Shikiya Ronald, Marky Luis A
Department of Pharmaceutical Sciences and Biochemistry and Molecular Biology, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198, USA.
J Phys Chem B. 2005 Sep 29;109(38):18177-83. doi: 10.1021/jp052327y.
DNA triplexes have been the subject of great interest due to their ability to interfere with gene expression. The inhibition of gene expression involves the design of stable triplexes under physiological conditions; therefore, it is important to have a clear understanding of the energetic contributions controlling their stability. We have used a combination of UV spectroscopy and differential scanning calorimetric (DSC) techniques to investigate the unfolding of intramolecular triplexes, d(A(n)C5T(n)C5T(n)), where n is 5-7, 9, and 11, and related triplexes with a single AT --> TA substitution in their duplex stem. Specifically, we obtain standard thermodynamic profiles for the unfolding of each triplex in buffer solutions containing 0.1 M or 1 M NaCl. The triplexes unfold in monophasic or biphasic transitions (triplex --> duplex --> coil) depending on the concentration of salt used and position of the substitution, and their transition temperatures are independent of strand concentration. The DSC curves of the unsubstituted triplexes yielded an unfolding heat of 13.9 kcal/mol for a TAT/TAT base-triplet stack and a heat capacity of 505 cal/ degrees C.mol. The incorporation of a single substitution destabilizes triplex formation (association of the third strand) to a larger extent in 0.1 M NaCl, and the magnitude of the effects also depends on the position of the substitution. The combined results show that a single AT --> TA substitution in a homopurine/homopyrimidine duplex does not allow triplex formation of the neighboring five TAT base triplets, indicating that the in vivo formation of triplexes, such as H-DNA, is exclusive to homopurine/homopyrimidine sequences.
由于DNA三链体具有干扰基因表达的能力，因此一直备受关注。基因表达的抑制涉及在生理条件下设计稳定的三链体；因此，清楚了解控制其稳定性的能量贡献非常重要。我们结合使用紫外光谱和差示扫描量热法（DSC）技术来研究分子内三链体d(A(n)C5T(n)C5T(n))的解折叠情况，其中n为5 - 7、9和11，以及在其双链茎中具有单个AT→TA取代的相关三链体。具体而言，我们获得了在含有0.1 M或1 M NaCl的缓冲溶液中每个三链体解折叠的标准热力学曲线。根据所用盐的浓度和取代位置，三链体以单相或双相转变（三链体→双链体→单链）解折叠，并且它们的转变温度与链浓度无关。未取代的三链体的DSC曲线显示，对于TAT/TAT碱基三联体堆积，解折叠热为13.9 kcal/mol，热容为505 cal/℃·mol。在0.1 M NaCl中，单个取代的引入在更大程度上使三链体形成（第三条链的缔合）不稳定，并且影响的程度也取决于取代的位置。综合结果表明，在同型嘌呤/同型嘧啶双链体中单个AT→TA取代不允许相邻的五个TAT碱基三联体形成三链体，这表明体内三链体（如H-DNA）的形成仅限于同型嘌呤/同型嘧啶序列。
