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Abstract Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show thatE. coliRNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition. 

For half a century, the possibility of organic molecules in sulfuric acid droplets in the clouds above Venus has been largely discounted. Here, we report the first results from an experimental exploration of this possibility, of primary interest to astronomers but also uncovering reactions that are remarkable to organic chemistry. This work provides a detailed mechanism of how small organic molecules might be generated in the sulfuric acid (H2SO4) aerosol droplets that form the clouds above Venus, starting from formaldehyde (HCHO), a simple one carbon species produced photochemically in the gas phase. Laboratory 13C and 1H nuclear magnetic resonance studies detail processes by which dissolved HCHO reacts with dissolved carbon monoxide (CO) to produce a two-carbon organic species, glycolic acid (HOCH2COOH). They show that glycolic acid is surprisingly stable, for days or longer, depending on temperature, in concentrated H2SO4. However, glycolic acid slowly reacts further to give higher molecular weight organic materials, including colored and fluorescent species. These may contribute to the UV and visible light astronomy of Venus, and are guiding the design of an autofluorescence nephelometer scheduled to fly on a Rocket Lab mission to Venus in 2025. 

: One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson−Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that “polyphosphate kinases” can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α32P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.