Joining Natural and Synthetic DNA Using Biversal Nucleotides: Efficient Sequencing of Six-Nucleotide DNA

By rearranging hydrogen bond donor and acceptor groups within a standard Watson-Crick geometry, DNA can add eight independently replicable nucleotides forming four additional not found in standard Terran DNA. For many applications, the orthogonal pairing of standard and nonstandard pairs offers a key advantage. However, other applications require standard and nonstandard nucleotides to communicate with each other. This is especially true when seeking to recruit high-throughput instruments (e.g., Illumina), designed to sequence standard 4-nucleotide DNA, to sequence DNA that includes added nucleotides. For this purpose, PCR workflows are needed to replace nonstandard nucleotides in (for example) a 6-letter DNA sequence by defined mixtures of standard nucleotides built from 4 nucleotides. High-throughput sequencing can then report the sequences of those mixtures to bioinformatic alignment tools, which infer the original 6-nucleotide sequence by analysis of the mixtures. Unfortunately, the intrinsic orthogonality of standard and nonstandard nucleotides often demand polymerases that violate pairing biophysics to do this replacement, leading to inefficiencies in this "transliteration" process. Thus, laboratory <i>in vitro</i> evolution (LIVE) using "anthropogenic evolvable genetic information systems" (AEGIS), an important "consumer" of new sequencing tools, has been slow to be democratized; robust sequencing is needed to identify the AegisBodies and AegisZymes that AEGIS-LIVE delivers. This work introduces a new way to connect synthetic and standard molecular biology: <b>biversal nucleotides</b>. In an example presented here, a pyrimidine analogue (pyridine-2-one, <b>y</b>) pairs with Watson-Crick geometry to both a nonstandard base (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 of 6-amino-5-nitro-[1<i>H</i>]-pyridin-2-one, <b>Z</b>) and a base that completes the Watson-Crick hydrogen bond pattern (2-amino-2'-deoxyadenosine, <b>amA</b>). PCR amplification of GACT<b>ZP</b> DNA with d<b>y</b>TP delivers products where <b>Z</b>:<b>P</b> pairs are cleanly transliterated to A:T pairs. In parallel, PCR of the same GACT<b>ZP</b> sample at higher pH delivers products where <b>Z</b>:<b>P</b> pairs are cleanly transliterated to C:G pairs. By allowing robust sequencing of 6-letter GACT<b>ZP</b> DNA, this workflow will help democratize AEGIS-LIVE. Further, other implementations of the biversal concept can enable communication across and between standard DNA and synthetic DNA more generally.