通过表面等离子体共振成像测量特定序列DNA与药物结合的动态区分,并与溶液相测量进行比较
Lauren K Wolf1, Yang Gao, Rosina M Georgiadis
1Department of Chemistry, Metcalf Center for Science and Engineering, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Journal of the American Chemical Society
|August 10, 2007
概括
表面等离子体共振 (SPR) 成像直接检测小分子DNA结合. 这种方法量化了与DNA的药物相互作用,揭示了药物发现的结合率和亲和力.
科学领域:
- 生物物理学的生物物理.
- 分子生物学分子生物学
- 分析化学 分析化学
背景情况:
- 直接检测与DNA结合的小分子对药物发现至关重要.
- 了解DNA与药物相互作用需要定量动力学和热力学数据.
- 表面等离子体共振 (SPR) 成像为生物分子相互作用分析提供了一个无标签的方法.
研究的目的:
- 为了证明SPR成像用于直接检测与表面结合的DNA探头结合的小分子的实用性.
- 量化区分模型药物与不同固定DNA结合部位的相互作用.
- 阐明DNA与药物相互作用的机制细节,特别是具有明显DNA序列的actinomycin-D (ACTD).
主要方法:
- 使用SPR成像,设计了数量分析的阵列表面.
- 测量了actinomycin-D (ACTD) 与双链DNA序列 (5'-TGCT-3'和5'-GGCA-3') 的关联和解离率.
- 在表面和溶液中确定DNA-ACTD相互作用的动力和热力学常量.
主要成果:
- ACTD对特定DNA序列具有差异性的结合动力学,与高亲和度5'-TGCT-3'位点 (kd-1 = 3300秒) 的缓慢解离,与低亲和度5'-GGCA-3'位点 (kd-1 = 210秒) 的更快解离.
- 结合 afinities 确定为 8.8 x 10^6 M^-1 的 5'-TGCT-3' 和 1.0 x 10^6 M^-1 的 5'-GGCA-3'.
- 与溶液相比,表面环境抑制了约4倍的结合亲和力,并将DNA与药物关联速度减慢了100倍.
结论:
- SPR成像是有效的直接,定量检测的小分子结合DNA探头.
- 这项研究提供了对DNA-ACTD相互作用的机制性见解,突出了差异性的结合亲和力和动力学.
- 基于表面的生物传感器测量显示,与溶液相相互作用相比,结合动力学和亲和力发生了显著的变化,影响了药物发现应用.
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