PA1 (dIm-PyPyβPyPyPy-γ-PyPyβPyPyPyPyβ-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5’-W₂GW₇-3’, where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. However, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties. In ongoing attempts to understand the basis for these distinctions, we conducted thermodynamics studies of PA1-DNA interactions. The temperature dependence of binding affinity was measured using TAMRA-labeled hairpin DNAs containing a single target sequence. PA1 binding to either an ATAT/TATA or an AAAA/TTTT pattern is consistently entropically driven. This is in contrast to the A/T pattern-dependent driving forces for DNA binding by netropsin, distamycin, and smaller hairpin polyamides. Analysis of the salt dependence of PA1-DNA binding reveals that within experimental error, there is no dependence on ionic strength, indicating that the polyelectrolyte effect does not contribute to PA1-DNA binding energetics. This is similar to that observed for smaller PAs. PA1-DNA recognition sequence binding stoichiometries were determined at both nM (fluorescence) and μM (circular dichroism) concentrations. With all sequences and under both conditions, multiple PA1 molecules bind the small DNA hairpin that contains only a single recognition sequence. Implications for these observations are discussed.

PA1 (dIm-PyPyβPyPyPy-γ-PyPyβPyPyPyPyβ-Ta) 是一种大的（14 环）发夹聚酰胺，旨在识别 DNA 序列 5'-W ₂ GW ₇ -3'，其中 W 是 A 或 T。在较小的 6-8 环发夹聚酰胺 (PA) 中很常见，它以低 nM 亲和力结合其目标识别序列。然而，除了其大尺寸外，它与这些具有更广泛特征的 PA 的不同之处在于其对错配和抗病毒特性的高耐受性。在不断尝试理解这些区别的基础的过程中，我们对PA1 -DNA 相互作用进行了热力学研究。使用含有单个靶序列的 TAMRA 标记的发夹 DNA 测量结合亲和力的温度依赖性。PA1与 ATAT/TATA 或 AAAA/TTTT 模式的结合始终是熵驱动的。这与 netropsin、远霉素和较小的发夹聚酰胺对 DNA 结合的 A/T 模式依赖驱动力形成对比。对PA1 -DNA 结合的盐依赖性分析表明，在实验误差范围内，不依赖于离子强度，表明聚电解质效应对PA1 -DNA 结合能量学没有贡献。这类似于对较小的 PA 观察到的情况。在 nM（荧光）和 μM（圆二色性）浓度下测定PA1 -DNA 识别序列结合化学计量。在所有序列和两种条件下，多个PA1分子结合仅包含单个识别序列的小 DNA 发夹。讨论了这些观察结果的含义。