The overall goal of the following experiment is to demonstrate a single molecule mechanical unfolding experiment on an RNA structure. In the first step, a flow chamber is constructed and mounted to the instrument. In the second step, micron size beads are mixed with the RNA sample of interest, and then the two different sizes of beads are loaded into the flow chamber.
Finally, the flow chamber and the optical trap can be used to steer the beads into contact with one another to evaluate a variety of single molecule interactions. Ultimately, this nano manipulation technique can be used to study the folding of RNA at the secondary and tertiary structural levels. This method can help answer key questions in the RNA folding field, such as how does an RNA fold and unfold?
How does it transit between different confirmations in response to an environmental change? And how does it interact with other molecules? Today's experiment will be performed by my graduate student, Mr.Williams Stevenson, To make a flow chamber begin by drilling six two millimeter diameter holes into one number two cover slip.
Then use a laser engraver to cut three two millimeter wide slits into two pieces of double-sided polyamide tape. Attach one of the pieces of tape to the cover slip, making sure to align the holes in the tape with the holes in the cover slip. Attach the other piece of tape to a clean un drilled cover slip.
Remove the protective covering from both pieces of tape, and then place a glass pulled micro pipette and two bi tubes between each of the channels on one of the cover slips. Next sandwich, the two cover glasses, keeping the micro pipette and bypass tubes in place with the two layers of tape. To make a flow chamber, mount the flow chamber onto the backside of a metal frame, matching the holes in the chamber over the fluidic channels.
Then turn the frame over and connect the channels to 10 milliliter syringes filled with a buffer of choice via polyethylene tubing. Now mount the entire chamber onto the optical tweezers between the two objectives, ensuring that the front side of the chamber with the fluidic channels faces to the right. Then retract the left objective and plug the brass pins of the frame into the mounting holes on the tweezers and tighten the screws to fix the chamber into position.
Use a bungee cord to lift the instrument off the table. Enclose the instrument in an acoustic box before delivering beads to the reaction site of the flow chamber. First, mix an A kneeled RNA sample with anti-D, dig oxygen ENC coated beads or dig beads, and then incubate the mixture at room temperature for 15 minutes.
During the incubation, lotus suspension of stripped habit and coated beads into a one milliliter syringe, and connect the syringe to the fluidic tubing leading to the bottom channel of the flow chamber. Plunge the syringe to load the strep beads into the chamber, and then use the motor control software to place the optical trap near the opening of the bottom bypass tube and move the entire chamber. Then manipulate the track ball to operate the optical trap and capture a strep bead.
Use the motor control software to move the bead close to the tip of the micro pipette. Next, pull the syringe connected to the micro pipette to vacuum the bead onto the micro pipette, and then gently plunge the syringe to apply flow to the middle channel and flush away any remaining strep beads. Deliver the dig bead sample mixture to the top channel of the chamber and capture a dig bead as just demonstrated.
Then use the optical trap to bring the dig bead close to the strep bead. Apply flow to clean the middle channel. Then to fish a single molecule tethering between the pair of beads.
Place the captured dig bead vertically on top of the strep bead. Next, steer the trap to move the dig bead down towards the strep bead and open a force distance plot window on the monitor to visualize the force changes. Finally, upon contact, move the dig bead vertically away from the strep bead tracking the force of their separation.
Once an RNA molecule is tethered between beads by its five prime and three prime ends, it can be repeatedly stretched and relaxed. Elasticity of the double stranded handles is indicated by non-linear force extension curves. Cooperative folding and unfolding of the RNA structure between the handles are reflected by abrupt rips and zips in the force extension curve with negative slopes, the refolding of the hairpin is indicated by a zip on the force extension curve.
Importantly, the same molecule may be unfolded and refolded at different forces each time. Such a trend is evident in the distributions of the transition forces. A feedback mechanism can be used to maintain a constant force on the RN.A hairpin folding of an RNA hairpin is indicated by a shortening of extension, whereas unfolding is reflected by an increase in extension under the passive mode, the positions of the trap and the micro pipette are both fixed.
When an RNA folds into a hairpin and shortens its extension, the trapped bead moves away from the center of the trap increasing force. When the RNA is extended to a single strand, the bead moves towards the trap center decreasing force. Using this technique, RNA folding pathways can be observed and manipulated.
With pico Newton and nanometer resolution state lifetimes and transition forces of single molecules can be amassed to derive rate constants and free energies of RNA folding. While attempting this procedure, it's important to remember to be patient while phishing a single molecule. The chance to find a single molecule tether between two beads is very low.
Therefore, many pairs of beads may not stick together at all.
