Scintillation proximity assays for mechanistic and pharmacological analyses of HIV-1 integration.
Scintillation proximity assays for mechanistic and pharmacological analyses of HIV-1 integration.
复制标题
用于 HIV-1 整合机制和药理学分析的闪烁邻近分析。
DOI：
10.1016/j.ymeth.2009.03.002
复制
发表时间：
2009
期刊：
影响因子：
4.8
通讯作者：
D. Hazuda
中科院分区：
文献类型：
--
作者：
J. Grobler;K. Stillmock;D. Hazuda
HIV-1复制的早期事件通过逆转录和整合而突出，并且逆转录酶和整合酶是重要的治疗靶点。整合通过一系列步骤进行，包括整合酶在病毒供体DNA末端的组装、3′-加工和DNA链转移。第一代整合酶测定通常包括溶液中的所有生物化学试剂，其中过量的供体底物可以用作DNA链转移的靶标。这些条件虽然对于理解HIV-1整合的机制方面很有价值，但没有达到关键的药理学设计，因为发现大多数早期抑制剂阻断组装而不是酶功能。第二代设计，从DNA链转移解耦组装，提供了鉴定临床相关化合物所需的特异性。在这里，我们描述了通用的闪烁接近为基础的测定，其中整合酶组装到供体DNA上，固定在珠的表面上。过量供体DNA的固定化和随后的洗涤消除了其作为靶DNA的可能性，从而允许在分离中研究DNA链转移反应。如果采用放射性标记的靶DNA，则组装的复合物可用于高通量DNA链转移测定，或用于使用合适的放射性配体的整合酶结合测定。
The early events of HIV-1 replication are highlighted by reverse transcription and integration, and the reverse transcriptase and integrase enzymes are important therapeutic targets. Integration proceeds through a series of steps including assembly of integrase on the viral donor DNA ends, 3′-processing, and DNA strand transfer. First generation integrase assays typically included all biochemical reagents in solution where excess donor substrate could serve as the target for DNA strand transfer. These conditions, though valuable for understanding mechanistic aspects of HIV-1 integration, fell short of critical pharmacological designs as most early inhibitors were found to block assembly instead of enzyme function. Second generation designs, which decoupled assembly from DNA strand transfer, afforded the specificity required to identify clinically relevant compounds. Here, we describe versatile scintillation proximity-based assays whereby integrase is assembled onto donor DNA that is immobilized onto the surface of beads. Immobilization and subsequent washing of excess donor DNA eliminates its potential to serve as target DNA, allowing investigation of the DNA strand transfer reaction in isolation. Assembled complexes can be used in high-throughput DNA strand transfer assays if radio labeled target DNA is employed or in integrase binding assays using a suitable radioligand.
DOI：
10.1128/9781555817954
发表时间：
2002
期刊：
--
影响因子：
--
作者：
N. Craig;M. Chandler;M. Gellert;A. Lambowitz;P. Rice;S. Sandmeyer
通讯作者：
N. Craig;M. Chandler;M. Gellert;A. Lambowitz;P. Rice;S. Sandmeyer
DOI：
10.1073/pnas.93.18.9742
发表时间：
1996-09-03
影响因子：
11.1
作者：
Farnet, CM;Wang, BB;Bushman, FD
通讯作者：
Bushman, FD
DOI：
10.1006/meth.1997.0484
发表时间：
1997-08-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Chow, SA
通讯作者：
Chow, SA