250 12 Metabolic Channeling Using DNA as a Scaffold
DNA‐binding sequences in the host genome [29]. The binding affinity between
different TAL DNA‐binding domains is similar, and in the nanomolar range, as
for zinc finger proteins. Due to the practically unlimited number of different
combinations, there is no concern with running out of DNA‐binding sites,
regardless of the number of desired scaffolded enzymes.12.3.3 Other DNA‐Binding Proteins
In theory, practically any DNA‐binding protein could be used in DNA scaffold
applications. With the exception of zinc fingers and TALs, many of the charac
terized DNA‐binding proteins bind DNA as dimers or tetramers (TetR, CI, and
others), which would complicate the construction of DNA scaffold molecules if
one desires to bind enzymes in a predefined molar ratio. Nevertheless, they may
be useful for applications involving oligomeric enzymes.12.4 DNA Program
The DNA scaffold is, in principle, more flexible in scaffold designs than protein
or ssRNA scaffolds. Since dsDNA forms a helical turn approximately every 10 nt,
we can use this property to guide the relative orientation of the enzymes coupled
to the DNA‐binding domains. We can change (i) the spatial orientation of the
binding enzymes by changing the spacer length between the DNA‐target sites;
(ii) the number of DNA scaffold repeats, allowing us to additionally tune the
biosynthetic pathway; and last but not least (iii) the DNA scaffold, which enables
us to modify the enzymatic stoichiometry.12.4.1 Spacers between DNA‐Target Sites
The program DNA is designed to organize biosynthetic pathway enzymes into a
functional complex. Spacers in the DNA sequence separating the DNA‐target
sites on the program DNA determine the spatial orientation of chimeric bio
synthetic enzymes relative to each other (Figure 12.6). The binding sites for
three‐fingered zinc fingers span nine nucleotides but can be extended to 18‐bp
recognition motifs for longer zinc fingers, spanning from one to two DNA duplex
helical turns, respectively. The binding sites for DNA‐binding proteins are sepa
rated by spacers, which are nucleotides that are not occupied by DNA‐binding
proteins. The length of the spacer sequence is not coincidental, and the selection
follows the three‐dimensional structure of a DNA molecule. One turn of the
DNA helix is 10.5 bp long, which roughly overlaps with the length of a DNA mol
ecule encircled by one zinc finger domain recognizing and binding to 9 bp. In
order to have functional units on the same side of a DNA molecule serving as a
DNA program, it is of high importance to select the right spacer length. The
double helix of the DNA defines on which side of the helix the functional domain
will be attached, which is defined by the length of the spacer between the DNA‐
target sites: a spacer of one or two nucleotides positions them very close, while a