DNA robot

Question 1

DNA nanostructures/nanomachines

Answer 1

= 3D DNA origami

3D DNA components can specifically self-assemble in solution on the basis of shape complementarity, without base pairing

Question 2

Inspiration for assembly of DNA nanostructure/nanomachine

Answer 2

Interaction between RNase P and pre-tRNA
They interact through weaker interactions than base pairing
tRNA fits precisely into a correspondingly shaped binding pocket in RNase P and is held in place by a few nucleobase stacking/pi-pi stacking interactions (i.e. weak interactions)

Therefore hypothesised that stacking interactions might suffice to stabilise 3D higher-order complexes made from multilayer DNA objects in solution

Question 3

What were the authors attempting to do?

Answer 3

Translate non-hybridisation-based shape recognition principles from natural RNA to synthetic DNA objects

Question 4

Comparison between DNA bricks and tRNA/RNase complexes

Answer 4

Blunt-ended double helical DNA protrusions on one domain assume the role of the tRNA acceptor stem
The corresponding recessions on another domain mimic the RNase P binding pocket
Nucleobase stacking interactions engage at the double helical interfaces of the shape-complementary protrusions and recessions when the 2 domains are brought into contact
(but only upon the correct fit of the helices and the correct helical orientation of the interfacial nucleobase pairs)

Question 5

How are the DNA building blocks used?

Answer 5

In a combinatorial fashion to create libraries of shape-complementary motifs
Shape-complementary partners are accepted and precisely oriented
Non-complementary partners will be sterically rejected

Question 6

What did the authors design?

Answer 6

Four multilayer DNA origami bricks that form the subunits of a tetrameric complex - this illustrates the shape selectivity and ability of the recognition scheme to constrain the position and orientation of individual DNA objects within larger complexes

The embossed surface of brick A fits precisely into the recessed surface of brick B
And likewise for combinations of B with C and C with D
The bricks can self-assemble into all possible multimeric complexes, including dimers, trimers and a tetramer

Question 7

How can the bricks be identified by TEM?

Answer 7

Bricks B, C and D exhibit characteristic asymmetric features that enable their orientation to be identified
These asymmetrical features are indicated in dark grey

Question 8

How did the authors illustrate the ability of the click-in shape recognition scheme for precisely defining conformational states?

Answer 8

They designed a switch-like DNA object consisting of 2 rigid beams connected by a pivot
One rotational degree of freedom

Question 9

What does the switch-like DNA object consisting of 2 rigid beams connected by a pivot illustrate?

Answer 9

The ability of the click-in shape recognition scheme to precisely define conformational states within a multidomain DNA object

Question 10

DNA switch

Answer 10

Can dwell either in an ensemble of open states or in a structurally well-defined closed state

Question 11

Structure of the closed state of the DNA switch

Answer 11

Governed by the shape-complementary patterns of double-helical DNA domains that can click into each other when the 2 beams draw together

Question 12

Why is the conformational equilibrium of objects that utilise shape-complementary interactions sensitive to the concentration of counter ions in solution?

Answer 12

Due to repulsions between the negatively charged surfaces of the DNA binding partners

Question 13

What is the ‘tiered hierarchy’ in the shape recognition scheme?

Answer 13

There is a tiered hierarchy between intradomain stability and interdomain interaction, as it is based on a few nucleobase stacking interactions rather than the many base pairing interactions that stabilise entire DNA objects

Question 14

How can the conformational equilibrium of objects that utilise shape-complementary interactions be altered?

Answer 14

The conformational equilibrium can be adjusted rapidly and reversibly by global parameters such as cation concentration and solution temperature
These options can be tested using both ensemble and single-molecule FRET experiments, as well as TEM imaging

Question 15

FRET

Answer 15

Fluorescence Resonance Energy Transfer

Question 16

Effect of increasing cation concentration on both the switch and dimeric bricks

Answer 16

Increasing cation concentration shifted the conformational equilibrium from the open/monomeric states to the closed/dimeric states, for the switch and dimeric bricks respectively
Occurred with both mono- and divalent cations
Transitions were reversible upon cyclic changes in the concentration of cations

Question 17

The greater the strength of the designed interaction between the shape-complementary interfaces of the switch…

Answer 17

…the lower the cation concentration necessary for stabilising the closed state

For strong hybridisation-based interactions at all complementary sites (instead of the minimal stacking interactions), the open state could not be prepared without compromising the overall structural integrity of the switch

Question 18

Applications of the self-assembly of higher-order DNA objects

Answer 18

Can constrain the position of binding partners with sufficient rigidity so that self-complementary bricks can seamlessly self-assemble into homomultimeric filaments of up to hundreds of monomers with no bending deformations
Simply decreasing or increasing [cation] allows recovery of constituent monomers or restoration of the growth of filaments - this ability to reversible shrink/grow filaments is of interest for creating active materials
There is the opportunity for creating various reversibly reconfigurable DNA devices with arbitrary shapes

Question 19

Nanorobot

Answer 19

Heterotrimeric 15 MD complex comprising 3 asymmetric subunits that assemble specifically on the basis of shape recognition
2 modules form the robot's torso, 1 module forms its legs
The 2 torso modules each have an 'armlike' domain hooked up to a shoulder-like protrusion via a pivot (similar to the pivot in the switch)
The arms can therefore switch between open and closed states, where the closed states are stabilised by shape-complementary stacking bonds between the forearm and the hip of the robot

Question 20

Major advantage of DNA nanotechnology

Answer 20

The complexity of the structures used in DNA nanotechnology provides the freedom to attach a plethora of functionalities in any desired pattern/orientation

Question 21

Limitations/disadvantages of 3D DNA origami

Answer 21

Low yield (typically 1 % after several days of thermal annealing)
Small production scale
Cost of synthetic DNA
DNA nanostructures are often labile and highly sensitive to ion strength, temperature and nucleases - not good for their potential applications as drug delivery agents

Question 22

Potential improvements for DNA nanotechnology

Answer 22

Proper analysis of the folding process to reduce folding time from days to hours/minutes (Dietz et al)
Protecting strategies e.g. coatings, chemical modifications to increase the structural stability of the DNA
Larger scale purification methods
Enzymatic production methods of staple strands