Joining\nNatural and Synthetic DNA Using Biversal Nucleotides:\nEfficient Sequencing of Six-Nucleotide DNA

By\nrearranging hydrogen bond donor and acceptor groups within a\nstandard Watson–Crick geometry, DNA can add eight independently\nreplicable nucleotides forming four additional not found in standard\nTerran DNA. For many applications, the orthogonal pairing of standard\nand nonstandard pairs offers a key advantage. However, other applications\nrequire standard and nonstandard nucleotides to communicate with each\nother. This is especially true when seeking to recruit high-throughput\ninstruments (e.g., Illumina), designed to sequence standard 4-nucleotide\nDNA, to sequence DNA that includes added nucleotides. For this purpose,\nPCR workflows are needed to replace nonstandard nucleotides in (for\nexample) a 6-letter DNA sequence by defined mixtures of standard nucleotides\nbuilt from 4 nucleotides. High-throughput sequencing can then report\nthe sequences of those mixtures to bioinformatic alignment tools,\nwhich infer the original 6-nucleotide sequence by analysis of the\nmixtures. Unfortunately, the intrinsic orthogonality of standard and\nnonstandard nucleotides often demand polymerases that violate pairing\nbiophysics to do this replacement, leading to inefficiencies in this\n“transliteration” process. Thus, laboratory <i>in vitro</i> evolution (LIVE) using “anthropogenic evolvable\ngenetic information systems” (AEGIS), an important “consumer”\nof new sequencing tools, has been slow to be democratized; robust\nsequencing is needed to identify the AegisBodies and AegisZymes that\nAEGIS-LIVE delivers. This work introduces a new way to connect synthetic\nand standard molecular biology: <b>biversal nucleotides</b>.\nIn an example presented here, a pyrimidine analogue (pyridine-2-one, <b>y</b>) pairs with Watson–Crick geometry to both a nonstandard\nbase (2-amino-8-imidazo-[1,2<i>a</i>]-1,3,5-triazin-[8<i>H</i>]-4-one, <b>P</b>, the Watson–Crick partner\nof 6-amino-5-nitro-[1<i>H</i>]-pyridin-2-one, <b>Z</b>) and a base that completes the Watson–Crick hydrogen bond\npattern (2-amino-2′-deoxyadenosine, <b>amA</b>). PCR\namplification of GACT<b>ZP</b> DNA with d<b>y</b>TP delivers\nproducts where <b>Z</b>:<b>P</b> pairs are cleanly transliterated\nto A:T pairs. In parallel, PCR of the same GACT<b>ZP</b> sample\nat higher pH delivers products where <b>Z</b>:<b>P</b> pairs are cleanly transliterated to C:G pairs. By allowing robust\nsequencing of 6-letter GACT<b>ZP</b> DNA, this workflow will\nhelp democratize AEGIS-LIVE. Further, other implementations of the\nbiversal concept can enable communication across and between standard\nDNA and synthetic DNA more generally.