Figure 4.

Monitoring G4 structures disrupted by antisense c12 oligo using single-molecule TPM in real time. (a). The h22L substrates were prepared at 150 mM NaCl (folded state, green bar) and subjected to buffer change (gray area) with various concentrations of c12. The recordings were continuous during the buffer change, so it is certain that the same DNA molecules were followed. The dead time of the experiment (∼80 s) was due to the buffer change and restabilization of the stage for imaging. The recordings were then taken for another 8 min (red bar) to monitor the unfolding process. We rarely observe unfolding process in the presence of 400-fold excess c12 within 8 min of recording (1/26, 3.8%). More unfolded events were observed under high c12 concentrations: 27/107, 25.2% for 10,000-fold. (b) The averaged folded fraction of h22L at various time points after adding 10,000-fold antisense c12 shows biexponential decay kinetics. Different fields of DNA tethers were sampled at specified time points to estimate the folded fraction. The unfolding lifetimes were fitted to 82 ± 43 s (46%) and 3152 ± 1138 s (54%), with R² = 0.99. (b, inset) Histograms of the unfolding times observed in the time-course experiments shown in panel a, where the unfolding time is defined as the dwell time between the end of buffer exchange (gray area) and the beginning of the unfolded state. Note that it does not contain the ∼80 s dead time mentioned above. The histogram is fitted to a single exponential decay of 5.5 s unfolding time. Considering the experimental dead time, this is consistent with the 82 s unfolding time measured from panel b.