Development of a novel nonradiometric assay for nucleic acid binding to TDP-43 suitable for high-throughput screening using AlphaScreen technology.
Development of a novel nonradiometric assay for nucleic acid binding to TDP-43 suitable for high-throughput screening using AlphaScreen technology.
复制标题
DOI：
10.1177/1087057110382778
复制
发表时间：
2010-10
影响因子：
--
通讯作者：
Pawlyk AC
中科院分区：
文献类型：
--
作者：
Cassel JA;Blass BE;Reitz AB;Pawlyk AC
TAR DNA结合蛋白43（TDP-43）是与囊性纤维化和神经退行性疾病如肌萎缩侧索硬化和额颞叶痴呆的病理学相关的核酸结合蛋白。我们开发了一种稳健的、定量的、非放射性高通量测定法，使用AlphaScreen®技术测量寡核苷酸与TDP-43的结合。生物素化的单链TAR DNA（bt-TAR-32）和6个TG重复序列（bt-TG 6）与TDP-43以高亲和力结合，KD值分别为0.75 nM和0.63 nM。两种寡核苷酸均表现出缓慢的解离速率，bt-TAR-32的半衰期为750分钟，bt-TG 6的半衰期为150分钟。通过bt-TAR-32或bt-TG 6的置换测定的未标记寡核苷酸的亲和力与先前关于核酸与TDP-43相互作用的报道一致，其中增加TG或UG重复产生更大的亲和力。对7360种化合物的多样性文库进行了筛选，以抑制TDP-43与bt-TAR-32的结合，发现了一系列化合物，其新生SAR和IC 50值范围为100 nM至10 μM。这些化合物可以证明是有用的生物化学工具，以阐明TDP-43的功能，并可能导致新的治疗适应症的TDP-43核酸相互作用是因果关系的相关病理。
TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of cystic fibrosis and neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have developed a robust, quantitative, nonradiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen® technology. Biotinylated single-stranded TAR DNA (bt-TAR-32) and 6 TG repeats (bt-TG6) bound with high affinity to TDP-43, with KD values of 0.75 nM and 0.63 nM, respectively. Both oligonucleotides exhibited slow dissociation rates, with half-lives of 750 min for bt-TAR-32 and 150 min for bt-TG6. The affinities of unlabeled oligonucleotides, as determined by displacement of either bt-TAR-32 or bt-TG6, were consistent with previous reports of nucleic acid interactions with TDP-43, where increasing TG or UG repeats yield greater affinity. A diversity library of 7360 compounds was screened for inhibition of TDP-43 binding to bt-TAR-32, and a series of compounds was discovered with nascent SAR and IC50 values ranging from 100 nM to 10 μM. These compounds may prove to be useful biochemical tools to elucidate the function of TDP-43 and may lead to novel therapeutics for indications where the TDP-43 nucleic acid interaction is causal to the associated pathology.
登录
查看更多内容
影响因子：
14.9
作者：
Kuo PH;Doudeva LG;Wang YT;Shen CK;Yuan HS
通讯作者：
Yuan HS
DOI：
10.1073/pnas.0800546105
发表时间：
2008-03-11
影响因子：
11.1
作者：
Ayala, Youhna M.;Misteli, Tom;Baralle, Francisco E.
通讯作者：
Baralle, Francisco E.
影响因子：
2.5
作者：
Banks, Gareth T.;Kuta, Anna;Isaacs, Adrian M.;Fisher, Elizabeth M. C.
通讯作者：
Fisher, Elizabeth M. C.
影响因子：
--
作者：
Zhang, JH;Chung, TDY;Oldenburg, KR
通讯作者：
Oldenburg, KR
影响因子：
3.5
作者：
Ayala, YM;Pagani, F;Baralle, FE
通讯作者：
Baralle, FE