Introduction
DNA Nanotechnology
Towards the Development of Nanomachine Connectors
Survey of Studies on Base Stacking
Thesis Outline and Contributions
Design and Implementation of Stacking Bonds
Introduction
In addition to DNA, other information-containing heteropolymers such as proteins can be used for various combinatorial connections. Pure base stacking can be studied by examining the binding between open ends, the ends of a DNA duplex.
Design, Results and Discussion
- Stacking of origami rectangles
- DNA sequence at blunt ends
- Global twist
- Crossover geometry at blunt ends
- Two approaches towards specific stacking bonds
- Binary codes
- Shape coding
- Summary and comparison of the two approaches
- Programming of cis-trans isomeric structures
- Thermodynamic measurements
- Measurement of the free energy of stacking bonds
- First energy model: assuming loop-loop interactions are neutral
- Second energy model: fitting with non-zero loop-loop interactions
Instead, we simply assume here that the multiplicity of strongest partial bonds is equal for different sequences. For edges of the tall rectangle system (which has a total of 16 available faces), there are 4614 different binary sequences with (p, i) or (7,4), as shown in Table II-1.
Conclusion and Future Directions
It is interesting to ask whether the average loop interaction is typical or whether most loop interactions are neutral and only a few inactive patches contribute most of the binding energy. More experiments will be needed to answer this question, particularly measurements of the binding energy for folding bonds that have the same folding sequence but loops with different base sequences. For a stacking sequence of 16 patches with 7 active patches, the 9 inactive patches will contribute a binding energy roughly equivalent to two active patches and about one quarter (by chance) of the total binding free energy.
Our binary coding approach allows the binding type to be easily and inexpensively reprogrammed post-synthesis simply by pipetting a different subset of edge clamps. A disadvantage of stacking bonds is that the total binding energy is limited by the size of the origami: a larger range of binding energies can be achieved in a sticky-end-based approach by varying the lengths of the sticky ends. In order for DNA structures linked by DNA hybridization to rearrange, DNA helices must be unwound and then rewound; this may involve a high activation energy, depending on the number and strength of the bonds involved.
But base pairing has a few components: In addition to the interaction between hydrogen bond donors and acceptors, there is the geometric complementarity of the base pairs.
Materials and Methods
- General
- Design of origami
- Preparation of origami
- Atomic force microscopy (AFM)
- Detail aspects in the design process
- Searching for large orthogonal sets of sequences
- Warnings: potential technical issues in designing DNA origami
- Supplementary materials
Effect of the concentration of Mg2+ in the Na+ solution during the surface diffusion step. Step (2): we then exchange the buffer on the mica substrate by pipetting out ~20 ul of the TAE/Mg2+ buffer on top (we expect the majority of origami to be strongly bound to the surface), which ~5 ul of the buffer distribution leaves on the mica surface. The sequence direction of the parC DNA may impose some structural or functional polarity in the ParR/parC complexes.
Furthermore, the asymmetry of the polar ParM filament itself may also impose potential polarity in the ParR/parC complex. 95 A detailed look at the images roughly reveals the structure of the ParR/parC complexes (Figure IV-9d). The loops are about the same height as those of the rectangular origami (based on the color profile), so it is very likely that they are single DNA double strands, and in particular the promoter portion (~59 bps) between the two sets of five iterons (ParR binding sites) in the parC sequence.
The majority of origami containing ParR/parC complexes (highlighted in red) appear at the ends of ParM filaments (highlighted in green), suggesting the binding of ParM filaments to ParR/parC complexes.
Two-Dimensional Crystallization of DNA Origami Checkerboards via Cation-
Introduction
At the same time, the use of DNA origami allows higher complexity and access to unique addressability in each structural unit. 63 patterned crystals based on surface diffusion, where the diffusion of DNA origami occurs on the substrate surface and is dynamically controlled by changing the cationic state of the system. Unlike previous surface-based self-assembly studies of DNA nanostructures77-79, our protocol can operate at an isothermal condition without the need for a thermal annealing process, giving a further practical advantage.
Monovalent cations weaken the binding of DNA to mica surfaces and have often been observed to release DNA previously adsorbed onto mica. It has been shown that Ni2+ ions enable permanent binding of DNA to mica, even at high Na+ concentrations83. Such direct binding of DNA to mica can be regarded as a short-range attraction82, which induces strong adsorption of DNA to mica and enables practically stable imaging of DNA by AFM.
We discuss in detail the factors that enable the surface diffusion and two-dimensional crystallization of DNA origami rectangles.
Results and Discussion
- Two-dimensional assembly by surface diffusion
- Factors that allow two-dimensional assembly
- Why checkerboards, not linear chains?
- Effects of the strength of stacking interaction
- Effects of the concentration of divalent cation
- Crystal size is limited by irregularity of mica
We had originally thought that it could be attributed to non-uniform base sequences at the open ends. 70 caused severe distortion along the edges, such as bending, preventing the edges of the uncorrected origami from meeting in the linear configuration even in solution. After the surface diffusion protocol, some of the rectangles still formed checkerboard patterns, and some of them remained as linear chains.
We varied the number of stacking blunt ends at each corner to see the effect of stacking interaction strength. First, again some of the linear chains formed in solution seem to break during the diffusion step, especially in the case of N=5. Presumably, linear chains already formed in solution with a bond strength of N=7 may have a lower tendency to break at the surface, thus maintaining a linear shape.
As already mentioned, during step (2) of the protocol, we still have Mg ions in the system, especially pre-adsorbed on the mica surface.
