James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA.
James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA.
J Mol Biol. 2014 Apr 17;426(8):1629-50. doi: 10.1016/j.jmb.2014.01.009. Epub 2014 Jan 31.
Sequence analogs of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K(+)-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of "prefolded" hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1=0.1s at 25 °C in 25 mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intramolecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2≈3700s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3≈750s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies {e.g. d[TTGGG(TTAGGG)3A] and d[TAGGG(TTAGGG)3TT]}. Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway.
序列类似物的人类端粒 DNA 如 d[AGGG(TTAGGG)3]（Tel22） 折叠成单体四链体结构在合适的阳离子存在下。为了研究单分子四链体形成的途径，我们通过圆二色性（CD）、固有 2-氨基嘌呤荧光和荧光共振能量转移（FRET）监测 K（+）诱导 Tel22 折叠的动力学。结果与四步途径 U↔I1↔I2↔I3↔F 一致，其中 U 和 F 代表未折叠和折叠构象的集合，I1、I2 和 I3 是中间体。先前的动力学研究表明，I1 是在快速预平衡中形成的，可能由 DNA 阳离子诱导的静电坍塌引起的“预折叠”发夹结构的集合组成。当前的研究表明，在 25mM KCl 中，I1 在 25°C 下以松弛时间 τ1=0.1s 转化为 I2。I2 的 CD 光谱特征为反平行四链体，可能是由于 I1 发夹的分子内折叠而形成。I3 形成相对较慢（τ2≈3700s），并且 CD 和 FRET 性质与平衡熔融研究中先前观察到的三链体结构一致。I3 以 τ3≈750s 转化为 F。用形成混合平行/反平行杂交-1 和杂交-2拓扑结构的寡核苷酸（例如 d[TTGGG（TTAGGG）3A]和 d[TAGGG（TTAGGG）3TT]）观察到具有不同动力学常数的相同途径。上述光谱方法和使用互补链捕获测定的时间分辨荧光寿命测量也监测了解折叠途径的动力学。这些实验揭示了沿解折叠途径的一个缓慢的、限速步骤。
