Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region

Abstract

When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye-DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0 degrees +/- 3.2 degrees). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.

Figure 1.

Pictorial representations of different forms of dsDNA. (A) B-DNA which is the structure of the dsDNA below 50 pN. (B) Stretched DNA (S-DNA) and (C) partially melted DNA are suggested structures of the dsDNA in the overstretching region.

Figure 2.

Single λ-DNA molecule tethered between two 2.6 µm-sized streptavidin coated polystyrene beads. One of these beads is placed on a micropipette (which can be moved with nanometer accuracy), while the second bead is held with the optical tweezers. YOYO-1 molecules which are present in the surrounding buffer bind the dsDNA through bisintercalation. Excitation of the dsDNA–YOYO-1 complex is achieved by scanning the dsDNA–dye complex with a line-shaped (0.3 × 3.5 µm) excitation beam (488 nm). Line frequency is 12.5 Hz.

Figure 3.

Force extension curves of dsDNA molecules in the presence of various YOYO-1 dye concentrations in the surrounding buffer. Closed and open symbols represent the extension and relaxation part of the cycle, respectively. The solid gray lines present the curve-fitted extensible WLC model (5) (for 0 and 40 nM). Pulling speeds are 3 µm/s.

Figure 4.

Total fluorescence intensity of the dsDNA–YOYO-1 complex and the force as a function of the extension. Panel A, B and C present the total intensity of the dsDNA–YOYO-1 complex at 100, 100 and 10 nM YOYO-1, respectively. Panel D, E and F present the corresponding force extension curves of dsDNA, respectively. The molecule in A and D did break during the stretching procedure, probably due to photo-induced damage. B and E presents the data on a molecule that, although stretched to only 60 pN, did not break and revealed a relaxation curve as well. Black and gray squares present the stretching and relaxation part of the force extension cycle. Area between two gray dotted lines marks the overstretching region (in C and F).

Figure 5.

Total fluorescence intensity of the dsDNA–YOYO-1 complex (at 10 nM YOYO-1) as function of extension, recorded at different polarization settings. Black and gray symbols represent extension and relaxation intensities, respectively. Polarization orientation for excitation (Ex) and emission (Em) are indicated for each graph.

Figure 6.

Schematic picture of the YOYO-1 molecule intercalated in dsDNA. (A) YOYO-1 molecules intercalating the dsDNA where YO moieties interact with the adjacent base pairs. Interactions are indicated with dotted lines. (B) YOYO-1 molecules bound to the dsDNA that is partly melted. The center YOYO-1 molecule only interacts with one of the two strands, allowing the other YO moiety in the YOYO-1 molecule to freely rotate.

Figure 7.

(A)Fluorescence images of the dsDNA–YOYO-1 complex at different forces in the presence of 100 nM YOYO-1. Scale bar is 5 µm (B). Fluorescence intensity of the dsDNA–YOYO-1 complex at 73.2 pN compared with the background signal.

Figure 8.

Force extension curve of denatured DNA–YOYO-1 complex at 1 nM YOYO-1. Black and gray symbols present extension and relaxation, respectively. Gray lines presents the calculated curve for 100% dsDNA, 100% ssDNA and 40% ssDNA + 60% dsDNA, which best fitted the experimental data. Image panel presents the image of the combined ssDNA and dsDNA at 18.2 µm extension (10 pN), scale bar 5 µm.

Figure 9.

Kymograph created from a sequence of fluorescence images recorded simultaneously while recording a force extension curve of dsDNA–YOYO-1 complex at 10 nM YOYO-1. The top axis is time, the left axis of the kymograph indicates the extension of the complex, and the lower axis represents the force on the complex. The two extremely bright lines in the kymograph represent the streptavidin coated polystyrene beads. The top bright line indicates bead in the trap and other bright line indicates bead on the micro pipette.

Figure 10.

Average orientation of YOYO-1 molecules with respective dsDNA helical axis as function of extension, calculated from the fluorescence polarization data recorded during the stretching of DNA–YOYO-1 in presence of 1 nM YOYO-1. Black and gray symbols present average (effective) angle while extension and relaxation of the complex, respectively. Area between two dotted gray lines indicated the overstretching region.