We report DNA- and RNA-like systems built from eight nucleotide “letters” (hence the name “hachimoji”) that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson-Crick geometry. These were added to lies in a ibrarlaboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Z’s and P’s. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system, and that GACTZP libraries are richer reservoir of functionality than standard libraries.

The extent to which synthetic biology can be used to expand genetic information systems compatible with natural enzymes and cells will depend on the extent to which multiple and contiguous non-natural nucleobase pairs fit within the standard double helical conformations of DNA. Toward this goal, two non-standard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) were designed to form a Z:P pair with a standard “edge on” Watson-Crick geometry, but with rearranged hydrogen bond donor and acceptor groups. Here, we present the crystal structures of two self-complementary 16-mer oligonucleotides containing Z:P pairs. The first contained two consecutive Z:P nucleobase pairs and was found to crystallize within a host-guest complex in B-form. The second contained six consecutive Z:P pairs; it was found to crystallize as an A-form DNA duplex, although it can adopt B-form in solution as inferred from circular dichroism spectra. Although Z:P pairs have some structural properties that are similar to those of G:C pairs, unique features include stacking of the nitro group on Z with the adjacent heterocyclic nucleobase ring in A-DNA. In both B-and A-DNA, major groove widths associated with the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs potentially due to the presence of the nitro group in Z. Thus, our structural studies suggest that multiple and consecutive Z:P pairs are readily accommodated in DNA duplex structures recognized by natural polymerases, and therefore the GACTZP synthetic genetic system has the requisite properties to expand sequence space.