A capillary electrophoretic assay for ribonuclease H activity

被引：28
作者：
King, KC
Budihas, SR
Le Grice, SFJ
Parniak, MA
Crouch, RJ
Gaidamakov, SA
Isaaq, HJ
Wamiru, A
McMahon, JB
Beutler, JA [1 ]
机构：
[1] NCI, Mol Targets Dev Program, Ctr Canc Res, Frederick, MD 21702 USA
[2] SAIC Frederick Inc, NCI, Ctr Canc Res, Lab Proteom & Analyt Technol, Frederick, MD 21702 USA
[3] NCI, HIV Drug Resistance Program, Ctr Canc Res, Frederick, MD 21702 USA
[4] Univ Pittsburgh, Sch Med, Div Infect Dis, Pittsburgh, PA 15213 USA
[5] NICHHD, Mol Genet Lab, Bethesda, MD 20892 USA
来源：
ANALYTICAL BIOCHEMISTRY | 2004年 / 331卷 / 02期
关键词：
capillary electrophoresis; ribonuclease H; HIV-1; reverse transcriptase; fluorescence;
D O I：
10.1016/j.ab.2004.05.017
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type I reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme. Published by Elsevier Inc.
引用
收藏
页码：296 / 302
页数：7
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