DNA origami

Question 1

DNA origami

Answer 1

= 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

Question 2

Geometric model of DNA structure

Answer 2

Draw

Question 3

How are the DNA helices held together?

Answer 3

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

Question 4

Why are the parallel DNA helices in DNA lattices not close-packed?

Answer 4

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)

Question 5

Single long scaffold strand

Answer 5

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”)

Question 6

What is the fundamental constraint on a folding path?

Answer 6

The scaffold can only form a crossover at locations where the DNA twist places it at a tangent point between helices

Question 7

Seam

Answer 7

A contour which the path does not cross

Question 8

What are staple strands?

Answer 8

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

Question 9

What happens wherever 2 staples meet?

Answer 9

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)

Question 10

How are seams strengthened?

Answer 10

By imposing an additional pattern of breaks and merges

Question 11

Secondary structures found in M13mp18 DNA

Answer 11

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

Question 12

Why was a 100-fold excess of staple strands used?

Answer 12

To stop any kind of secondary structure in the viral DNA from forming

Question 13

Process for forming structure

Answer 13

In one pot there is:
100-fold excess of staple strands
100-fold excess of remainder strands
Scaffold

1. Strands are annealed at 95 degrees and then the mixture cooled to 20 degrees over 2 h
2. Samples deposited on mica - only folded DNA structures stick to the surface while excess staples remain in solution
3. AFM imaging without prior purification

Question 14

Remainder strands

Answer 14

<= 25 nt

Complement unused sequence of the 7176 nt long viral DNA

Question 15

Mica

Answer 15

Shiny silicate mineral with applications in atomic force microscopy

Question 16

When was a particular structure considered qualitatively ‘well-formed’?

Answer 16

If it had no defect (hole or indentation in the the expected outline) greater than 15 nm in diameter

Question 17

Square fold

Answer 17

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

Question 18

What did sequential imaging of formation of the square fold show?

Answer 18

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

Question 19

What did subsequent designs after the square have?

Answer 19

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

Question 20

Rectangle fold

Answer 20

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

Question 21

How was stacking abolished?

Answer 21

By omitting staples along vertical edges

Question 22

Five-pointed star fold

Answer 22

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

Question 23

How was the low yield of stars improved?

Answer 23

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

Question 24

Three-hole disk

Answer 24

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

Question 25

Triangle form

Answer 25

Created by combining distinct raster fill domains in non-parallel arrangements
Triangle = built from 3 separate 2.5-turn spacing rectangular domains, with only single covalent bonds (i.e. phosphodiester bonds) along the scaffold holding the domains together

Question 26

Why was the yield of the desired triangles so low (<1 % well-formed structures)?

Answer 26

Due to stacking, which caused the rectangular domains of separate triangles to bind
May also be due to the flexibility of the single-bond joints at the vertices of the triangles

Question 27

How was the low yield of the triangle form improved?

Answer 27

By building ‘sharp triangles’ from trapezoidal (rather than rectangular) domains with 1.5-turn spacing
The slanted edges of the trapezoids meet at the triangle vertices, allowing the addition of bridging staples along these interfaces
88 % of sharp triangles were well-formed
55 % when bridging staples at the vertices were not used

Question 28

Function of staples

Answer 28

Bind to the DNA scaffold and hold it in shape
Also provide a means for decorating shapes with arbitrary patterns of binary pixels - using labelled staples e.g. “dumbbell hairpins” that are added to the middle of 32-mer staples

Question 29

How are dumbbell hairpins designed?

Answer 29

To avoid dimerisation at high concentration

Question 30

AFM images with labelled staples

Answer 30

Labelled staples (3 nm above mica) give greater height contrast than unlabelled staples (1.5 nm above mica), resulting in a pattern of light (labelled) an dark (unlabelled) pixels

Question 31

Ways to avoid stacking of structures

Answer 31

Removing staples on the edge of a rectangle
4-T hairpin loops (i.e. 4 thymines in a row) or 4-T tails can be added to edge staples
Stacked chains of 3-5 monomers still formed but 30 % of rectangles occurred as monomers

Question 32

How was controlled combination of shapes achieved?

Answer 32

By designing ‘extended staples’ that connected shapes along their edges
In order to create a binding interaction between 2 particular edges, extended staples were designed by merging and breaking normal staples along these edges
This approach can be used to create both finite and periodic structures
Note - very sensitive to the concentrations of extended staples

Question 33

Reasons for the success of this DNA origami approach

Answer 33

1. Strand invasion/staple excess - allows correct binding of excess full-length staples to displace any unwanted secondary structures/incorrect staples
2. Cooperative effects - each correct addition of a staple organises the scaffold for the subsequent binding of adjacent staples
3. Staples are not designed to bind to one another so their relative concentrations do not matter

Question 34

Application of patterned DNA origami

Answer 34

Creation of a ‘nano breadboard’, to which e.g. proteins can be attached