DNA in nanotechnology

Question 1

Why is DNA useful for building nanoscopic machines/structures?

Answer 1

Because DNA has a nanoscopic structure and can be assembled (and disassembled) in a highly specific manner

Question 2

DNA walker 1

Answer 2

A synthetic, chemically driven molecular motor that can step autonomously along DNA
The catalytic cycles of the ‘feet’ are coordinated to create a Brownian ratchet with the characteristics required for directional and progressive motion
Autonomous

Question 3

How is movement of the lifted foot in DNA walker 1 driven?

Answer 3

Movement of the lifted foot is driven solely by thermal fluctuations
The added fuel is used to provide the energy necessary to rectify this motion and break up the detailed balance between lifting and replacing the front and back feet

Question 4

How do synthetic machines made from DNA, e.g. walkers, generate motion?

Answer 4

Using energy provided by DNA base pairing/hybridisation of a DNA fuel

i.e. they are powered by DNA hybridisation

Question 5

What is required for directional motion?

Answer 5

Free energy

Question 6

Kinesin/myosin V

Answer 6

Protein motors with two feet (“heads”) driven along cytoskeletal filaments by ATP hydrolysis

Question 7

Feet of the DNA walker

Answer 7

Coordinated by means of competition where their binding sites on the track overlap
This competition exposes different ends of the identical feet
This means each foot interacts with the fuel at different rates
A fuel molecule can bind to either foot to displace it from the track, but the catalytic activity of the foot in the left position is much greater than that of the right foot
This means motion is always unidirectional

Question 8

Components of the fuel

Answer 8

The fuel consists of 2 DNA hairpin loops (i.e. H1 and H2) with complementary 18 nt loop domains held closed by the hybridisation of 9 nt neck domains

Question 9

Complementarity between the fuel components

Answer 9

Hairpin H1 is complementary to hairpin H2, except H1 also has a 6 nt ‘tail’ (“toehold domain”) at its 3’ end

Question 10

Structure of feet

Answer 10

Feet are single stranded

They are attached to an 18 bp double-stranded spacer via 4 nt linkers

Question 11

Motor design

Answer 11

Competition between the feet for binding to the track can lift part of the left foot from the track to reveal a “toehold domain”
This can bind to the complementary “toehold domain” of H1
This binding initiates a strand-displacement reaction that opens the neck of H1 and completely displaces the left foot from the track
Part of the open loop of H1 is free to act as a second toehold and initiate hybridisation with H2, eventually forming the H1-H2 duplex as a stable waste product
This displaces H1 from all but the initial toehold domain of the lifted foot, allowing the foot to rebind to the track to the left (‘idling’) or right (‘productive’) with equal probability

Question 12

Why can both components of the fuel (i.e. H1 and H2) be added simultaneously?

Answer 12

Because their spontaneous hybridisation is inhibited by closure of their necks

Question 13

How is the catalytic activity of the left foot activated?

Answer 13

When its toehold domain is exposed by competition from the right foot

Question 14

What is each motor step of the DNA walker coupled to?

Answer 14

One H1-H2 hybridisation reaction

Question 15

Which steps of the DNA walker 1 motion is irreversible?

Answer 15

The breaking of the H1 neck and its binding to the left foot as well as the hybridisation of H1 to H2
These steps are enthalpy-driven due to the formation of all the H-bonds

Question 16

What can DNA nanostructures help to achieve?

Answer 16

The precise transport of a nanoscale object from one location on a nanostructure to another location along a designated path

Question 17

Why is DNA such an excellent building material for nanoconstruction?

Answer 17

Due to its immense information-encoding capacity and well-defined Watson-Crick base pair complementarity

Question 18

DNA walker II overview

Answer 18

The self-assembled track contains 3 anchorages at which the walker can be bound
At each step, the walker is ligated to the next anchorage by a DNA ligase and then cut from the previous one by a restriction endonuclease
Each cut destroys the previous restriction site and each ligation creates a new site in such a way that the walker cannot move backwards
This is the autonomous, unidirectional motion of a DNA motor along a DNA track

Draw roughly

Question 19

Walker

Answer 19

A 6 nt DNA fragment

Question 20

Anchorages

Answer 20

The DNA track consists of 3 evenly spaced DNA double helical ‘anchorages’ (A, B and C)
Each anchorage is tethered to the DNA duplex track by means of a 4 nt flexible ‘hinge’
Each anchorage consists of 13 bp with a 3 nt single-stranded ‘overhang’ = sticky end

Question 21

How far away is each anchorage from its nearest neighbours?

Answer 21

3 helical turns (31/32 base pairs) away from each neighbour

Question 22

Why do the duplex segments of the backbone track and the anchorages behave like rigid rods?

Answer 22

Because they are much shorter than the persistence length of duplex DNA

Question 23

Why is the 4 nt single stranded ‘hinge’ flexible?

Answer 23

Because the persistence length of a single DNA strand is 3 nucleotides

Question 24

Persistence length

Answer 24

A basic mechanical property that quantifies the stiffness of a polymer
Persistence length of dsDNA is approx 390 A

Question 25

How is the autonomous motion of the walker initiated?

Answer 25

By the addition of ATP = energy source

Question 26

What does the motion of DNA walker 2 depend on?

Answer 26

Alternate enzymatic ligation and restriction (cleavage)

Question 27

Which steps of DNA walker 2 are irreversible?

Answer 27

Ligation of the two complementary sticky ends to seal the nicks and join the anchorages together
Irreversible step that consumes energy provided by the hydrolysis of ATP

Question 28

Why is the motion of the walker unidirectional?

Answer 28

Because the product of ligation between 2 neighbouring anchorages can only be cleaved such that the walker moves onto the downstream anchorage
Idling is possible, in which B* can re-ligate to A and C* can re-ligate to B
These idling steps do not block or reverse the overall unidirectional motion of the walker - once B* has been ligated to C, the walker can never return to A

Question 29

How can the autonomous and unidirectional motion of the walker be verified?

Answer 29

By denaturing polyacrylamide gel electrophoresis (PAGE) to track the motion of the radioactively labelled walker
5’ end of walker labelled with gamma-P32
The completion of each step of the process can be detected by the appearance of bands corresponding to radioactively labelled DNA fragments of various lengths
The appearance of each band corresponds to the transfer of the radioactively labelled fragment between the anchorages

Question 30

What are possible reasons for the low measured yields for some of the processes in DNA walker 2?

Answer 30

Imprecise stoichiometry
Low ligation/restriction (cleavage) efficiency, potentially due to steric constraints imposed by the design of the motor - each substrate for the enzymes is created by the hybridisation of 2 anchorages, which are also linked by the backbone of the track

Question 31

Restriction endonuclease

Answer 31

Cuts dsDNA to give a 3’ OH and a 5’ phosphate