Chapter 9 – Emerging Biophysics Techniques  407

complexes bound to the DNA act as buffers to prevent propagation of phonons across them

into adjacent domains. This is important since it implies that a simple snap-​fit process may

not work but requires thought as to where specifically gene circuits are engineered in a

genome relative to other gene circuits.

Another biological challenge to the design of artificial gene circuits is epigenetics.

Epigenetic changes to the expression of genes occur in all organisms. These are heritable

changes, primarily by changes to the state of chemical methylation of the DNA, which propa­

gate to subsequent generations without a change of the actual nucleotide sequence in the

DNA (see Chapter 2). The effect of such epigenetic modifications is often different to predict

from base principles, especially for an extensive network of coupled gene circuits, resulting

in nontrivial design issues.

9.3.3  DNA ORIGAMI

DNA origami is a nanotechnology that utilizes the specific nucleotide base pairing of the

DNA double helix to generate novel DNA nanostructures. This DNA nanotechnology was

first hypothesized by Nadrian “Ned” Seeman in the 1980s (Seeman, 1982), though it was two

decades later that the true potential of this speculation was first empirically confirmed using

a range of biophysics tools among others. Double-​stranded DNA has a persistence length of

~50 nm (see Chapter 8), implying that over a length scale of ~0–​50 nm, it is in effect a stiff

rod. Also, DNA has base pairing that is specific to the nucleotide types (C base pairs with

G, A with T, see Chapter 2). These two factors make DNA an ideal construction material for

structures at or around the nanometer length scale, in that, provided the length of each “rod”

is less than ~50 nm, complex structures can be assembled on the basis of their nucleotide

sequence. Since its inception toward the end of the twentieth century, these physical prop­

erties of DNA have been exploited in DNA origami to create a wide range of different DNA

nanostructures. Biophysics tools that have been used to characterize such complex DNA

nanostructure structures include fluorescence microscopy, AFM, and electron microscopy

(EM) imaging, though the workhorse technique for all of this work is gel electrophoresis,

which can at least confirm relatively quickly if key stages in the nanostructure assembly pro­

cess have worked or not.

DNA origami offers the potential to use artificial DNA nucleotide sequences designed

with the specific intention for creating novel synthetic nanoscale structures that may have

useful applications (see Turberfield, 2011). It has emerged into a promising area of research

both in applying single-​molecule biophysics tools in characterizing DNA nanostructures,

and in also utilizing the structures for further single-​molecule biophysics investigations. An

advantage with such structures is that, in general, they self-​assemble spontaneously with high

efficiency from solutions containing the correct relative stoichiometry of strand components

at the correct pH and ionic strength, following controlled heating of the solution to denature

or “melt” the existing double strands to single strands, and then controlled cooling allowing

stable structures to self-​assemble in a process called thermal annealing. Usually, sequences

are designed to minimize undesirable base pairing, which can generate a range of different

suboptimal structures.

In their simplest forms, DNA nanostructures include 2D array (Figure 9.3a). For example,

four DNA strands, whose nucleotide sequences are designed to be permutationally com­

plementary (e.g., strand 1 is complementary to strand 2 and 3, strand 2 is complementary

to strand 3 and 4) can self-​assemble into a stable square lattice 2D array, and similarly a

three-​strand combination can give rise to a hexagonal 2D array (Figure 9.3a). By using more

complex complementary strand combinations, basic geometrical 3D nanostructures can be

generated, such as cubes, octahedra, and tetrahedra (Figure 9.3b), as well as more complex

geometries exemplified by dodecahedra and icosahedra. Typically, a range of multimers are

formed in the first instance, but these can be separated into monomer nanostructure units

by using repeated enzymatic treatment with specific restriction nuclease enzymes to control­

lably break apart the multimers (see Chapter 7).