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as has already been exposed in the preceding sections. Another motivation that should not
be left out here is the topic of finding alternative base pairing schemes which are orthogo-
nal to the natural Watson-Crick base pairing and therefore might be used to expand the
natural genetic code [27]. The emerging field of synthetic biology might benefit from such
a development in future.
9.2.1 Modifications of the Hydrogen Bonding Pattern
The first approaches aiming at expanding the genetic code by using artificial base pairs
were based on modifications of the hydrogen bonding patterns between the nucleobases
of strand and counterstrand. Therefore, numerous heterocyclic nucleobase-like com-
pounds were synthesized having hydrogen bond donor and acceptor sites similar to the
natural nucleobases but arranged in other positions. Pairs of these artificial nucleobases
were then tested for their propensity to undergo heterodimerization and for their orthogo-
nality to the natural nucleobases, both in the form of their monomers and in an oligo-
nucleotide context. From these works, important knowledge was derived about the role of
hydrogen bonding for the sequence-specific hybridization of single strands [28].
9.2.2 Shape Complementarity
Later studies concentrated on the role of the nucleobases' shape and their ability to inter-
act by p-stacking along the double helical structure. By using flat, aromatic nucleobase
analogs devoid of any hydrogen donor and acceptor sites (called “hydrophobic bases”) it
was found that the role of hydrogen bonding should not be overestimated, since already
the shape complementarity of the pairing bases in strand and counterstrand allowed spe-
cific base pairing in short duplexes. Furthermore, using DNA replicating and transcribing
enzymes (polymerases), it was found that the shape of the bases seems to play a very
important role in the base recognition in enzyme-catalyzed processes of templated oligo-
nucleotide polymerization [29].
9.2.3 Metal Coordination
Early experiments to introduce metal ions into the center of the double helix made use of
unmodified DNA strands at high pH (thereby facilitating the deprotonation of the nucleo-
bases) and metal ions such as Zn(II), Co(II) and Ni(II) [30]. The structure and conductive
properties of this so-called M-DNAwere, however, controversially discussed and, despite
some structural proposals, the exact positions of the metal ions inside or around the DNA
strands remain unclear [20]. In contrast, it was unambiguously shown that Hg(II) ions
coordinate strongly between two thymine bases oppositely arranged in a DNA double
strand [31]. Whereas such T-T mismatches lead to a significant destabilization of the
duplex structure in the absence of Hg(II), the addition of one equivalent of Hg(II) leads to
the formation of a duplex stabilizing T-Hg(II)-T base pair in which the imide protons are
removed. Likewise it was found that two oppositely arranged cytosine bases can bind Ag
(I) to form the C-Ag(I)-C base pair (Figure 9.4) [32]. Since these two coordination events
were found to proceed largely in an orthogonal manner, systems containing both C-C mis-
matches and T-T mismatches can be individually addressed by Ag(I) or Hg(II), respec-
tively. This principle has been used to generate DNA-based logical switches that react on
the input of Ag(I) and/or Hg(II) (see Section 9.6.2).
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