Biomod/2011/Caltech/DeoxyriboNucleicAwesome/SPEX Experiments

Tuesday, April 16, 2024 Home Members Project Protocols Progress Discussion References

SPEX Experiments Verification of Overall Mechanisms in Solution See also: SPEX Results Several strands were labeled with fluorophores and quenchers to study there interactions in solution. Walkers were tagged with quenchers while goals for walkers were tagged with fluorophores. The expected reaction is shown below. Walkers are expected to walk from tracks to walker goals. Cargo strands were tagged with fluorophores and goals for cargo were tagged with quenchers. The expected reaction is shown below. Cargo strands are expected to be transferred from walkers to goals. Verification of Random Walking Mechanism on Origami Figure 1. The irreversible binding between walker (W) and walker goal (WG). A ROX fluorophore (red star) was attached to the 5' end of each probe for walker goal (PWG) and a quencher (black cross) was attached to the 3' end of each walker. TR1, track 1. PTR1, probe for track 1. Curved regions shown on the probes were inserted into the origami. Fluorescent spectroscopy (SPEX) was used to verify random walk on the origami. We designed a strand named walker goal (WG) which can bind to walkers irreversibly due to its perfect complementarity with walkers. A ROX fluorophore was attached to the 5' end of the probe for the walker goal (PWG) and the corresponding quencher was attached to the 3' end of the walker (Figure 1). When the walker binds to the walker goal, its quencher is directly above the fluorophore of the walker goal, hence quenching it. Thus we can setup experiments where the fluorescent level slowly decreases as more and more walkers reach the walker goal. The overall origami layout was shown in Figure 2. To verify that the walker walks on the origami using the intended mechanisms, we need a control where there are no tracks, except for the one that the walker is bound to, to see if the walker can simply dissociate off its track and bind to the walker goal, and another control where no walker trigger is released to see if the walker will start moving without the walker trigger. SPEX Results show that there is no decrease in fluorescence in the absence of tracks or walker trigger, but there is a decrease in fluorescence when both tracks and walker trigger are present. Figure 2. The overall origami layout used in SPEX experiments. Each type of track is indexed with a unique number. Three different starting positions were chosen, shown in the figures below. Since it is possible for the walkers to undergo space walking (SW), namely dissociating from the origami, binding to free floating tracks in the solution and rebinding to origami, we added a control group in which only one track at the starting position 10 was planted on the origami. Excess of walker triggers were added in the beginning of the experiment to activate the walkers and excess of walkers with quenchers attached were added in the end to stop the reactions.SEPX Results show that fluorescent signals decreased faster when the walkers were planted nearer to goals. White dots: regular rectangle Light-blue dots: marker to distinguish the orientation of the origami for AFM images Dark Blue dots: track 1 Red dots: track 2 Yellow star: walker goal Green dot: biotinylated control staple used for AFM imaging (that staple is not biotinylated in SPEX experiments) Longest track Middle track Shortest track Space Walk