寡核苷酸是短核酸,其充当最有前途的药物形式类别之一。然而,建立寡核苷酸物理化学评估平台以全面了解其性质的尝试受到限制。由于在高浓度下的化学稳定性和功效以及溶液性质应与它们的高阶结构和分子内/分子间相互作用有关,他们的详细了解使有效的配方开发。这里,凝血酶结合适体(TBA)和四个修饰的TBA的高阶结构和热力学稳定性,它们具有相似的序列,但预期具有不同的高阶结构,使用紫外光谱(UV)进行评估,圆二色性(CD),差示扫描量热法(DSC),核磁共振(NMR)。然后,高阶结构和溶液性质之间的关系,包括溶解度,粘度,并对稳定性进行了研究。也证实了高级结构对抗凝血酶活性的影响。由于钾浓度不同,寡核苷酸的高阶结构和分子内/分子间相互作用受到缓冲液类型的影响,这对于G-四链体结构的形成至关重要。因此,解决方案属性,如溶解度和粘度,化学稳定性,和抗凝血酶活性,也受到了影响。每种仪器分析在研究TBA和修饰的TBA的高阶结构中都具有补充作用。还讨论了临床前发育阶段每种物理化学表征方法的实用性。
Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.