Single-molecule TPM studies on the conversion of human telomeric DNA.
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.
Chu
Jen-Fei
JF
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan.
Chang
Ta-Chau
TC
Li
Hung-Wen
HW
D001703:Biophysics / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D016373:DNA, Antisense; D006801:Humans; D009038:Motion; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D016615:Telomere / Q000737:chemistry* / Q000378:metabolism*
D016373:DNA, Antisense; D012965:Sodium Chloride; D004247:DNA; D012964:Sodium; D011188:Potassium
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