Synthetic Biology Parts, Devices and Applications

12.1 Introduction 241

Niemeyer and coworkers [13] were the first to in vitro assemble enzymes on a
DNA scaffold. They arranged NADH:FMN oxidoreductase and luciferase onto a
double‐stranded DNA scaffold using the biotin streptavidin linkage and showed
that the immediate spatial proximity of the enzymes enhances the coupled activ­
ity. Later, they showed the operational DNA scaffold using glucose oxidase and
horseradish peroxidase covalently linked to the DNA [14]. This system was fur­
ther evolved by Wilner et al. [15], using a supramolecular DNA scaffold, who
linked glucose oxidase and horseradish peroxidase via a lysine residue to the
DNA oligonucleotides that hybridized onto the DNA nanostructures.
The DNA scaffold with conjugated oligonucleotides onto enzymes and assem­
bled to DNA nanostructures is impractical to use in vivo. Conrado and cowork­
ers [11] were the first to demonstrate the functional DNA scaffold in bacteria,
where the enzymes were attached to the DNA‐binding domains and scaffolded
onto the DNA program. The principle of the DNA scaffold has some advantages
in comparison with protein scaffolding (Figure 12.1c, Table 12.1). A DNA pro­
gram sequence requires no maturation, and an ordered nucleotide binding motif
can be selected at will, which provides huge orthogonality. The docking of the

Table 12.1 Advantages and disadvantages between DNA, protein, and RNA scaffolds.

Scaffold DNA Protein RNA

Spatial

orientation

Linear Bundled Linear

Order Highly predictable Unpredictable Predictable, however

less than for the DNA

scaffold

Localization in

eukaryotes

Nuclei No limitation Cytosol

Scaffold–

enzyme ratio

Difficult to achieve

substantial amount of

scaffold, ratio in favor of

enzymes

Easy to achieve

favorable ratio with

gene expression

regulation

Easy to achieve

favorable ratio with

gene expression

regulation

Scaffold–

enzyme

interactions

Similar, well‐characterized,

predictable interactions

Variations in

strength, limited

number of well‐

characterized

interactions

Limited number of

well‐characterized

RNA binding domains

Variability,

number of

available

elements

Large number of zinc

fingers and other DNA‐

binding domains is readily

available, engineered zinc

finger domains

Limited number of

protein dimerization

domains

Limited number of

well‐characterized

RNA binding domains

Interference

with cellular

metabolism

May bind to chromatin;

selecting sequences that do

not affect growth

Signal transduction

domains usually do

not interfere in

bacteria

May bind to

endogenous RNA

molecules; selecting

sequences that do not

affect growth