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Abstract Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ⁻:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ⁻:dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ⁻:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC⁺-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis. 

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. 

Abstract Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas. 

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. 

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host–guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5′-CTTBPPBBSSZZSAAG, 5′-CTTSSPBZPSZBBAAG and 5′-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, andB:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters ofP:ZandB:Spairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further,Zcan participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.