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  • Introduction In vitro synthetic biology has received conside

    2020-03-24

    Introduction In vitro synthetic biology has received considerably less attention than in vivo processes so far (Foster and Church, 2007). However, cell-free biosynthetic production is very likely to become important for many biotechnological challenges for reasons such as (energy) efficiency, system simplicity, and easier quality control. The aspect of establishing fully synthetic biosystems has only more recently come into focus. A basic motivation behind such projects is the ambition to understand biological processes to an extent that will permit their re-creation. This ability would be documented best, if functioning molecular systems could be established that are entirely independent of any natural compounds and their assembly into artificial molecular systems, thus evaluating biological models experimentally. Re-creation of molecular biosystems by means of enantiomeric molecules offers an obvious route toward this end. However, the availability of complex biomolecules in their unnatural enantiomeric conformation is still limiting, although chemical synthesis processes have significantly improved in recent years. For a while, relatively little work had been done on enantiomeric nucleic acids or proteins. The L-form of DNA has been described as a tool for few biotechnological processes (Hauser et al., 2006, Lin et al., 2009, Ke et al., 2012). Also, an 83-nucleotide (nt) RNA polymerase ribozyme made of D-RNA could be selected that catalyzes the ligation of L-RNA oligonucleotides on an L-RNA template (Sczepanski and Joyce, 2014). At the level of proteins, pde inhibitor in D-configuration were incorporated during chemical syntheses of proteins for purposes such as their stabilization toward enzymatic degradation (Mitchell and Smith, 2003). The group of Stephen Kent chemically synthesized the full enantiomer of the HIV-1 protease (99 amino acids) and demonstrated that the D- and L-molecules were mirror-images of one another in all elements of three-dimensional structure and displayed reciprocal chiral specificity in their biochemical interactions (Milton et al., 1992). Using a small DNA-binding peptide, based on the zinc finger domain of the GAGA transcription factor, and a synthetic DNA-oligonucleotide, Sugiura and colleagues could demonstrate that the conformations and recognition of the reciprocal molecule pairs were exact mirror-images of each other in structure and biological activity (Negi et al., 2006). For few D-peptides, it was shown that immunogenicity was much reduced. By administering a mixture of D- and L-peptides into mouse T cells, only an L-peptide-specific immunoglobulin G antibody response was observed (Chong et al., 1996). Similarly, D-proteins have been shown to be markedly less immunogenic in mice than their L-counterparts (Dintzis et al., 1993, Uppalapati et al., 2016). In an approach termed mirror-image phage display, selected D-peptide ligands were isolated on the basis of analyzing an L-peptide phage display library against a small D-form target peptide; the selected L-peptide was then synthesized as its mirror-image D-ligand peptide that exhibited the same specificity to the natural L-target (Schumacher et al., 1996). Finally, the interplay between D- and L-proteins to access a wider array of crystal packing space groups has been exploited through racemic protein crystallography (Yeates and Kent, 2012). In recent years, advances in the field of D-protein synthesis accelerated. The chaperone GroEL/ES was found to catalyze similarly the folding of both the D- and the natural L-form of the E. coli protein DapA (Weinstock et al., 2014). The mirror-image D-version of the ribonuclease barnase was used for screening an aptamer library of natural RNA molecules (Olea et al., 2015). The identified aptamers were prepared as L-RNA copies and in turn acted as inhibitors of the barnase in its natural protein conformation. A crucial step toward the establishment of a self-replicating in vitro system was the synthesis of a chirally mirrored version of the 174-residue African swine fever virus polymerase X, which acts as both an RNA and a DNA polymerase (Wang et al., 2016). This was complemented by a D-version of a heat-stable mutant of Sulfolobus solfataricus DNA polymerase IV (Pech et al., 2017), by which PCR could be performed. This development could have consequences in practical biotechnology applications, such as the selection of aptamers from libraries made of synthetic L-DNA molecules.