Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Methods Appl Fluoresc. 2016 Sep 6;4(3):034009. doi: 10.1088/2050-6120/4/3/034009.
G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1 × 10 M. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.
G-四链体 DNA（GqDNA）结构在许多特定的细胞功能中发挥着重要作用，并且是小分子（配体）抗肿瘤的有前途的靶点。在这里，我们测量了配体（Hoechst）与平行 c-Myc（mPu22）GqDNA 结合的动态斯托克斯位移，时间范围从 100 fs 到 10 ns，与之前报道的 DAPI 与反平行人类端粒（hTelo22）GqDNA（Pal 等人，2015 年 J. Phys. Chem. Lett. 6 1754）的动力学进行了比较。荧光上转换和时间相关单光子计数实验的斯托克斯位移数据相结合，覆盖了广泛的动态范围。结果表明，Hoechst 在平行 mPu22 GqDNA 中的溶剂化动力学遵循幂律松弛，外加快速 2 ps 指数松弛，时间范围从 100 fs 到 10 ns，两个配体-GqDNA 系统中的幂律指数仅有细微差异（Hoechst-mPu22 为 0.06，DAPI-hTelo22 为 0.16）。我们测量了稳态荧光光谱和时间分辨各向异性衰减，这证实了 Hoechst 与平行 mPu22 的紧密结合，结合常数约为 1×10 M。通过对 Hoechst 在平行 GqDNA 中的分子对接，然后对 Hoechst-GqDNA 复合物进行 50 ns 的分子动力学（MD）模拟，揭示了 Hoechst 通过末端堆积靠近 G13 和 G4 与外 G-四联体之一结合，这与 DAPI 在反平行 hTelo22 GqDNA 沟槽中的结合位点不同。综合以前的实验和模拟结果，我们将 2 ps 分量分配给 mPu22 附近仅受轻微扰动的水的水合动力学，将幂律松弛分配给水和 DNA（即 DNA 骨架、未配对碱基和连接 G-四联体的环）的耦合运动，该运动靠近平行 GqDNA 内的 Hoechst。
