Unnatural Base Pairs
The idea to expand the DNA by the incorporation of an unnatural base pair (UBP) was already born in 1962. Since then, much effort has been done to engineer UBPs that function as an orthogonal system to create a semi-synthetic DNA (xenogenic DNA or XNA). Besides hydrogen bonding, researchers also investigated UBPs with different chemical properties. Usage of an UBP creates several challenges like the adaption of the whole transcriptional and translational machinery. When dealing with a semisynthetic organism, additional tasks arise, e.g. the biosynthesis of the "base" as well as the synthesis of the corresponding nucleosides and nucleotides. The de novo synthesis as well as the salvage pathway of nucleotides is a very complex metabolism, which includes a lot of different enzymes. To obtain a fully autonomous semi-synthetic organism, the easiest path is the incorporation of UBPs that are similar to canonical nucleotides using hydrogen bonding. This brings up isoguanosine (isoG) and isocytosine (isoC) with conceivable biosynthesis pathways.
Background to the Unnatural Base Pairs (UBPs)
Figure 1: Unnatural bases.
All amino acids are encoded by codons, which are defined by three base pairs. This information is encoded in the genome of an organism and since the origin of life every natural genome has consisted of the two-base-pair genetic alphabet dA-dT (adenine-thymine) and dG-dC (cytosine-guanine). There are strong efforts to replace a canonical base pair or expand the genetic code by a third unnatural base pair (UBP) (Martinot and Benner, 2004; Jiang and Seela, 2010; Kwok, 2012; Zhang et al., 2017; Yamashige et al., 2012; Seela et al., 2005; Switzer et al., 1989; Yang et al., 2011).
So far, the modification of sugars and phosphates for nucleotides with important applications have been explored. First experiments with unnatural bases extended the nucleotide alphabet by replacing thymine with 5-chlorouracil in E. coli over a period of 25 weeks (Dunn and Smith, 1957; Marlière et al., 2011). But for an UBP, two modified nucleobases are needed. A. Rich discussed the extension of the DNA by two additional bases already in 1962 (Rich, 1962). An additional UBP can be interesting for physiochemical properties if the nucleobases can be site-specifically derivatized with linkers for chemical groups. Furthermore, the availability of an UBP in vivo would be a milestone in the field of synthetic biology. This would mean the creation of a semi-synthetic organism with altered storage capabilities for genetic information that leads to new and useful functions and applications (Malyshev and Romesberg, 2015).
UBPs with hydrogen bonding
Another aspect is the similarity of the unnatural bases isoG und isoCm to the natural bases guanine and cytosine while being an orthogonal system at the same time. Due to the structural similarity, there is better chance for compatibility with interacting enzymes. In 1992 the Benner lab showed that the in vitro translation of mRNA containing disoC worked with a non-standard tRNA containing the purine complementary disoG inside the anticodon (Bain et al., 1992). Their cell free experiments showed a high specifity for the incorporation of a non-canonical amino acid by the ribosome using this unnatural base. With these stereoisomer of the natural bases it is more likely to achieve an optimized replication, transcription or translation with less adaption of the correspondent enzymes than with hydrophobic UBPs. On top of that, the hydrophobic UBPs are very expensive, because of their complex synthesis. Looking forward to create an autonomous synthetic organism it seems to be impossible to create a biosynthetic pathway for unnatural bases that differ a lot from natural bases. Whereas isoG is already known to be metabolic substance of the plant Croton tiglium. Revealing this metabolic pathway can make it usable for any synthetic organism and therefore stepping forward towards a fully autonomous synthetic organism.
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