The overall goal of this experiment is to directly measure the folding and unfolding of G-quadruplexes, abbreviated G4, and the regulation by binding proteins at the single-molecule level using magnetic tweezers. This message can help answer key questions in G4 field, such as floating dynamics of G4, and the regulation by binding proteins. The main advantage of this technique, is that magnetic tweezers, allow stable measurement, of nanometer sized floating dynamics of a single-molecule over days, by using anti-drift technique.
Let's start this with this message, can provide the inside the molecule mechanisms of G4 stability, the helicase activities on G4, it can also use to probe the al-a-do-gees related to G4 binding proteins, or ligans, which may have potential therapeutic applications. In addition, it can also used to probe the outer protein DNA interactions, or protein protein interactions. Begin this procedure with preparation of G4 DNA in the flow channel, as described in the text protocol.
G4 DNA was prepared by ligate G4 forming single stranded DNA, two flank single stranded DNA, and a double stranded DNA handle, using T4 DNA ligates. For the magnetic tweezers setup, start the magnetic tweezers program. Here, the magnetic tweezers are controlled by an in-house written lab view program.
Next, align the magnet centers before mounting the channel. Use a 10 times objective lens to adjust the X and Y axis of the magnetics, in the optical access of the microscope. Use a computer controlled motorized manipulator to move the magnets along the Z direction, and set the distance between the magnets and coverslip as D equals zero, when the magnetics attach to a coverslip on the microscope.
Program the movement of the magnetics through the manipulator to achieve control of the force, including constant force and time varying force. Use a bright light emitting diode light source for back scattered illumination of the bead through the objective. Collect the bead images and a sampling rate of 100 hertz, by a change coupled device camera.
For tether formation, gently flow 200 X diluted M280 para magnetic beads in as-say solution into a channel. Incubate the mixture for 10 minutes to allow the beads to bind to biotin labeled DNA molecules immobilized on the SMCC coded surface through the thiol labeled ent. Gently wash away untethered beads using 200 microliters of standard reaction solution.
Next, mount the channel onto the microscope stage. Search for the beads on the bottom surface using the 100 times oil immersion objective. Select a reference bead on the surface, and a moving tethered bead.
Build the initial image libraries, of both the reference bead and tethered bead and different de-focused plains. Before experiments, use a objective Piezo actuator to obtain images of both reference and tethered beads at different de-focused plains, spaced at 20 to 50 nanometers. Store the images as two separate bead image libraries that are respectively indexed with the de-focused distance.
During the experiments, determine the position of the bead in the XY plain, by the bead centroid. Also determine the height change of the tethered bead by comparing the current bead image with those stored in the library. Use the auto correlation function analysis of the power spectrum of the Fourier transform of the bead images.
During experiments, use the Pizeo to lock the distance between the objective in a specific reference bead image stored in the library through a low frequency feedback control, so that the stuck bead image, has the best correlation with the specific image stored in the library. Determine whether the tether is a single double stranded DNA molecule, by applying approximately 65 piconewtons of force. A tether is a single double stranded DNA molecule if it undergoes the characteristic DNA overstretching transition.
Repeat the process until a single double stranded DNA tether is found. The relationship of force and magnet position, or so called, the force distance curve, was calibrated based on bead fluctuation. Furthermore, the overstretching transition of double stranded DNA that occurred near 65 piconewtons, could also be used to calibrate the force.
For force ramp experiments, perform a force increase scan at a loading rate of 0.2 piconewtons per second, followed by a force decrease scan at minus 0.2 piconewtons per second. After each stretching cycle, hold the DNA molecule at one piconewton for 30 seconds, to allow the single stranded DNA to refold to G4.For force-jump experiments, cycle the force between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds allowing a folded G4-15T to refold. Finally, analyze the unfolding force, using an in-house written mat lab program.
Shown here, is a typical example of a force increase and force decrease curve of a tethered G4 quadruplex. The extension jump in the force increase scan indicated an unfolding transition of G4, at approximately 52 piconewtons. However, in the presence of RHAU, G4 remained folded during the force increase scan for up to 60 piconewtons, indicating G4 force stabilization by RHAU binding.
In the presence of RHAU and ATP, the unfolding force distribution of G4, was shifted to a lower force, indicative of an ATP depended destabilization of G4 by RHAU. Representative traces of the bead height during force-jump cycles, shows that the force applied to the molecule was cycled between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds, under which it could refold. The average lifetime a folded G4 at 54 piconewtons, was approximately 6.4 seconds, estimated by fitting the lifetime histogram with an exponential decay function.
When RHAU helicase was introduced, the extension of tethered DNA remained at folded level throughout the 30 seconds holding time at 54 piconewtons, indicated that the RHAU strongly stabilizes G4 against mechanical unfolding. Upon addition of RHAU and ATP, the extension was at the level of unfolded single stranded DNA, indicating a destabilization of G4.After watching this video, you should have a good understanding of how to measure the unfolding and refolding dynamics of nuclear ACS junctions using this ultra stabled magnetic tweezers. The allotment of this technique paved the way for researchers in field of nucleic acid structure and binding proteins to explore the regression of structure stability and the folding kinetics, as well as the interaction of nucleic acid and binding proteins.
