DNA origami Flashcards
DNA origami
= folding long, ssDNA molecules into arbitrary 2D shapes
Involves using numerous short single strands of DNA to direct the folding of a long single strand of DNA into desired shapes
Geometric model of DNA structure
Draw
How are the DNA helices held together?
By a periodic array of crossovers
These crossovers designate positions at which strands running along one helix switch to an adjacent helix and continue there
Why are the parallel DNA helices in DNA lattices not close-packed?
Probably due to electrostatic repulsion
Gap depends on spacing of crossovers
(Gap ~1 nm with crossovers every 1.5 turns along alternating sides of a helix)
Single long scaffold strand
7249 nt long, ssDNA
From the virus M13mp18
Folded back and forth in a raster fill pattern so that it comprises one of the 2 strands in every helix
Progression of this scaffold from one helix to another creates an additional set of crossovers (“scaffold crossovers”)
What is the fundamental constraint on a folding path?
The scaffold can only form a crossover at locations where the DNA twist places it at a tangent point between helices
Seam
A contour which the path does not cross
What are staple strands?
They provide Watson-Crick complements for the scaffold strand and create periodic crossovers
Staples reverse their direction at these crossovers, meaning crossovers are antiparallel - this is a stable configuration that has been well characterised in DNA nanostructures
What happens wherever 2 staples meet?
There is a nick in the backbone
In the final step, pairs of adjacent staples are merged across nicks to yield fewer, longer staples
This gives the staples larger binding domains with the scaffold, leading to higher binding specificity and higher binding energy (and therefore higher melting temperatures)
How are seams strengthened?
By imposing an additional pattern of breaks and merges
Secondary structures found in M13mp18 DNA
Although naturally single-stranded, a hairpin with a 20 bp stem was found
It was unknown whether staples could bind at this hairpin, so a 73 nt region containing it was avoided - cut out by digestion with BsrBI restriction enzyme
Why was a 100-fold excess of staple strands used?
To stop any kind of secondary structure in the viral DNA from forming
Process for forming structure
In one pot there is:
100-fold excess of staple strands
100-fold excess of remainder strands
Scaffold
- Strands are annealed at 95 degrees and then the mixture cooled to 20 degrees over 2 h
- Samples deposited on mica - only folded DNA structures stick to the surface while excess staples remain in solution
- AFM imaging without prior purification
Remainder strands
<= 25 nt
Complement unused sequence of the 7176 nt long viral DNA
Mica
Shiny silicate mineral with applications in atomic force microscopy
When was a particular structure considered qualitatively ‘well-formed’?
If it had no defect (hole or indentation in the the expected outline) greater than 15 nm in diameter
Square fold
26-mer staples (so bind two adjacent helices via 13 bases with each)
2.5 helical turns between crossovers
13 % of structures were well-formed squares
25 % rectangles
25 % hourglass shape with a continuous deformation of the crossover lattice
What did sequential imaging of formation of the square fold show?
It documented the stretching of a square into an hourglass
This suggests that hourglasses were originally squares that stretched upon deposition or interaction with the AFM tip
No subsequent designs exhibited stretching
What did subsequent designs after the square have?
Either a tighter 1.5-turn spacing with 32-mer staples spanning 3 helical domains
OR
Smaller domains that appeared to slide rather than stretch
Rectangle fold
Designed to test the formation of a bridged seam
32-mer staples
1.5 helical turns between crossovers
Circular scaffold
90 % yield of well-formed rectangles
Rectangles stacked along their vertical edges, often forming chains up to 5 uM long
How was stacking abolished?
By omitting staples along vertical edges
Five-pointed star fold
32-mer staples
1.5 helical turns between crossovers
Stars are somewhat squat
Many of the structures observed were star fragments
Only 11 % of structures observed were well formed
How was the low yield of stars improved?
It was thought that the low yield many be due to strand breakage during BsrBI digestion or subsequent steps to remove the enzyme
When the untreated circular scaffold was folded into stars, 63 % were well formed
Three-hole disk
Created to show that DNA origami doesn’t have to be topological disks, and that scaffolds can be routed arbitrarily through shapes
Approximated shape is highly symmetric, but the folding path is highly asymmetric and has 5 distinct seams to increase structural rigidity
Has several characteristic deformations unlike rectangles, but still 70 % well-formed
