸盐(PPi)的产生。 EnzChek焦磷酸测定试剂盒(Molecular Probes,Life Technologies,Carlsbad,CA)包括两种酶和用于显色以定量焦磷酸的足够材料。无机焦磷酸酶(试剂盒中的组分E)将焦磷酸盐降解为磷酸盐。嘌呤核苷磷酸化酶(PNP,组分B)利用磷酸将生色底物2-氨基-6-巯基-7-甲基嘌呤核糖核苷(MESG,组分A)切割成核糖-1-磷酸和2-氨基-6-巯基-7 - ...

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