Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA
Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA
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DOI：
10.1016/j.bpj.2009.12.4328
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发表时间：
2010-04-21
影响因子：
3.4
通讯作者：
Li, Hung-Wen
中科院分区：
文献类型：
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作者：
Chu, Jen-Fei;Chang, Ta-Chau;Li, Hung-Wen
人类端粒的3‘末端含有富含鸟嘌呤的单链DNA序列串联重复序列。在单价阳离子(如Na+和K+)存在下，富含G的序列可以通过Hoogsteen碱平折叠成各种二级结构，称为G-四链(G4S)。我们发展了一种单分子拴系粒子运动(TPM)方法来实时研究人端粒序列AGGG(TTAGGG)3中G4S的去折叠过程。TPM方法监测G4形成引起的DNA系链长度变化，从而允许在单分子水平上监测去折叠过程和结构转换。在其反义序列的存在下，折叠的G4结构可以被破坏并转变为未折叠的构象，其表观去折叠时间常数为82 S和3152 S。我们还观察到不同的单价阳离子对G4的稳定性有很大的影响。G4的折叠平衡常数强烈依赖于盐的浓度，在5 mM Na+时为1.75，在15 mM Na+时为3.40。早期对Na+和K+折叠态的光谱研究表明，这两种不同的折叠结构之间的光谱转换可能经历了结构上未折叠的中间态。然而，我们的单分子TPM实验在我们的实验分辨率范围内没有检测到任何完全未折叠的中间体，当钠折叠的G4DNA分子用高浓度的过量钾离子滴定时。这一观察表明，在几分钟的时间尺度上，完全展开的途径可能不是光谱转换的主要途径，折叠状态之间的相互转换可以通过环重排实现。这项研究还表明，TPM实验可以用于研究单链DNA分子的构象变化。
Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3' tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na+ and K+). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)3 in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na+ to 3.40 at 15 mM Na+. Earlier spectral studies of Na+- and K+-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.