Interdisciplinary Graduated Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkean, Bangkok 10900, Thailand.
J Virol Methods. 2011 Feb;171(2):381-7. doi: 10.1016/j.jviromet.2010.12.001. Epub 2010 Dec 15.
Highly sensitive fluorescence dye, PicoGreen, can detect a small amount of polynucleotide duplex. The DNA/RNA duplex is a reaction product of HIV-1 reverse transcriptase (HIV-RT), which demonstrated that the HIV-1 RT kinetics and anti-HIV agent efficiency could be determined rapidly by a fluorometric method using PicoGreen dye. A small amount of dye is enough to test a minimal quantity of substrate to rapidly determine the enzyme kinetic properties K(m) and V(max). This method was applied to screen HIV-RT inhibitors. The enzymatic inhibition of 11 dipyridodiazepinone derivative inhibitors was compared. The IC(50) of all compounds was determined and it was found that two showed high inhibition efficiency. In addition, this method is applied for screening of many natural agents belonging to diverse sources. The proposed fluorometric method using PicoGreen dye is an easy, rapid, and sensitive detection method to determine HIV-RT activity, monitor enzyme kinetics and perform high-throughput screening of anti-HIV-1 RT agents.
高灵敏度荧光染料 PicoGreen 可检测少量多核苷酸双链。DNA/RNA 双链是 HIV-1 逆转录酶（HIV-RT）的反应产物，这表明可以通过使用 PicoGreen 染料的荧光法快速测定 HIV-1 RT 动力学和抗 HIV 药物的效率。少量的染料足以检测最小量的底物，从而快速确定酶动力学特性 K(m)和 V(max)。该方法已用于筛选 HIV-RT 抑制剂。比较了 11 种二吡啶并二氮杂卓衍生物抑制剂的酶抑制作用。测定了所有化合物的 IC(50)，发现其中两种具有很高的抑制效率。此外，该方法还可用于筛选来自不同来源的多种天然药物。使用 PicoGreen 染料的荧光法是一种简便、快速、灵敏的测定 HIV-RT 活性、监测酶动力学和进行高通量筛选抗 HIV-1 RT 药物的方法。
