THERMAL MEASUREMENTS OF BIOMOLECULAR SYSTEMS

生物分子系统的热测量

• 批准号: 6432114
• 负责人: PHILIP D ROSS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432114
• 关键词: CHO cells; acetylcholine; bioenergetics; calorimetry; chemical structure function; glycolysis; heat; inhibitor /antagonist; intermolecular interaction; muscarinic receptor; receptor binding; thermodynamics; transfection

Triple-stranded nucleic acid complexes are of interest due to potential application in biotechnology and medicine. A site-specific binding oligonucleotide that forms a triple helix may be capable of blocking a regulatory or endonuclease cleavage site, blocking transcription at a DNA duplex target site or block translation at an m-RNA target site. To achieve these aims, high binding affinity and base-sequence specificity are required; these are primarily thermodynamic properties. Thermodynamic information about nucleic acid helicies can provide a rational approach toward the design of oligonucleotides with the requisite properties. We have reviewed our complete thermodynamic profiles of the interaction of the complexes of poly(dT) and poly(dU) with poly(dA) to ascertain the effects of the T to U substitution. Poly(dU) exhibits different changes in heat capacity upon dissociating from the poly(dA + dU) and poly(dA + dT) duplexes. This suggests a difference between the two double helical structures contributing to the heat capacity change arising either through differing internal motions of the polymer chains and/or presentation of a different array of charges, hence a different electrostatic field to the surrounding electrolyte. Either or both contributions to the heat capacity change could arise from an underlying difference in structure. Comparison of the dissociation of poly(dT) and poly(dU) from the same poly(dA + dT) double helical core indicated a free energy of stabilization from introduction of the 5-methyl group into the third polypyrimidine strand of only 135 cal/mol at 37 degrees C. The same change in heat capacity accompanied both reactions over a wide temperature range, suggesting similar structures of the poly(dA + 2dT) and poly(dA + dT + dU) triple helicies. The poly(dA +2 dU) triplex was markedly (nearly 1kcal/mol in free energy)less stable to total disruption than poly(dA+ 2 dT). This manifested itself through a 3 kcal/mol difference in the melting enthalpy, which reflects very different interactions in the two triplex structures. Again, these differences most likely arise from an underlying difference between the poly(dA + dU) and poly(dA + dT) duplex structures. This work illustrates the extreme sensitivity of thermodynamic probes in indicating significant differences in nucleic acid complexes that are beyond the current resolution of structural methods.

由于潜在的生物技术和医学中的应用，三链核酸复合物引起了人们的关注。形成三重螺旋的位点特异性结合寡核苷酸可能能够阻止调节性或内核酸酶裂解位点，阻止DNA双链靶位点的转录或在M-RNA目标位点进行阻止翻译。为了实现这些目标，需要高结合亲和力和基础序列特异性；这些主要是热力学特性。有关核酸螺旋的热力学信息可以为具有必要特性的寡核苷酸设计提供合理的方法。我们已经回顾了聚（dt）和poly（du）与poly（da）的络合物相互作用的完整热力学特征，以确定t对U取代的影响。从聚（DA + DU）和Poly（da + dt）双链体分离后，聚（DU）在热容量中表现出不同的变化。这表明两个双螺旋结构之间存在差异，这些结构导致了通过聚合物链的内部运动和/或不同电荷阵列的呈现引起的热容量变化，从而与周围电解质有不同的静电场。对热容量变化的贡献或两种贡献可能是由于结构的潜在差异而产生的。 比较聚（DT）和聚（DU）与相同的聚（DA + DT）双螺旋芯的解离表明，从将5-甲基引入到第三个polypyrimidine链中，仅在37度下仅135 cal/mol的第三个polypyrimidine链，在37度C中，两种反应均伴随着 + da + da + da + da + da + da + 2.三层螺旋。 与poly（da + 2 dt）相比，poly（da +2 du）三重（在自由能中的近1kcal/mol）对总干扰稳定。这通过熔化焓的3 kcal/mol差异表现出来，这反映了两个三元结构中的相互作用非常不同。同样，这些差异很可能是由于poly（da + du）和poly（da + dt）双链结构之间的基本差异引起的。 这项工作说明了热力学探针在表明核酸络合物的显着差异时的极端敏感性，而核酸复合物的显着差异超出了当前结构方法的分辨率。