Conclusion and Future Directions
Materials and Methods
- Surface diffusion assembly
- Atomic force microscopy (AFM)
The subnanometer resolution of the present reconstruction confirms the polar double-helix structure of ParM filaments. The outer diameter of a ParM filament matches the inner diameter of the ring formed by a ParR/parC complex. The detailed design of the parC strand and the origami linker is described in the Materials and Methods section.
The distance should be the lateral distance between the bottom of the well (energy minimum) and the top of the well where it plateaus near 0 (theoretically it approaches 0 at infinity). When we mixed the origami chains with ParR and ParM, we could observe a unique behavior of the ParM filaments. Since we do not know the exact shape of the ParR/parC complexes and the binding mode of.
Due to the large buffer volume, ParR took some time (on the order of minutes) to diffuse and bind to the parC sites on origami, which was another factor that enabled AFM films (aside from high-speed scanning) .
ParMRC and Expandable DNA Nanostructures
Introduction
- Using DNA origami to study biophysical questions
- The ParMRC system
- Open questions
- DNA origami design: proposed and actual
- Building expandable structures using growing biopolymers
One is to use DNA origami as a personal tool to study some biophysical aspects of the ParMRC machinery. We attempt to couple the dynamic behaviors of the ParMRC system with DNA origami nanostructures and demonstrate expandable dynamic nanostructures. This moves the ParR/parC complexes, and thus each of the two plasmid copies, to opposite poles of the cell.
A high-resolution cryoEM reconstruction of the ParM filament ( Fig. 2A and B ) was obtained as previously reported ( Gayathri et al., 2012 ). Conflicting models and supporting evidence for the geometry of ParR/parC complexes. a) The "Ring" model and the corresponding EM data. The upper strand of double-stranded parC was extended at the 5' end by one.
87 triangle expands through the polymerization of ParM and the stabilization of the growing filaments by ParR/parC complexes at each edge near the bonds of origami.
Results and Discussion
- Biophysical studies of the ParMRC system
- AFM studies of ParR binding to parC on origami
- Origami with multiple parC strands
- Expandable nanostructures
- Model system: origami as ParR/parC cores stabilizing ParM filaments 101
- Control system: non-expanding DNA nanotubes
As seen in Figure IV-10c, the parC strands (199 bps, double-stranded portion, including the origami linker) extend from the triangular origami shape. Nevertheless, these movies provide interesting snapshots of the dynamic behavior of the DNA-binding protein, including some transient binding of ParR molecules to the parC strand. The blue dotted boxes highlight the different mobility shifts of the origami gel containing multiple strands of parC.
The most influential variable was the concentration of the crowding agent, methylcellulose, in our system. Design and microscope images of DNA origami rectangle chains. a) Schematic diagram of the DNA origami rectangle chains used for the study. Each of the parC strands was fluorescently labeled with Cy3. b–e) AFM images and (f) a fluorescence microscope image showing the rectangle chains.
By selecting a subset of key sites, we can control the total number and locations of parC strands. Each of the parC strands was fluorescently labeled with Cy3 (hybridizing (CTT)5-Cy3 to the terminus of the parC “down” strand). Design and microscopy imaging of DNA nanotubes. a) Schematic diagram of the DNA nanotubes used for our experiments.
Conclusion and Future Directions
- Expandable triangles
- Effects of distance between ParR/parC complexes on ParM filament
- Effects of the number of parC sites on single origami structures on the
- Cup experiments
- Is it just the geometry that really matters?
- Would the active site in ParR alone stabilize filaments?
Also, as mentioned earlier, to be able to capture early moments in a dynamic system (ideally from t = 0), the construction of a fluidic chamber system that allows mixing of the components under the microscope would be helpful. Examining how the efficiency of the search process varies with controlled distances between ParRC complexes would reveal interesting insights and expand our understanding of system dynamics. Effects of the number of parC sites on individual origami structures on the growth rate of ParM filament bundle structures on the growth rate of ParM filament bundles.
Another question we are interested in is how the number of parC sites on a single clustered structure (origami in our case) would affect the growth rate of the ParM filament bundles. Or we can leave out some staple strands on part of the loop, allowing for possible open clamp binding. We can synthesize a hybrid of a DNA strand and the C-terminal domain and place it on the inside of the cup.
By placing a large number of them, we can create a high local concentration of the domain within the cup, and active domain peptides can successfully stabilize ParM filaments without the need for the well-defined ParR loop structure, much less the parC strand.
Materials and Methods
- Preparation of DNA origami
- DNA origami with a single parC
- DNA origami with multiple parC strands
- Preparation of microbeads with parC
- Preparation of proteins
- Preparation of ParM polymerization buffer
- AFM of parC-origami, ParR, and ParM
- Gel electrophoresis of parC-origami with ParR
- Preparation of glass slides
- Sample preparation for fluorescence microscopy
The upper strand of double-stranded parC was extended at the 5' end with a 20 A overhang that can bind to the 20-T anchors on the origami. For the triangular origami, 177 clips out of a total of 228 were replaced with those containing a 20-T tail. Each strand of parC was designed to contain a biotin at the 5' end of the "bottom" strand.
The bead solution and parC solution were mixed at a ratio of 1 mg of beads to 1.40 nmol of parC, according to the manufacturer's instructions. The solution was then subjected to a dialysis assay with a 300k cut-off membrane in a two-liter reservoir of 1× TAE/Mg2+ buffer containing a stir bar for ∼12 h, to remove extra parC strands if present. A slow withdrawal of the slides and covers from the water tank can indicate how well the silanization is done (the water should completely repel the surface and the slides and covers should come out dry).
High-throughput, real-time monitoring of the self-assembly of DNA nanostructures by FRET spectroscopy.
