SYNTHESIS AND ACTIVITY OF MODIFIED CYTIDINE 5′-MONOPHOSPHATE PROBES FOR T4 RNA LIGASE 1
SYNTHESIS AND ACTIVITY OF MODIFIED CYTIDINE 5′-MONOPHOSPHATE PROBES FOR T4 RNA LIGASE 1
复制标题
DOI：
10.1080/15257770902946181
复制
发表时间：
2009-04-01
影响因子：
1.3
通讯作者：
Kuersten, Scott
中科院分区：
文献类型：
--
作者：
Kore, Anilkumar R.;Charles, Irudaya;Kuersten, Scott
我们描述了一系列独特的碱基修饰的连接探针如p（5 ′）C-4-乙二胺3、P（5 ′）C-4-生物素4和预腺苷酸化形式A（5 ′）pp（5 ′）C-4-生物素6的合成并进行了测试。其生物活性与T4 RNA连接酶I使用的标准。pCp探针I作为对照。用5 ′-FAM标记的24聚体单链（ss）RNA建立了分子间连接试验，发现pCp 1、p（5 ′）C-4-亚乙基二氨基3、p（5 ′）C-4-生物素4和预腺苷酸化形式A（5 ′）pp（5 ′）C-4-生物素6的平均连接效率分别为44%、81%、39%和16%。如使用变性凝胶分析测定的。此外，确认了生物素化探针与RNA底物的连接活性。通过链霉亲和素缀合和非变性凝胶电泳分析。这些结果强烈表明，新的探针是T4 RNA连接酶I的有效底物，因此可以用于开发一种miRNA检测系统，包括快速分离，有效标记和检测灵敏度增强的微阵列上的miRNA。
We describe the synthesis of a series of unique base modified ligation probes such as p(5')C-4-ethylenediamino 3, P(5')C-4-biotin 4, and pre-adenylated form A(5')pp(5')C-4-biotin 6 and tested. their biological activity with T4 RNA ligase I using a standard. pCp probe I as a control. The intermolecular ligation assay was developed, using a 5'-FAM labeled, 24 mer single-stranded (ss) RNA and the average ligation efficiencies for pCp 1, p(5')C-4-elhylenediamino 3, p(5')C4-biotin 4, and pre-adenylated form A(5')pp(5')C-4-biotin 6 were found to be 44%, 81%, 39% and 16% respectively, as deter-mined using a denaturing gel analysis. Furthermore, confirmation of the ligation activity of the biotinylated probes to the RNA substrate was confirmed. by streptavidin conjugation and analysis by nondenaturing gel electrophoresis. These results strongly suggest that the new probes are valid substrates for T4 RNA ligase I and therefore could be useful for developing a miRNA detection system that includes rapid isolation, efficient labeling and detection of miRNAs on sensitivity-enhanced microarrays.