Department of Nucleic Acids Bioengineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego, Poznan, Poland.
Department of Structural Chemistry and Biology of Nucleic Acids, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego, Poznan, Poland.
PLoS One. 2018 May 24;13(5):e0197835. doi: 10.1371/journal.pone.0197835. eCollection 2018.
Thrombin binding aptamer (TBA), is a short DNA 15-mer that forms G-quadruplex structure and possesses anticoagulant properties. Some chemical modifications, including unlocked nucleic acids (UNA), 2'-deoxy-isoguanosine and 2'-deoxy-4-thiouridine were previously found to enhance the biological activity of TBA. In this paper, we present thermodynamic and biological characteristics of TBA variants that have been modified with novel isoguanine derivative of UNA as well as isoguanosine. Additionally, UNA-4-thiouracil and 4-thiouridine were also introduced simultaneously with isoguanine derivatives. Thermodynamic analysis indicates that the presence of isoguanosine in UNA or RNA series significantly decreases the stability of G-quadruplex structure. The highest destabilization is observed for substitution at one of the G-tetrad position. Addition of 4-thiouridine in UNA or RNA series usually decreases the unfavorable energetic cost of the presence of UNA or RNA isoguanine. Circular dichroism and thermal denaturation spectra in connection with thrombin time assay indicate that the introduction of UNA-isoguanine or isoguanosine into TBA negatively affects G-quadruplex folding and TBA anticoagulant properties. These findings demonstrate that the highly-ordered structure of TBA is essential for inhibition of thrombin activity.
凝血酶结合适体（TBA）是一种短链 DNA，由 15 个碱基组成，形成 G-四链体结构，具有抗凝血特性。先前发现一些化学修饰，包括非锁定核酸（UNA）、2'-脱氧异鸟苷和 2'-脱氧-4-硫代尿苷，可以增强 TBA 的生物活性。在本文中，我们介绍了经过新型 UNA 异鸟嘌呤衍生物以及异鸟苷修饰的 TBA 变体的热力学和生物学特性。此外，还同时引入了 UNA-4-硫代尿嘧啶和 4-硫代尿苷。热力学分析表明，UNA 或 RNA 系列中异鸟苷的存在会显著降低 G-四链体结构的稳定性。在 G-四联体位置之一上的取代会观察到最大的去稳定化。在 UNA 或 RNA 系列中添加 4-硫代尿苷通常会降低 UNA 或 RNA 异鸟嘌呤存在的不利能量成本。圆二色性和热变性光谱结合凝血酶时间测定表明，将 UNA-异鸟嘌呤或异鸟苷引入 TBA 会对 G-四链体折叠和 TBA 抗凝血特性产生负面影响。这些发现表明，TBA 的高度有序结构对于抑制凝血酶活性至关重要。
