Kinetics of the triplex-duplex transition in DNA

Abstract

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.

Figure 1

(A) Triplex-forming DNA construct composed of a long mirror-repeat homo-pyrimidine sequence and a homo-purine sequence complementary to the 5′ side of the former. When the triplex folds, the energy transfer will occur between a donor (green, left-hatched) and an acceptor dye (red, solid). (B) Typical triplex-forming situation from a duplex DNA. Watson-Crick pairs (dashes). Hoogsteen pairs (stars). 3′ ends of the strands (gray arrowheads). In panels A and B, the triplex-forming (third) strand is shown (red base letters and red line, respectively).

Figure 2

FRET efficiency histograms at various pH conditions. (A) 50 mM MES at pH 6.5, (B) 50 mM HEPES at pH 7.5, and (C) 50 mM Tris-HCl at pH 8.5. A triplex stabilized at low pH exhibits high FRET values.

Figure 3

Calibration of the FRET efficiency with respect to the location of the donor dye. (A) The donor is next to the acceptor, (B) 6 nts away (HDNA-6), and (C) 9 nts away (HDNA-9). (Inset) Molecular arrangement for each measurement. The high FRET peak shifted according to the shift of donor position.

Figure 4

The conformational distribution of the triplex-forming molecule and the (un)folding kinetics under various salt concentrations. Here pH was fixed to be 7.5 by 50 mM HEPES. The data in the first row (A, red, right-hatched) were obtained with [Na⁺] = 26 mM. The data in the second (B, blue, left-hatched) and the third (C, green, cross-hatched) rows show similar data with [Na⁺] = 50 and 100 mM, respectively. The first, second, third, and fourth columns show the FRET efficiency histograms, the dwell-time histograms of the low- and the high-FRET states, and representative FRET time traces of each corresponding condition, respectively. The time constants obtained for this set of data and other experimental data sets are summarized in Table S1. In the dwell-time distributions, we used a bin with the same size (0.1 s).

Figure 5

(A) Optimal shape of the single strand of length S at the moment of contact. (B) Zipped configuration where the zipped section is indicated (dot-dashed line). (C) Optimal shape of the single-stranded loop at equilibrium with loop length S^∗. The character O represents the origin of the XY coordinate system. In panels (B and C), the bound ssDNA strand is represented as if absorbed on the centerline of the duplex.

Figure 6

A possible mechanism for spreading. (A) Before spreading. A loop inserted is located at the first (B) and second (C) base of the triplex. (Ladder) Duplex DNA. (Red circles) Nucleotides of the third strand.