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  • The ribosomal synthesis of D

    2020-10-10

    The ribosomal synthesis of D-proteins is not currently feasible; the best effort undertaken so far was limited to the translational cno stock incorporation of two D-amino acids into the nascent protein chain by means of modified ribosomes (Dedkova et al., 2006). We therefore apply solid-phase peptide synthesis (SPPS) in concert with native chemical ligation for the synthesis of D-proteins. Conceptually developed in the 1960s, SPPS routinely allows the synthesis of peptides of about 30–50 cno stock in length, depending on the actual sequence (Kent, 2009). Native chemical ligation (Dawson et al., 1994) permits the synthetic merger of unprotected peptide fragments in aqueous solution, enabling the synthesis of entire protein domains (Dawson and Kent, 2000, Bondalapati et al., 2016, Kulkarni et al., 2018) and has been used to synthesize proteins of up to some 300 amino acids in length (Wang et al., 2016, Xu et al., 2017). Here, we report the synthesis of a DNA-ligase activity in D-conformation for the production of long stretches of L-DNA from synthetic oligonucleotides. It represents another important piece to the molecular puzzle needed for the establishment of an orthogonal self-replicating system. We did not aim at the production of an enantiomeric molecule for comparative studies of the structures or biochemistry of the D- and L-form, the utility of which has been discussed (Siegel, 1992), but went for the activity mainly.
    Results
    Discussion Synthesis of a DNA-ligase in D-conformation adds another component to the portfolio of proteins required for the assembly of an orthogonal self-replicating system. Currently, chemical synthesis is the sole means available for producing such molecules. While chemical synthesis is rather complex in comparison with the production of recombinant enzyme, it could nevertheless be sufficient for many applications. Relatively large amounts of protein could be produced that could last for multiple reactions in molecular biology systems. Also, once produced, the enzyme is expected to be covalently robust, since it is not susceptible to enzymatic degradation. Any unfolding that occurs over time could be reversed by the process used for protein folding in the first place. However, the chemical synthesis of the large proteins required for ribosomal activity, for example, could be a challenge; enzymes such as aminoacyl-tRNA synthetases are more than 1,000 residues in size. Still, chemically, the task is overall more the number of proteins needed rather than their actual length. Another problem is appropriate protein folding to yield functional proteins. However, it may not be that much of an obstacle for many proteins. In vitro synthesis of very large numbers of proteins yielded a surprisingly high percentage of molecules that were functional or recognized their partners specifically in interaction studies (e.g., Syafrizayanti et al., 2017). Still, assembling a structure such as a ribosome and keeping it in its functional state is likely to be a major aspect of getting a self-replicating molecular system going. In vitro ribosomal assembly and function has been studied (Jewett et al., 2013, Sashital et al., 2014, Earnest et al., 2015), optimizing the yields to be comparable with components purified in vivo. Ultimately, it may be possible to produce mirror proteins through the synthesis of a fully functional mirror ribosome. In principle, an alternative for production could be non-ribosomal peptide synthetase enzymology, which permits incorporation of D-amino acids (Hur et al., 2012). However, this approach is currently still lagging well behind chemical synthesis in terms of yield and length of the products.
    Significance Systems and synthetic biology have become key research areas. What is missing is an experimental system in which the knowledge gained from molecular analyses could be reproduced in an artificial setting that is void of the risk of natural contamination. We work at setting up a self-replicating molecular system that forms the basis toward the establishment of an artificial biology that is independent from Nature but identical in terms of biophysical and biochemical parameters. Various enzymes are required to achieve such an end. The DNA-ligase described here represents an important piece in the puzzle of enzymatic processes needed for such an artificial biological system that—in the very long run—may lead to an archetypical model of a cell.