Since at least the last common ancestor of all life on earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. The central goal of synthetic biology is to create new life forms and functions1, and the most general route to this goal is the creation of semi-synthetic organisms (SSOs) whose DNA harbors two additional letters that form a third, unnatural base pair (UBP). Previously, our efforts to generate such SSOs culminated in the creation of a strain of Escherichia coli that by virtue of a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2), imports the requisite unnatural triphosphates from the media and then uses them to replicate a plasmid containing the UBP dNaM-dTPT3 (Fig. 1a)2. While the SSO stores increased information, retrieval of the information requires in vivo transcription of the UBP into mRNA and tRNA, aminoacylation of the tRNA with a non-canonical amino acid (ncAA), and finally, efficient participation of the UBP in decoding at the ribosome. Here, we report the in vivo transcription of DNA containing dNaM and dTPT3 into mRNAs with two different unnatural codons and tRNAs with cognate unnatural anticodons, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or ncAAs into superfolder green fluorescent protein (sfGFP). The results demonstrate that interactions other than hydrogen bonding can contribute to every step of information storage and retrieval. The resulting SSO both encodes and retrieves increased information and should serve as a platform for the creation of new life forms and functions.
Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 pair to form an unnatural base pair (UBP) and allow for the creation of semi-synthetic organisms (SSOs) with additional sense codons. Here we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded non-canonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to site-specifically incorporate multiple, different ncAAs into a protein should now allow for the development of proteins with novel activities and possibly even SSOs with new forms and functions.
How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation: most mutations were located in just a few hotspots in the genome, and over time, mutations increasingly were consistent with the involvement of 8-oxo-guanosine, formed exclusively on the transcribed strand. This work provides strong support for retromutagenesis as a general process creating adaptive mutations during ageing.
We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding, but by hydrophobic and packing forces. These UBPs enabled the creation of the first semi-synthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing non-canonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than dNaM-dTPT3 in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and especially dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA containing TPT3 and that their improved retention in DNA results in more efficient production of proteins with non-canonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biology “parts” in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen-bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and especially dPTMO-dTPT3 represents significant progress towards the development of SSOs with an unrestricted ability to store and retrieve increased information.
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