The expanded genetic alphabet

Abstract

All biological information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.

Keywords: DNA; base pairs; hydrophobicity; semi-synthetic organism; unnatural nucleotides.

Figure 1

Natural dG-dC base pair and dZ-dP, dDs-dPx (R = H or -CH(OH)-CH₂OH), and dNaM-d5SICS UBPs. Sugar and phosphate are omitted for clarity.

Figure 2

disoC-disoG and dX-dκ UBPs. Sugars and phosphates are omitted for clarity.

Figure 3

dx-dy and ds-dz UBPs. Sugars and phosphates are omitted for clarity.

Figure 4

dMMO2-dSICS UBP and d5SICS and dTPT3 unnatural nucleotides.

Figure 5

dNaM–dTPT3-like family of UBPs

Figure 6

dQ-dF UBP and dDs, dPa, and dPn unnatural nucleotides.

Figure 7

Structures of UBPs.

Figure 8

KlenTaq polymerase induces the dNaM-d5SICS UBP to adopt a natural, Watson-Crick-like structure. Structure of the polymerase-template binary complex (a), polymerase-template-d5SICSTP ternary complex (b), a superposition of binary and ternary complexes (c), and a superposition of the ternary complex where d5SICSTP is paired with dNaM and the ternary complex where dGTP is paired with dC (d). Reproduced from Betz, et al.^[156]

Figure 9

Proposed mechanism of replication of d5SICS-dNaM UBP. Intermediates based on solved structures are shown in darker color and those not yet validated by structural studies are shown in lighter color. The steps corresponding to incorporation of the unnatural triphosphate and subsequent extension of the nascent UBP are indicated. Phosphates are indicated with open circles, natural nucleotides are indicated with open rectangles, and the unnatural nucleotides are indicated with grey and black boxes. Reproduced from Betz, et al.^[167]

Figure 10

Recently developed semi-synthetic organism. Porins in the outer membrane and PtNTT2 in the inner membrane are illustrated in blue and green, respectively.