Nanomolar Binding of an Antibiotic Peptide to DNA Measured with Raman Spectroscopy
Nanomolar Binding of an Antibiotic Peptide to DNA Measured with Raman Spectroscopy
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DOI：
10.1021/acs.langmuir.3c00099
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发表时间：
2023-03-08
期刊：
影响因子：
3.9
通讯作者：
Harris, Joel M.
中科院分区：
文献类型：
--
作者：
Myres, Grant J.;Harris, Joel M.
将DNA固定到表面提供了一种方便的手段来筛选潜在的小分子治疗候选分子的结合亲和力和选择性。不幸的是，大多数检测这些结合作用的表面敏感方法并不能提供关于分子结构的信息，这些信息对于理解稳定结合的非共价相互作用是有价值的。在这项工作中，我们报告了一种通过使用共聚焦拉曼显微镜来定量结合小槽结合的抗菌肽Netropsin与固定在多孔二氧化硅颗粒内表面的双链DNA发夹序列的关联来应对这一挑战的方法。为了评估结合选择性，将带有不同DNA序列的功能化颗粒与100 nM的netropsin溶液平衡，并基于颗粒中netropsin的拉曼散射检测选择性结合。选择性研究表明，netropsin与具有丰富AT识别区的双链DNA序列结合。为了定量结合亲和力，将这些富含AT的DNA序列与一定范围的netropsin溶液浓度(1到100 nM)进行平衡。Netropsin的拉曼散射强度随溶液浓度的变化可用单结合位的朗缪尔等温线和纳摩尔解离常数很好地描述，这与先前的等温量热法和表面等离子体共振结果一致。靶序列结合伴随着Netropsin和DNA振动模式的变化，这与Netropsin和腺嘌呤的酰胺基团之间的氢键以及DNA小沟中的胸腺嘧啶之间的氢键一致。Netropsin与缺乏富含AT识别区的对照序列的结合显示出比目标序列弱近4个数量级的亲和力。与此对照序列相互作用的netropsin的拉曼光谱显示，在与自由溶液相似的频率下，有较宽的吡咯和酰胺模振动，与观察到的与富含AT的序列的特定结合作用相比，揭示了较少的约束构象。
Immobilization of DNA to surfaces offers a convenient means of screening the binding affinity and selectivity of potential small-molecule therapeutic candidates. Unfortunately, most surface-sensitive methods for detecting these binding interactions are not informative of the molecular structure, information that is valuable for understanding the non-covalent interactions that stabilize binding. In this work, we report a method to meet this challenge by employing confocal Raman microscopy to quantify the association of a minor-groove-binding antimicrobial peptide, netropsin, to duplex DNA hairpin sequences immobilized on the interior surfaces of porous silica particles. To assess binding selectivity, particles functionalized with different sequences of DNA were equilibrated with solutions of 100 nM netropsin, and selective association was detected based on the presence of netropsin Raman scattering in the particles. The selectivity study revealed that netropsin binds to sequences of duplex DNA having AT-rich recognition regions. To quantify binding affinities, these AT-rich DNA sequences were equilibrated with a range of netropsin solution concentrations (1 to 100 nM). Raman scattering intensities of netropsin versus solution concentration were well described by single-binding-site Langmuir isotherms with nanomolar dissociation constants, in agreement with previous isothermal calorimetry and surface plasmon resonance results. Target sequence binding was accompanied with changes in netropsin and DNA vibrational modes consistent with the hydrogen bonding between the amide groups of netropsin and adenine and thymine bases in the DNA minor groove. The binding of netropsin to a control sequence lacking the AT-rich recognition region exhibited an affinity nearly 4 orders of magnitude weaker than found for the target sequences. The Raman spectrum of netropsin interacting with this control sequence showed broad pyrrole and amide mode vibrations at frequencies similar to a free solution, revealing less constrained conformations compared with the specific binding interactions observed with AT-rich sequences.