Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements.
Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements.
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DOI：
10.1021/ja072401l
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发表时间：
2007-08
影响因子：
15
通讯作者：
L. Wolf;Yang Gao;R. Georgiadis
中科院分区：
文献类型：
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作者：
L. Wolf;Yang Gao;R. Georgiadis
我们演示了使用表面等离子共振 (SPR) 成像来直接检测小分子与表面结合 DNA 探针的结合。使用精心设计的阵列表面，我们定量区分模型药物与不同固定 DNA 结合位点的相互作用。具体来说，我们测量了放线菌素-D (ACTD) 与双链 5'-TGCT-3' 和 5'-GGCA-3' 结合位点的结合和解离嵌入率。测量的速率提供了有关 DNA-ACTD 相互作用的机制信息； ACTD 最初与 DNA 非特异性结合，但通过从强亲和力位点缓慢解离来发挥其活性。我们观察到与强亲和力位点 5'-TGCT-3' 结合的 ACTD 的缓慢解离时间为 kd-1 = 3300 +/- 100 s，而与弱结合位点 5'-GGCA-3' 的 ACTD 的解离时间要快得多（210 +/- 15 s）。这些解离速率相差一个数量级，决定了每个位点的结合亲和力（分别为 8.8 x 10(6) 和 1.0 x 10(6) M(-1)）。我们通过确定溶液中相同 DNA-ACTD 相互作用的动力学和热力学常数来评估表面环境对这些生物传感器测量的影响。该表面将两个结合位点的结合亲和力抑制大约 4 倍。这种抑制表明DNA-药物关联存在障碍； ACTD 与双链 DNA 的结合在表面比在溶液中大约慢 100 倍。
We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.