Figure 1.
Detection of DNA-bound proteins using a scanning DNA loop and magnetic and optical tweezers.
(A) A DNA loop is created by moving the optically trapped beads (blue) around the magnetic-bead DNA tether (red). Zoom in shows the intertwined geometry of the DNA loop. The green dot depicts a bound protein. (B) Top view image series showing the formation of the DNA loop. The loop is made by rotating the beads trapped by optical tweezers around the magnetic bead which is located in the center of the image. The position of the DNA molecule is indicated by the white dashed line. The bead in the upper left corner functions as a reference and is stuck to the surface of the flow cell. (C) The loop is scanned in the horizontal direction by moving both optically trapped beads in concert. Upon encountering a bound protein the DNA cannot slide through the loop anymore and the magnetic bead tether is deflected.
Figure 2.
Schematic illustration of the hybrid magnetic and optical tweezers setup.
A 4 W 1064 nm laser beam passes first trough a beam isolator and is expanded by a beam expander. Two half-wave plates (λ/2) and a polarizing beam splitter (PBS) are used to control the power in both polarizations of the beam. The beam is subsequently split and sent through a pair of acousto-optical-deflectors (AODs) to steer the beam. Lenses are used to further expand the beam and transfer the beam deflection from the conjugate plane of the AODs to the back focal plane of the objective. Two lenses are mounted on translation stages to allow adjustment of the position of the optical traps in the axial direction. 10% of the back reflected light is collected with a plate beam splitter and directed onto a separate position sensitive detector (PSD) for each trap. A vertical slit is used to block reflections from the surfaces of the flow cell and objective. Optical-trap stiffness was calibrated by using a square-wave method where the optical traps are quickly displaced and the return of the bead to the equilibrium position is monitored (see PSD signal top right). The positions of both optical beads, the magnetic bead and the flow cell surface was determined using video microscopy (bottom left image).
Figure 3.
Step by step assembly of DNA tethers using a laminar flow system.
(A) Schematic of the laminar flow cell showing four different inlets, that are combined into a central channel. Magnetic beads are flushed in via a side channel that exits perpendicular to the main flow direction. This allows for a very low flow rate (<10 µL/h) during experiments, while preventing diffusion into this channel. (B) Photograph of a flow cell using three channels, the central channel contains blue dye, showing clear separation by laminar flow from the other two flow lanes. (C) Illustration of the step-by-step assembly procedure to perform the scanning loop experiments. In step 1 two beads are caught by optical tweezers. In step 2 a biotin-labeled DNA molecule is tethered between the streptavidin-coated beads. In step 3 the presence of a single DNA molecule is confirmed by force extension analysis and EcoRI proteins are allowed to bind to the tethered DNA molecule. In step 4 the DNA tether is brought within close distance of a magnetic bead tether and a loop is formed. A low concentration of EcoRI proteins is present in this channel to assure that proteins remain bound to the DNA.
Figure 4.
DNA-bound proteins are detected by scanning a DNA loop.
(A) Scan position defined as the mean position of the two optically trapped beads (red lines are forward scans, black lines are backward scans). (B) Magnetic bead deflection showing three spikes in both the forward and backward scans indicating the presence of DNA-bound proteins. The tension in both the magnetic bead tether and optical bead tether was set at 12 pN.
Figure 5.
Detection of DNA-bound EcoRI proteins.
(A) 7 consecutive scans superimposed (red lines forward scans, black lines backward scans, data from a single molecule is shown). Protein positions (grey dashed lines) were determined from the intersection of linear fits to the baseline (green line) and individual identified spikes (blue dashed line). Expected positions based on the DNA sequence are indicated at the top, numbers indicate sequence position in bases. (B) Protein position determined from the forward (red crosses) and backward scans (black crosses) of panel A. EcoRI positions are systematically detected slightly to the left in forward scans compared to the positions detected in the backward scans.
Figure 6.
Calculated position and forces acting on the scanning DNA loop.
(A) schematic diagram, not to scale, showing the displacement of the DNA loop as the optically trapped beads are scanned to the right. Forces and positions were calculated iteratively for a 48 kb DNA molecule, at an initial loop height of 2 µm above the surface, initial tensions were 10 pN for both DNA molecules, and optical trap stiffness was set to 100 pN/µm. The elasticity of the DNA was modeled by the worm-like-chain model. As the loop encounters a bound protein or other obstacle during the scan, the DNA will no longer slide through the loop but displace the magnetic bead by dx. Due to the direction of forces acting on the loop it will move to the right and upward following the track depicted by the red dotted line. (B) Calculated upward movement, dz, of the loop as a function of trap displacement after encountering a protein. (C) Movement of the magnetic bead in the scan direction after the loop encounters a bound protein (blue points experimental data, red dashed line calculation). The displacement of the magnetic bead is almost equal to that of the scanning traps, inset shows the difference between the magnetic bead position, dx, and trap position, Xtrap; note the nm scale. (D) Calculated force acting on a protein blocking the DNA loop.