组成图示
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传感器类型
表面等离子共振(SPR)生物传感器
检测对象
发夹聚酰胺（hairpin polyamides, KA1002、KA1007、KA1055、KA1039）；样品基质：HBS缓冲液（10 mM HEPES、150 mM NaCl、3 mM EDTA、0.05% P20，pH 7.4）与HEPES缓冲液（10 mM HEPES、50 mM NaCl、1 mM EDTA，pH 7.4）
检测原理
链霉亲和素修饰的SPR芯片上通过生物素-链霉亲和素作用固定含TGGCTT位点的发夹DNA。注入不同浓度发夹聚酰胺（PA）后，PA以序列特异性方式进入DNA小沟，通过酰胺氢键、范德华作用和疏水堆积与碱基对识别。结合事件增加芯片表面质量与局部折射率，SPR共振信号以响应单位（RU）实时变化。稳态RU随游离PA浓度升高并趋于饱和，按单点结合模型RUobs=RUMax×KACfree/(1+KACfree)拟合得到平衡常数KA；连续结合-解离sensorgram用1:1动力学模型全局拟合得到ka和kd。低pH甘氨酸再生表面。该方法为无标记、实时、可重复的SPR传感检测，未使用酶或核酸信号放大。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或R^2。
效应效果
SPR显示KA1002对TGGCTT最强，KA=(37±7)×10^8 M−1；KA1007因N端β降低120倍至(0.29±0.01)×10^8 M−1，kd增大约15倍；KA1055仅降低3倍；KA1039弱25倍。ΔTm：KA1002对TGGCTT 6.8°C、TGCCTT 11.3°C，其他突变0.5–4.5°C；KA1055对TGGCTT 5.3°C、TGCCTT 6.2°C。实验至少重复三次且可重复，未报告RSD、回收率或ELISA/HPLC/qPCR对比。作者认为结果揭示β位置与DNA碱基位置对PA-DNA结合和选择性的序列依赖效应，可指导DNA靶向药物设计。
传感器的构成
- 换能器基底：streptavidin-derivatized SPR sensor chip（链霉亲和素修饰SPR芯片），提供表面捕获与SPR光学换能
- 捕获/识别层：biotinylated hairpin DNA 5′-biotin-CCTTGGCTTCTTTTGAAGCCAA-GG-3′，含TGGCTT位点，经生物素-链霉亲和素非共价固定
- 被测配体：hairpin polyamides KA1002、KA1007、KA1055、KA1039，序列特异性结合DNA小沟
- 流动相：HBS buffer（10 mM HEPES、150 mM NaCl、3 mM EDTA、0.05% P20，pH 7.4）及HEPES buffer（10 mM HEPES、50 mM NaCl、1 mM EDTA，pH 7.4）
- 抗非特异添加剂：0.05% (v/v) surfactant P20，降低非特异吸附
- 再生剂：10 mM glycine（pH 2.5），解离PA-DNA复合物并恢复基线
- 信号读出：Biacore T200/T100 optical biosensor，监测SPR响应单位RU并生成sensorgrams
中文摘要
为深入理解β-丙氨酸（β）取代及杂环数目对DNA结合亲和力与选择性的影响，作者研究了八环发夹聚酰胺（PA）及其两个β衍生物和一个六环类似物与同源DNA序列5′-TGGCTT-3′的相互作用，并利用同源DNA和五个突变DNA评估结合选择性与β效应。研究采用互补的能量与结构方法，包括紫外热熔融、生物传感器表面等离子共振（SPR）、等温滴定量热（ITC）、圆二色（CD）和DNA连接梯全局结构检测。六环PA因杂环减少而结合亲和力降低，但其聚集显著少于较大PA，从而可测定结合热力学。PA-DNA结合焓为较大负值，ΔCp也为较大负值，是Gibbs自由能的主要驱动项。完整SPR结果清楚表明，β取代可按位置依赖方式显著削弱发夹PA的结合亲和力；PA与突变DNA结合的变化进一步证实这种位置依赖效应。突变序列比较还显示T·A与A·T碱基对识别存在不同效应。DNA突变对单个PA结合以及β取代位置对结合的影响，共同揭示了PA-DNA序列依赖结合的规律。
英文摘要
To improve our understanding of the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.