IV. ParMRC and Expandable DNA Nanostructures
IV.2. Results and Discussion
IV.2.1. Biophysical studies of the ParMRC system
IV.2.1.2. Origami with multiple parC strands
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A detailed look at the images roughly reveals the structure of the ParR/parC complexes (Figure IV-9d). Each complex shows slightly different structure but has in common two oval parts and loops connecting the two. The loops have roughly the same height as that of the rectangle origami (based on the color profile), so it is highly likely that they are single DNA double strands, and in particular, the promoter part (~59 bps) between the two sets of five iterons (ParR binding sites) in the parC sequence. This structure is, in fact, in very good agreement with previous AFM data for short DNA fragments in the literature (shown in Figure IV-2c), where they claimed the ParR/parC complex to be “U-shaped” based on their AFM data103. However, one needs to be careful in interpreting AFM data, as the interaction between molecules and the mica surface can create various kinds of artifacts. For example, the ParR/parC complex may simply get squashed onto the surface, while adopting, for example, a helical or circular form in solution.In summary, using our single-molecule DNA origami templates, we were able to reveal that ParR can bind to non-recognition sequence DNA, in addition to the parC sequences, under high stoichiometric conditions of ParR. But in the right concentration regime, ParR did specifically bind to parC and discriminated non-recognition sequences. The ParR-parC interaction observed in our system in the recognition regime is consistent with previous studies in literature in terms of both stoichiometry99 and structure103.
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Figure IV-10. DNA origami triangle with multiple parC strands. (a) Model diagram of the triangle with anchors (T20) for parC strands, without and with parC strands bound. (a) AFM of DNA origami triangles without parC strands. (b) AFM of DNA origami triangles with parC strands. Scale: one side of a triangle origami is ~130 nm.
We decided to test DNA origami with multiple parC strands, to be able to examine more bulk interactions between parC strands, ParR and ParM, at the regime where distortion by surface to measurements is minimal. We created DNA origami with multiple parC strands by incorporating linear parC strands onto an origami structure via linkers extended from 5’ ends of staple strands.
parC strands were further modified for fluorescence labeling. The detailed design of the parC strand and the linker for origami are described in the Materials and Methods section. We used triangle origami and rectangle origami for the investigation.
Figure IV-10 shows the model diagram (Figure IV-10a) and AFM images of the triangle origami in the absence (Figure IV-10b) and the presence (Figure IV-10c) of the parC strands. As can be seen in Figure IV-10c, parC strands (199 bps, double stranded part, including the linker to origami) stretch out of the triangular shape of the origami. Since the parC strands were linked to staple strands that compose the body of origami, they make the origami body thicker, as compared to the single double-strand layer of parC strands, as shown by the colored height profile.
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97 IV.2.1.2.1. AFM movies of ParR-parC binding
Although the use of origami with multiple parC strands was intended for characterization using methods other than AFM, AFM is still a very powerful tool to reveal and confirm details of structures and phenomena at the molecular level. Using the triangle origami functionalized with multiple parC strands, and using a high-speed AFM, we could obtain AFM movies that show progressive binding of ParR onto parC strands on origami. Figure IV-11 shows the model diagram and frames taken from two independent movies of parC-functionalized origami, to which ParR molecules were binding progressively. ParR was added into a large (~100 ul) buffer bath in situ while scanning, so the progression of binding also reflects the diffusion of ParR molecules in solution, in addition to the potential binding, searching and unbinding process.
Figure IV-11. AFM movies of ParR binding to parC strands on origami. (a) Model diagram of the triangle with multiple parC strands, before and after binding of ParR. (b,c) Representative frames from two independent AFM movies that show the progressive binding of ParR to parC strands on origami. Time points are indicated at the top of each frame. Note that in (b) the AFM tip was doubled. Scale: one side of a triangle origami is ~130 nm.
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The ratio between the total numbers of molecules parC and ParR was between ~1:10 and~1:100, the stoichiometric ratios where binding was unsaturated in the single-parC origami experiments. These movies are in good agreement with the observation for single-parC origami in that the parC strands do not seem to be saturated and form loop-like structures. Although it looks as if a large number of ParR molecules are bound to origami in a single imaging field, it may be due to the high local density of parC strands (177 per origami). Since these high-speed AFM experiments were carried out during demo sessions at a conference away from the lab (see Materials and Methods), it was difficult to perform more experiments with careful control over the concentrations due to the lack of time and resources.
Nonetheless, these movies provide interesting footage of the dynamic behavior of the DNA- binding protein, including some transient binding of ParR molecules to the parC strand. The movie files can be downloaded from http://dna.caltech.edu/Woo-thesis-movies. The movies were obtained at a rate of 1 frame / 4 seconds (by taking an AFM image every 4 seconds), and played at a rate of 4 frames/sec (16× faster).
