IV. ParMRC and Expandable DNA Nanostructures
IV.3. Conclusion and Future Directions
We used DNA origami nanostructures as templates in attempt to study some structural aspects of the ParMRC system. We were able to reveal that ParR at high concentration binds to non-recognition sequence DNA, in addition to the parC sequences, both through AFM studies and gel electrophoresis analysis. In the right concentration regime, we observed that ParR recognizes parC and discriminates non-recognition sequences. The specific ParR-parC interaction observed in our system was consistent with previous studies in literature in terms of both stoichiometry99 and structure103.
Then we used the dynamic filament growing mechanism of the ParMRC system to engineer expandable nanostructures. We first placed parC sequences on origami structures and successfully
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reconstituted the ParMRC system: using fluorescence microscopy, we observed colocalization of origami and the ends of ParM filaments, and dynamically growing filaments whose ends are stabilized by ParR/parC complexes anchored on origami. By using chains of rectangular origami connected via stacking bonds, we demonstrated that bundles of growing ParM filaments could break stacking bonds and dynamically expand the structure.This work is in progress, and we discuss potential future directions in subsections below.
IV.3.1. Expandable triangles
As briefly discussed earlier in the discussion, we are planning to test expandable triangle structures, each made with three corner shape origami, connected via stacking bonds, to amplify the dramatic change in the overall structure; from a small single bright dot (due to three corners being unresolvable by light) to a large, growing triangle with red dots at each corner. Figure IV-24a shows the schematic of the triangle design. Within the shaded area, there are ~100 staple strands, each of which we can extend to anchor a parC strand. The number of parC strands per origami (a single corner) is comparable to the number we had per origami in the origami chain case, so this triangle design would be appropriate for proof-of-concept experiments and for comparison with the chain case.
Figure IV-24. Schematic diagrams of (a) a three-corner triangle as a substrate for expandable nanostructure with the shaded area indicating the anchor sites for parC strands and (b) a cup-shaped origami design that might allow the creation of an artificial (parC-free or ParR/parC-free) ParMRC system.
Also, as mentioned earlier, to be able to capture the early moments in the dynamic system (ideally from t=0), construction of a fluid chamber system that enables mixing of the components while under the microscope would be helpful. However, careful control over the concentration of the components would be a challenge, and proper surface treatment protocol that can hold origami loosely in place on the substrate while preventing binding of protein components should be
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developed. Or, introduction of photo-activatable ATP might be a useful alternative. With photo- activatable ATP, all other the components can be added into the closed system without any change from the systems we have tested, and with an external stimulus in the form of light irradiation of specific wavelength, we may be able to watch the reaction from t=0.IV.3.2. Effects of distance between ParR/parC complexes on ParM filament connection
Besides engineering expandable nanostructures, we would still pursue the direction of studying biophysics of the ParMRC system, using DNA origami as a custom tool. As it has been the case that it is hard to study the system at the single parC regime (mostly because of technical reasons, e.g., difficulty of taking AFM of filaments, whether dynamic or not, and too low signal-to- background ratio of a single filament under fluorescence microscopes), we seek other questions that we could answer about the biophysical behavior of the ParMRC system using our DNA origami designs.
One question is: how would the distance between ParRC complexes affect the efficiency of ParM filament connection between the two positions? In a living bacterial cell, the actual distance relevant to the search process of ParM filaments for two ParRC complexes seems to be on the order of about hundred nanometers; within a cell whose size is ~1 um in diameter and ~2-3 um in length, the plasmids do not diffuse much and show a confinement radius of ~0.28 um, in the absence of (i.e., before getting pushed by) any ParM filaments111. Examining how the efficiency of the search process changes depending on controlled distances between ParRC complexes would reveal interesting insights and expand our understanding on the dynamics of the system.
