Chapter 9 – Emerging Biophysics Techniques  409

For example (see Goodman et al., 2005), a tetrahedron can be made from four 55 nucleo­

tide base DNA strands. Each of the six edges of the tetrahedron is composed of one of six

17-​base “edge subsequences” (edge length ~7 nm), which is hybridized to its complementary

segment. Each DNA strand contains three of these subsequences, or their complements,

which are separated by short sequences specifically designed not to hybridize with a com­

plementary strand, and thus act as a “hinge,” to ensure that the tetrahedron vertices have

flexibility to accommodate a 60° kink. Each strand runs around one of the four faces and is

hybridized to the three strands running around the neighboring faces at the shared edges,

and each vertex is a nicked three-​arm junction, and can exist as two stereoisomers (see

Chapter 2). Such a structure has the potential for acting as nanoscale brick for more exten­

sive synthetic 3D structures.

A valuable lesson to learn for the student is the importance of basic, rudimentary thought,

when it comes to the intellectual process of designing such nanostructures. When published

in research articles in their final form, fancy graphics are inevitably employed. However, the

initial intellectual process is often far more basic, down-​to-​earth, and human than this, as can

be seen wonderfully exemplified from the right panel of Figure 9.3b.

It is also possible to engineer larger DNA nanostructures than the simple 3D geometrical

shapes. These can include structures that are more complex than simple geometrical objects.

In principle, a single strand of DNA can be used to generate such structures, referred to as

the scaffold, though to hold it stably in place often requires several short sequences known as

staples, which pin down certain duplex regions relative to each other, which might be liable

to move relative to each other significantly otherwise. Such exotic structures have included

2D tiles, star shapes and 2D snowflake images, smiley faces, and embossed nanolettering,

even a rough nanoscale map of North and South America. Many of these exotic designs, and

the engineering principles used in their formulations, can be seen in the work of Caltech’s

Paul Rothemund in a pioneering research paper that was cited roughly 3000 times in the first

10 years since its publication, which says a great deal about the huge impact it has had to this

emerging field (Rothemund, 2006).

One limit to the size of an artificial DNA structure is mismatched defects. For example,

base pair interactions that do not rely on simple Watson–​Crick base pairing. Although rela­

tively uncommon, the effects over larger sections of DNA structures may be cumulative.

Also, the purification methods are currently relatively low throughput, which arguably has

limited extensive commercial exploitation for “useful” structures beyond the satisfaction of

designing nanoscale smiley faces, though it seems tempting to imagine that these techno­

logical barriers will be reduced by future progress.

“Useful” DNA nanostructures include nanostructures that can be used as calibration tools

or standards for advanced fluorescence microscopy techniques. For example, since optimized

DNA nanostructures have well-​defined atomic coordinates, then different color dyes can be

attached as very specific locations and used as a calibration sample in FRET measurements

(see Chapter 4).

Also, DNA origami can generate valuable 2D arrays that can be used as templates for the

attachment of proteins. This has enormous potential for generating atomic level structural

detail of membrane proteins and complexes. As discussed previously (see Chapter 7), there

are technical challenges of generating stable lipid–​protein interactions in a large putative

crystal structure from membrane proteins, making it difficult to probe structures using x-​ray

crystallography. The primary alternative technique of nuclear magnetic resonance (NMR)

(see Chapter 5) has associated disadvantages also. For example, it requires purified samples

>95% purity in the concentration of several mg mL⁻¹ typically prepared from recombinant

protein to be prepared by time-​consuming genetic modification of bacteria such as E. coli

(see Chapter 7). NMR is also relatively insensitive for small proteins whose molecular weight

is smaller than ~50 kDa.

The main alternative structural determination technique for membrane proteins is elec­

tron cryo-​EM that allows direct imaging of biomolecules from a rapidly frozen solution

supported on an electron-​transparent carbon film and circumvents many of the problems

associated with NMR and x-​ray crystallography (see Chapter 5). However, high electron

current flux in EM imaging can damage samples. Also, there are increased risks of protein