Hachimoji DNA and RNA: A genetic system with eight building blocks

Abstract

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.

Figure 1.

The eight nucleotides of hachimoji DNA (left) and hachimoji RNA (right) are designed to form four size- and hydrogen bond-complementary pairs. Hydrogen bond donor atoms involved in pairing are blue; hydrogen bond acceptor atoms are red. The left two pairs in each set are formed from the four standard nucleotides (note missing hydrogen bonding group in the A:T pair, a peculiarity of standard terran DNA/RNA). The right two pairs are formed from the four new non-standard nucleotides. Notice the absence of electron density in the minor groove of S, which has a NH (green) moiety.

Figure 2.

(left) Plot of experimental vs. predicted free energy changes (ΔG°₃₇) for 94 SBZP- containing hachimoji DNA duplexes (Tables S3, S6, and S9). (right) Plot of experimental vs. predicted melting temperatures of 94 SBZP-containing hachimoji DNA duplexes (Tables S3, S6, and S9). The outlier is a sequence embedded in the PP guest (Fig. 3E).

Figure 3.

Crystal structures of PB, PC, and PP hachimoji DNA. (A) The host-guest complex with two N-terminal fragments from Moloney murine leukemia virus reverse transcriptase in green and cyan bound to a 16mer PP hachimoji DNA; in the duplex sphere model, Z:P pairs are green, S:B pairs magenta. The asymmetric unit includes one protein molecule and half of the 16mer DNA as indicated by the line. (B) Hachimoji DNA structures PB (green), PC (red), PP (blue) are superimposed with GC DNA (gray). (C) Structure of hachimoji DNA with self- complementary duplex CTTATPBTASZATAAG (“PB”). (D) Structure of hachimoji DNA with self-complementary duplex 5’-CTTAPCBTASGZTAAG, (“PC”). (E) Structure of hachimoji DNA with self-complementary duplex with six consecutive non-standard components 5’-CTTATPPSBZZATAAG (PP). DNA structures are shown as stick models with P:Z pairs (C, green), B:S pairs (C, magenta), and natural pairs (C, gray). Examples of largest differences in detailed structures: (F) The Z:P pair (from the PB structure) is more buckled than corresponding G:C pair in (G). (H) The S:B pair (from the PB structure) exhibits a propeller angle similar to that in the corresponding G:C pair shown in (I).

Figure 4.

(Left) Schematic showing the full hachimoji spinach variant aptamer; additional nucleotide components of the hachimoji system are shown as black letters at positions 8, 10,76, and 78 (B, Z, P, and S respectively). The fluor binds in loop L12 (25). (Right) Fluorescence of various species in equal amounts as determined by UV. Fluorescence was visualized under a blue light (470 nm) with an amber (580 nm) filter. From left to right: (a) Control with fluor only, lacking RNA, (b) hachimoji spinach with the sequence shown in the left panel (c) native spinach aptamer with fluor, and (d) fluor and spinach aptamer containing Z at position 50, replacing A:U pair at positions 53:29 with G:C to restore the triple observed in the crystal structure. This places the quenching Z chromophore near the fluor; CD spectra suggest that this variant had the same fold as native spinach (Fig. S8).