Zozulya V, Blagoi Yu, Dubey I, Fedoryak D, Makitruk V, Ryazanova O, Shcherbakova A
Institute for Low Temperature Physics and Engineering, NAS of Ukraine, 47 Lenin Avenue, Kharkov, 61103, Ukraine.
Biopolymers. 2003;72(4):264-73. doi: 10.1002/bip.10403.
UV absorption and fluorescence techniques with a thermal denaturation procedure were used in studies of the anchorage of an oligonucleotide hybridization by a covalently tethered nucleoside analogue of an intercalating imidazophenazine derivative (Pzn). The formation by the (dT)(10)Pzn conjugate of the duplex complex with (dA)(15) and the triplex complex with (dA)(15) or poly(dA).poly(dT) was studied in buffered solutions with 0.11 and/or 1M sodium ions at the oligomer strand concentration of 10 microM. Because of the Pzn emission sensitivity to the interaction with adenine bases, a fluorescence technique was found to be effective in the detection of melting transitions. The attached Pzn substantially enhanced the thermal stability of complexes formed by (dT)(10) because of the intercalation mechanism, which increased the temperature of half-dissociation of the duplex by 10-12 degrees C and of the triplexes by approximately 13 degrees C. With the assumption of a two-state model of transition, the thermodynamic parameters for duplex formations were derived. The investigated variant of conjugation has a certain advantage over the widely used attachment via a flexible linker, consisting of a predetermined location of the Pzn chromophore in target sequences that makes it useful as a fluorescent reporter of the hybridization correctness. Molecular modeling was used to construct the geometries of the intercalation sites that turned out to be in conformity with the behavior of the Pzn fluorescence.
采用紫外吸收和荧光技术并结合热变性程序，对一种嵌入型咪唑并吩嗪衍生物（Pzn）的共价连接核苷类似物介导的寡核苷酸杂交锚定进行了研究。在寡聚物链浓度为10微摩尔的缓冲溶液中，研究了（dT）（10）Pzn共轭物与（dA）（15）形成双链复合物以及与（dA）（15）或聚（dA）·聚（dT）形成三链复合物的情况，缓冲溶液中含有0.11摩尔和/或1摩尔的钠离子。由于Pzn发射对与腺嘌呤碱基相互作用的敏感性，发现荧光技术在检测熔解转变方面是有效的。由于嵌入机制，连接的Pzn显著提高了（dT）（10）形成的复合物的热稳定性，使双链的半解离温度提高了10 - 12摄氏度，三链的半解离温度提高了约13摄氏度。在假设转变为二态模型的情况下，推导了双链形成的热力学参数。所研究的共轭变体相对于广泛使用的通过柔性接头连接具有一定优势，其优势在于Pzn发色团在靶序列中的预定位置，这使其可用作杂交正确性的荧光报告分子。使用分子建模构建了嵌入位点的几何结构，结果发现其与Pzn荧光行为相符。
