Force-induced melting of DNA--evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Abstract

Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.

Figure 1.

Overstretching mechanisms, DNA constructs and experimental setup. (A) The three proposed mechanisms for overstretching. (i.) Peeling, where the DNA progressively melts from free ends. (ii.) Internal melting, where the melting is initiated in regions of low stability forming ‘bubbles’. (iii.) B-to-S transition, where the base-pairing remains intact as the duplex is extended into a longer form. (B) Schematic description of the DNA constructs and the covalent inter-strand linkage. 5′AT: 64 bp with a terminal inter-strand cross-link. The inter-strand linker is formed by joining alkyne and azide modified bases on opposite strands together using the Cu⁺-catalyzed click reaction. 3′5′AT: 64 bp with one inter-strand linker at each end of the duplex region. ATGC: 122 bp duplex region consisting of the AT-core sequence shared with 5′AT and 3′5′AT, and a previously studied single-clicked GC-rich sequence (23). For bead attachment, each construct is extended in the 3′-ends with single-stranded DNA containing digoxigenin or biotin modified bases. (C) Experimental setup. The DNA constructs are tethered to the streptavidin and anti-digoxigenin coated beads by biotin and digoxigenin modified bases incorporated in the single stranded handles, respectively. One of the beads (left) is immobilized by suction onto a pipette, while the other (right) is manipulated by the optical trap.

Figure 2.

Melting and rehybridization of the AT-rich oligonucleotide in the single (5′AT) or double-clicked form (3′5′AT). (A) Pull and relax cycle of a 5′AT molecule in 1 M NaCl showing a melting transition at about 62 pN (a. and inset i.) during pulling (force is increased) and rehybridization (b. and inset ii.) at 24 pN during relaxation (force is decreased). Prior to the melting transition the molecule displays a region of reversible extension and contraction (c. in inset i.) likely due to partial melting of the double-stranded region. This intermediate state is observed for many but not all transitions and always coexists with the B-form state at any given trap position. (B) Pull and relax cycle of a 5′AT molecule in 5 mM NaCl. The melting force is reduced to 42 pN (a. and inset i) and the rehybridization force (b. and inset ii.) is also reduced by the decreased salt concentration but to lesser extent. The same type of bistability as in high salt is observed prior to complete melting of the duplex (c.). (C) Multiple pull (blue) and relax (red) cycles of a 3′5′AT molecule in 5 mM NaCl showing the melting (a. and inset i) and rehybridization (b. and inset ii.) transitions. The arrows in the insets mark the variation in melting and rehybridization forces within the same molecule during repeated cycles. (D) Histograms of melting (blue) and rehybridization (red) forces for (i.). Single-clicked 5′AT in 1 M NaCl, (ii.) 5′AT in 5 mM NaCl and (iii.) double-clicked 3′5′AT in 5 mM NaCl.

Figure 3.

Stretching of a double-clicked 3′5′AT construct in 1 M NaCl. (A) Plot of force versus trap position during one cycle of pulling (blue) and relaxation (red). The DNA duplex exhibits a continuous transition over a broad force interval (60–70 pN) with no detectable hysteresis between the pulling and relaxation parts of the cycle. Dashed lines show least-mean-square linear fits to the force versus distance data below and above the transition region. (B) Pooled data from nine pull and relax cycles of the molecule in panel (A) showing the relative extension of the duplex versus the applied force. Experimental data (circles with error bars ± 1 s.d.) and fit to a two-state model (red curve) to estimate the force F_tr at the midpoint of the transition (see Supplementary information). (C) Multiple pull and relax trajectories of a 3′5′AT molecule exhibiting hysteresis during the relaxation. Fifteen out of 31 studied 3′5′AT molecules had one or several relaxation curves of this hysteretic type. (D) Pull experiments on a 3′5′AT molecule in the presence of 0.5 M glyoxal (1 M NaCl, pH 7.9) before and after reaction. The blue curve is the pull trace before the DNA was exposed to glyoxal, and shows how the molecule is gradually extended as in panel (A). The molecule was then kept at 74 pN in order to expose the construct to the glyoxal for certain period of time, after which the force was reduced to 20 pN (relaxation curve not shown) and a new pulling experiment was performed to evaluate the effect of the glyoxal. The green and red curves show the pulling of the same molecule after an exposure time of 75 and 124 s, respectively. The transition force is reduced to about 65 pN (green curve in inset i.) and 57 pN (red curve in inset ii.), respectively, indicating the formation of glyoxal adducts.

Figure 4.

Force-induced melting and rehybridization of the ATGC construct. (A) Force versus trap position trajectories during multiple pull–relax cycles on an ATGC molecule in 1 M NaCl. The two distinct transitions (a. and b.) are reversible. Inset i. shows that the two transitions are well separated in force and that both exhibit bistability. Inset ii. shows a single pull trajectory for the same molecule and the linear fits (dashed) used to assign the data points to the three states at low (blue), intermediate (green) and high force (red). (B) Population probabilities of the three states as a function of force for the molecule in (A), based on pooled data from the pull and relax trajectories. The fitted curves (black) are used to estimate the transition forces , and . (C) Force versus trap position trajectory during a pull–relax cycle on an ATGC molecule in 5 mM NaCl. During pulling (blue), ATGC exhibits four transitions (a.–d.) The first three transitions display bistability (a. and a′. in inset ii, b. in inset i) while the fourth transition is irreversible (c. in inset i). During relaxation (red), the molecule rehybridizes at about 20 pN (d.). (D) Pull trajectories of an ATGC molecule in the presence of 0.5 M glyoxal (1 M NaCl, pH 7.9) before (t₀; blue) and after glyoxal reaction for 30 s (t_30s; green) and 55 s (t_55s; red) while kept in the fully extended state at 74 pN (gray dotted line). The first transition (a.) at about 65 pN (see also a. in panel (A)) occurs at lower forces after glyoxal exposure, at 58 pN after 30 s and at 47 pN after 55 s, respectively.