IV.2.1.2.2. Gel electrophoresis study of concentration dependence of ParR binding
Binding of ParR to the multiple parC sites on origami was also confirmed by gel electrophoresis. Different extents of gel mobility shift depending on the concentration of ParR revealed concentration dependence of ParR binding to parC sites on origami. Figure IV-12 shows the gel data. There are a few additional interesting points to note.
First, the ParR concentration dependence of ParR-parC binding can be recognized in a few different ways. The blue dotted boxes highlight the different gel mobility shifts of the origami that contain multiple parC strands. From the relatively fast migrating origami-only band (lane 2)§, the origami band loses its mobility when bound by multiple parC strands (lane 3) and subsequently by ParR (lanes 4-7). The mobility decreases progressively with increasing ParR concentrations from 10×, to 20×, 50×, and 100×. Near the highest concentration of ParR (50× and 100×), the mobility reduced significantly and a lot of materials were left unmigrated into the gel in this particular experiment. Those materials might have migrated a bit if the gel had been kept running longer, but it may be the case that either the origami-parC-ParR complexes with increasing occupancy of ParR
§ In lane 2, there are two bands for origami within the blue dotted box, the higher band of which was later found to be structurally disrupted origami because of the absence of Mg2+ in the gel-running buffer. Those disrupted origami are believed to have failed to overcome the high strain in origami crossover structures, thus, at least for this gel mobility experiment purposes, the parC strands are expected to be still incorporated and function similarly to those on normal origami.
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Figure IV-12. Gel electrophoresis data of ParR binding to parC strands on origami, which revealed both the concentration-dependent binding of ParR to parC and nonspecific binding of ParR to non- recognition DNA. (L: 1kb DNA ladder) The ratios given for ParR are relative to the parC concentration of each lane. For lanes 12-15, the same amounts of ParR were added as in the cases with parC.
on each origami just became too bulky to go through the gel (1% agarose), or ParR may be positively charged and may have neutralized DNA, substantially reducing the mobility in gel electrophoresis.
The ParR concentration dependence can also be observed in the free parC bands. The wide band in the lower area (highlighted by the orange box) in lane 3 corresponds to free parC strands that did not bind to origami (initially added in ~1600× excess of the origami scaffold, ~2× of total staple concentration), as verified by comparing it with the band in lane 1 (free parC only band).
Since parC strands were added in ~2× concentration of total staples, about half of the population in the band in lane 3 are bound to free staple strands, but the change in molecular weight is small (52
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nucleotides) compared to the weight of parC (total ~370 nucleotides) and does not get resolved in agarose gel. The mobility of those parC strands get shifted by ParR, with the extent increasing with increasing concentration of ParR (lanes 4-7). The same pattern can be observed for free parC strands mixed with ParR in lanes 8-11, with the relative gel heights between corresponding bands agreeing quite well.An interesting observation is made for the lanes for origami that do not contain any parC strands (lanes 12-15). ParR was found to bind to the non-recognition sequences on the origami (higher bands, highlighted by green boxes) and on the free staples as well (lower bands, highlighted by yellow boxes; note that free staples in lane 2 appear in a similar position to free parC strands because of the low resolution of agarose gel), consistent with our earlier observations on origami containing single parC under AFM. Origami bands appear to migrate slower by the binding of ParR, and such non-recognition binding also occurs in a concentration-dependent manner (lower mobility with higher ParR concentration). Note that the ratios of ParR given are relative to parC strands as if there were parC strands in the system, to keep the condition the same as the cases with parC strands (lanes 4-7). The concentration ratios between origami and ParR become ~16,000×,
~32,000×, ~80,000×, and 160,000× for 10×, 20×, 50×, and 100×, respectively, all of which are enormously excessive amounts from origami’s point of view. These ratios between origami and ParR were still the same for the cases with parC (lanes 4-7). Note also that the bands of nonspecific complexes, origami-ParR (green boxes) and staple-ParR complexes (yellow boxes) are located at higher positions than their counterparts of specific complexes, origami-parC-ParR (blue boxes) and parC-ParR (orange boxes)—compare lane 12 with lane 4, and lane 13 with lane 5, etc., despite the absence of parC in each complex. Ironically, this is in fact because of the absence of parC; the ParR molecules, which would have been bound to the parC strands both anchored on origami and free in solution in the presence of parC (lanes 4-7) and hence would have been “consumed”, do not have the specific targets and instead would extensively bind to the bare origami and staple strands. Note that parC strands were added at 2× the concentration of staples in the cases of lanes 4-7, so the origami and staple strands in lanes 12-15 have roughly double the amount of ParR to
“accommodate” per each origami or staple, compared to the cases in lanes 4-7. In addition to that, the high excess amount of ParR and extensive binding to origami and free staples might cause them to aggregate, thereby yielding larger and less mobile structures. Also, potential spurious interactions between free staple strands via partial complementarity may allow transient formation of duplex regions, which may get bound and stabilized by the high excess amount of ParR, which then may propagate into large network structures.