Synthetic biology

Six pack and stack

A pair of artificial DNA bases have now been shown to adopt an edge-to-edge geometry in DNA which is similar that found in Watson–Crick base pairing. Aptamers containing these bases have also been shown to bind more strongly to a target than those developed using only the four naturally occurring bases.

The Watson–Crick base pairing of naturally occurring nucleotides has long been a subject of fascination amongst chemists, in that it represents one of only a few examples of extensive isosteric and isomorphic supramolecular chemistries1. It is not clear, however, whether any reliable genetic system would require these extensive isosteric and isomorphic properties as there have been few opportunities to examine whether the uniqueness of Watson–Crick base pairing is a result of chemical or biological rarity: that is, whether there are few such systems within the space of chemical compounds, or many such systems, all of which could have been equally successful if biology had happened to chance upon them. Two recent papers2,3 published in the Journal of the American Chemical Society, now provide insights into whether other genetic alphabets may be equally pliable.

Steven A. Benner and co-workers have previously developed orthogonal, unnatural base pairs4, including 6-amino-5-nitro-2(1H)-pyridone (Z) and 2-aminoimidazo[1,2-a]-1,3,5-triazin-4(8H)-one (P) (Fig. 1a) — which combine with the four naturally occurring DNA bases to make a six-letter DNA system. DNA containing these Z and P unnatural bases can be amplified by polymerase chain reaction with high fidelity (99.8% per cycle)4. Millie M. Georgiadis and colleagues have now shown2 that this is in part because the Z and P nucleotides form a relatively 'natural' stack, that is similar to that adopted by Watson–Crick pairs. This similarity is such that multiple unnatural base pairs can be incorporated adjacent to one another without disrupting the basic structure of DNA. This similarity also enables ZP-containing DNA to adopt canonical B and A helical forms. Since the NO2 group in the Z nucleobase can stack with the heterocyclic ring of an adjacent nucleobase, multiple adjacent Z–P nucleobase pairs seem to have a higher propensity to form A-DNA, which may enable facile B-to-A structural transitions that are sometimes required for protein interactions5.

Figure 1: Comparison of the structures of the expanded genetic alphabets.

ac, Molecular structures (top) and space-filling models (bottom) of Z–P (a), Ds–Px (b) and 5SICS–NaM (c) base pairs. d, Movement over a putative fitness landscape, showing pathways for the natural genetic alphabet and expanded genetic alphabets. The addition of unnatural bases could potentially enable greater optimization of catalysts through the addition of new hydrophobic or hydrophilic interactions. However, the increase in the number of possible mutations could also lead to a change in the folded structure thereby hindering the evolution towards a better catalyst. Panels ac are reproduced from ref. 3, ACS.

Most importantly, though, the Z-nitro group imparts new properties to the major groove of DNA that can potentially be exploited for recognition by proteins. Furthermore, the sequence space available to the six-letter genetic alphabet is considerably larger than of the four-letter system. To create useful DNA molecules that take advantage of these properties a team led by Zhen Huang, Weihong Tan and Steven A. Benner adapted the six-letter genetic alphabet for in vitro evolution experiments3. Aptamers that could bind HepG2 liver cancer cells were selected, and the affinities of binders with Z and/or P were stronger than those of binders lacking both Z and P. The selected aptamers tightly bound to the target cells with dissociation constants in the range of 10–100 nM, and were selective relative to fifteen other types of tissue culture cells.

These two papers add to a growing wealth of compounds that have been used to expand genetic alphabets. Ichiro Hirao and co-workers have developed highly specific hydrophobic base pairs, by combining hydrophobicity and shape-complementarity (Fig. 1b)6. Aptamers containing 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) bases were generated against two human proteins, VEGF-165 and interferon-γ, and these were shown to have 100 times the affinity of aptamers formed from only natural bases that had been separately generated via conventional in vitro selection experiments7. The remarkable improvement in affinity was attributed to the hydrophobic properties of the Ds base, since proteins frequently have hydrophobic patches on their surfaces.

A further team, led by Floyd E. Romesberg, has also developed a base pair that interacts through hydrophobic interactions (5SICS–NaM; Fig. 1c). Structural studies in duplex DNA demonstrated that this unnatural base pair forms via cross-strand intercalation8,9. Although the intercalation improves the packing interactions between the unnatural hydrophobic bases, this shifting stack can induce significant distortion in double stranded DNA and potentially prevent the incorporation of multiple adjacent unnatural bases. In contrast, the base pairing reported by Georgiadis et al.2 forms an 'edge-on' geometry similar to that seen in natural base pairing and therefore the Z–P better resembles that observed in a 'natural' helix. DNA incorporating Romesberg's unnatural bases has, however, been shown to be replicated in vivo, creating a semi-synthetic organism with an expanded genetic alphabet10. This bodes well for in vivo incorporation of other non-standard alphabets.

While these results are all promising, the overall consequences of expanded alphabets on biopolymer functionality cannot be fully predicted. The expanded alphabets should be extremely useful for expanding the genetic code for protein production, as they will provide wholly new codon blocks. However, several theoretical papers11,12 have worried that nucleic acids with expanded genetic alphabets might be intrinsically more error-prone during replication, because of the increased propensity for mismatches (Fig. 1d).

In terms of nucleic acid functionality, the teams led by Benner and Hirao have both shown that augmented alphabets may have binding and catalytic payoffs, while Joyce has conversely shown that a more limited genetic alphabet reduces function13. But there may be limits to this, as well. For example, Watson–Crick base pairing amongst the natural nucleotides works very well for the formation of functional nucleic acid structures, from aptamers to ribosomes, in part because it is almost impossible not to make a folded secondary structure, especially given the possibility of wobble pairings. The same feature that leads to mutation (mispairing) can potentially enhance folding. However, it is not clear how increasing the number of different base pairs with different degrees of orthogonality will affect the ensemble of available shapes.

One way to think about this is to envision the available paths on a putative fitness landscape (Fig. 1d). When there are only three other mutational possibilities per base (and some of these lead to wobble or Hoogsteen pairings) then mutational paths that can connect structures via neutral networks can be readily envisioned14,15. Will this still be true with six-letter alphabets? Eight letters? At what point do mutational paths separate structures by such a distance that any individual change in base pairing is likely to alter the tertiary structure and thereby lead to a diminution of fitness, rather than allow it to move to higher fitness via a neutral network? To reliably answer these issues will require real experimental data. Future data from all of the novel genetic alphabets should provide a sounder basis for understanding how chemistry gives rise to genetics and eventually phenotype.


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Correspondence to Andrew D. Ellington.

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Jung, C., Ellington, A. Six pack and stack. Nature Chem 7, 617–619 (2015).

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