REMOVAL OF DOUBLE-STRANDED CONTAMINANTS FROM RNA TRANSCRIPTS - SYNTHESIS OF ADENOVIRUS VA RNAI FROM A T7 VECTOR
REMOVAL OF DOUBLE-STRANDED CONTAMINANTS FROM RNA TRANSCRIPTS - SYNTHESIS OF ADENOVIRUS VA RNAI FROM A T7 VECTOR
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DOI：
10.1093/nar/18.18.5401
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发表时间：
1990-09-25
影响因子：
14.9
通讯作者：
MATHEWS, MB
中科院分区：
文献类型：
--
作者：
MELLITS, KH;PEERY, T;MATHEWS, MB
噬菌体 RNA 聚合酶广泛用于在体外大规模合成特定的 RNA。不幸的是，RNA 产品含有一小部分污染 RNA，包括互补物种，这可能会导致解释错误。我们将编码 Ad2 VA RNA1 的基因克隆到含有 T7 RNA 聚合酶启动子的载体中，以产生大量 VA RNA，用于研究其与 dsRNA 依赖性蛋白激酶 DAI 的相互作用。 VA RNA1 的精确副本被有效合成，但被少量 dsRNA 污染，这会激活 DAI 并混淆激酶测定的解释。因此，我们开发了一种去除 dsRNA 污染物的方法，从而可以测试 VA RNA1 和突变体激活或抑制 DAI 的能力。看来这个方法是普遍适用的。
Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNA1 into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNA1 were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNA1 and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.