We can precisely control the positions of parC sites on a single origami, and hence the distance between parC sites on neighboring origami structures in rectangle chains or the corner- origami triangles. For longer range distances, say, larger than ~100 nm, the length of a single origami rectangle, we can program the stacking bonds based on our orthogonal bond sets to create origami multimers with defined arrangement, and put parC sites at distances larger than a single origami. We also plan to examine much larger distance ranges, e.g., several microns to a few dozens of microns, using our origami surface placement technology80, varying the distance between the binding patches on patterned substrates. With surface-immobilized origami, we could use the total internal reflection microscopy (TIRF) technique, which might allow us to resolve a small number of filaments, perhaps down to the single-filament regime.
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IV.3.3. Effects of the number of parC sites on single origami structures on the growth rate of ParM filament bundles
Another question that we are interested in is how the number of parC sites on a single grouped structure (in our case, origami) would affect the growth rate of the ParM filament bundles.
In living cells, it is proposed that multiple plasmids often form clusters and segregate together as a single unit104,111, so it would be interesting to examine how the behaviors change depending on the number of parC sites grouped in a single cluster.
Again, we can precisely control the number of parC sites on a single cluster using our origami structures. We can use discrete origami structures, e.g., single-unit triangles, instead of chains connected by stacking bonds, and observe the dynamic expansion driven by growing filament bundles. By taking time lapse images, and measuring the rate of change in length of filament bundles between origami foci, we could potentially draw some interesting relationships between the number of parC sites and the growth rate.
IV.3.4. Cup experiments
IV.3.4.1. Is it just the geometry that really matters?
As discussed earlier, we observed strong binding of ParR to non-recognition sequences of dsDNA on origami under high ParR concentrations. That observation led us to ask the question whether a ParR loop created by artificial shapes with non-recognition sequences would stabilize ParM filaments. This is a question of whether it is just the geometry that really matters, or whether the parC sequence has any important role other than facilitating the cooperative binding of ParR into a complex.
For such a test, we can design a three-dimensional origami in the shape of a cup or cylinder, where helices are curved and aligned perpendicular to the cylindrical axis (Figure IV-24b). At high concentrations of ParR, ParR will bind to the helices that are exposed and relatively free, as found by AFM studies discussed earlier. Then we can detect and measure the association of ParM filaments with these origami-mediated ParR loops. If these “artificial” ParR caps do the job of the real ParR/parC complexes, even in the absence of parC, we would be able to see continuous growth of the ParM filaments, with their ends in association with the ParR-origami complex.
Since we do not know the exact shape of the ParR/parC complexes and the binding mode of
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ParM filaments—ring vs. helix shape, parallel vs. perpendicular binding, wrap-around vs. open- clamp binding, etc.—the geometry of the origami would have to be carefully designed to accommodate various possibilities. For example, the cylinder shape could be designed to be reconfigured by changing the subset of the staple strands to allow a helical contour of the DNA helices. Or we could leave out some staple strands at part of the loop to allow potential open-clamp type binding.IV.3.4.2. Would the active site in ParR alone stabilize filaments?
In fact, the active site in ParR that is responsible for stabilizing ParM filaments was identified to be the C-terminal domain (~17-33 residues) of the protein98,100, and it was hinted that the active site alone could stabilize ParM filaments; the presence of synthetic peptide with the C-terminal amino acid sequence in high excess (~100× of ParM) interfered with the ParM filament binding to regular ParR/parC complexes100.
This allows us to pose a question: could we construct a system just using the C-terminal domain that stabilizes ParM filaments? Beyond eliminating parC, could we also remove ParR, except the C-terminal domain, from the ParMRC system? The cup design may be useful for such a test as well. We can synthesize a hybrid of a DNA strand and the C-terminal domain, and place them on the inside of the cup. By placing a large number of them, we can create a high local concentration of the domain inside the cup, and the active domain peptides may successfully stabilize ParM filaments without the need of the well-defined ParR loop structure, much less the parC strand. If this approach is realized, based on a synthetic peptide, and a designed DNA origami structure, we will be the first to have created a nearly totally artificial ParR/parC complex—just except for the scaffold strand in origami.