Semi-synthetic organism with an expanded genetic code

[Ygggdrasil]

All life on Earth stores its genetic information in DNA using just four nucleotide letters: A, T, C, and G. Research published this week in the journal Nature describes how scientists engineered a bacterium to incorporate two new letters into their DNA (which they call X and Y, pictured below), and read those letters to introduce new amino acids into proteins.

(the X-Y base pair is shown on the left. A natural A-T base pair is shown for comparision on the right)

The work builds upon previous work by the same group. In 2014, the team reported that they could get bacteria to incorporate an unnatural base pair into DNA and RNA, and earlier this year, they reported improvements to enable the bacteria to more stably maintain the unnatural base pair in their DNA. Of course, while getting an unnatural amino acid into DNA and RNA is in itself an important achievement, the authors were missing the last step of biology. DNA gets transcribed into mRNA which then gets read by the ribosome to produce protein. While the bacteria contained the extra base pair, their ribosomes lacked the capability to read the new nucleotides.

The latest work involves engineering the tRNAs and related machinery so that the ribosome can recognize X in mRNA and incorporate unnatural amino acids into the resulting protein. Thus, these bacteria are now capable of containing extra information in their DNA and translating that information into an expanded genetic code that can encode more than the 20 amino acids typically made by life on Earth. It is worth noting, however, that they are many (much simpler!) approaches to introducing unnatural amino acids into protein. Furthermore, more work would need likely to be done for the bacteria to make more extensive use of the unnatural nucleotides and amino acids. Still, the work presents the first step towards potentially building new life forms in the lab that have an entirely different genetic system than any other life on Earth.

You can read the full paper here: https://www.nature.com/articles/nature24659
Popular press coverage:
https://www.npr.org/sections/health...s-move-a-step-closer-to-making-synthetic-life
https://www.technologyreview.com/s/609567/semi-synthetic-life-form-now-fully-armed-and-operational/

 

[BillTre]

So this should increase the number of possible codons (three bases in a row) from the original number of 64 (4 x 4 x 4 = 64) to 216 (6 x 6 x 6 = 216).
The original 64 codons are redundantly used to code the 22 amino acids ribosomes can incorporate into proteins (using tRNAs etc.).

This should greatly increase the number of possible amino acids that could be incorporated into proteins.

 

[Auto-Didact]

Yeah, I remember reading about that a few years ago, around 2011. If I remember correctly, XNA was what they called this generalized ribose-type nucleic acid genetic theory consisting of non-Watson-Crick basepairs.

In any case, from a theoretical perspective it would be quite interesting to create new non-Watson-Crick type proteins in this way and so also carry out natural selection experiments on these proteins, XNA or ribozymes using dynamic combinatorial libraries. Just imagine what this could teach us about abiogenesis, in both terrestrial and extraterrestrial settings, given the discovery of stable self replicating structures.

Moreover, on a more applied experimental side the link to a CRISPR/Cas9 type of implementation is of course a very natural one, and perhaps a strategy capable of very selectively delivering drugs to targeted cells which have been infiltrated and reprogrammed to make these synthetic proteins. Doubt this would be clinically relevant, in short-term practice or even in principle, but have no qualms being proven wrong.

 

[laymanB]

It seems to me that if there are other bonding mechanisms for holding together base pairs in DNA besides hydrogen bonds, this should make us examine the question of why hydrogen bonding is the one used by life on Earth? Is there something inherently more probable in the hydrogen bond and the natural bases, that we should find it was used in the only tree of life that we know? In other words, if other bonds and bases are possible, which seems to be confirmed by this paper, why don't we find them here on Earth?

 

[rootone]

Other bonding can exist. but seeing heavier elements bonding with hydrogen is far more likely just because there is more hydrogen than anything else.
It's Carbon that makes thing interesting.

 

[mfb]

You need a bond strong enough to keep the DNA strands together and to get selective matching with partners, but weak enough to open the structure to read it without risking a break in the strands.
They introduced the new base pair at a few selected locations as proof of concept, I don't know if replacing a significant fraction of all base pairs with it would lead to issues.

 

[Ygggdrasil]

It has been known for a few decades that DNA base pairs can form in the absence of hydrogen bonding. These conclusions come from the work of Eric Kool at Stanford who synthesized DNA bases lacking the ability to hydrogen bond and characterized the stability of DNA containing these non-hydrogen bonding base pairs. From the work of Kool and others, we now know that hydrogen bonding is not the main energetic force keeping the double helix together; rather, it is the energy from the pi-pi interactions that come about from stacking adjacent nucleotide bases in the double helix:

In general, recent data with DNA alone (in the absence of enzymes) suggests that hydrogen bonds contribute strongly to the selectivity of DNA base pairing in DNA alone. The bonds also appear to contribute to pairing energetics favorably, although with only moderate magnitude. It is possible to design nonhydrogen-bonded pairs that are somewhat selective and that are at least as stable as natural base pairs. From the steric standpoint, it appears that steric effects may affect base pairing preferences somewhat, though the influence may be moderate. Finally, stacking effects are probably the major influence of base pair stability and are the major force holding the double helix together. It is interesting to note that the DNA bases stack weakly to moderately, and scientists have demonstrated many nonnatural bases that stack more strongly than the natural ones.

http://www.annualreviews.org/doi/full/10.1146/annurev.biophys.30.1.1

As for why hydrogen bonds are not a big factor in the thermodynamic stability of the double helix:

To a first approximation, hydrogen bonding between two groups in water is not energetically favorable because roughly equivalent hydrogen bonds to water must be exchanged for one such new bond. Thus, in enthalpic terms, solvation effects will not favor a hydrogen bonded pairing of two nucleobases. The bases G and C must first lose several hydrogen bonds to water in order to form a triply-hydrogen bonded pair. In addition, the bases lose entropy of relative translation and rotation in order to form the complex, a destabilizing effect. However, other entropic effects favor this pairing: The entropy of the freed water molecules is likely to be favorable; moreover, the formation of the second and third H-bond in the base pair comes with little additional translational/rotational entropy penalty. This is also true as multiple pairs are formed between two strands. Thus, the hydrogen bonding in a pair does appear to be energetically favorable in the context of a larger double helix.

http://www.annualreviews.org/doi/full/10.1146/annurev.biophys.30.1.1

Hydrogen bonding does appear to play an important role in the fidelity of base pairing (though this requirement appears to be relaxed in the context of enzymatic DNA replication). Presumably, the contributions of hydrogen bonding to the fidelity of nucleic acid replication was likely important in the primordial RNA world which lacked highly evolved DNA and RNA polymerases.

I would definitely recommend the review quoted above from Eric Kool for those interested in the biophysics of DNA base pairing.

 

[Grinkle]

In any case, from a theoretical perspective it would be quite interesting to create new non-Watson-Crick type proteins in this way and so also carry out natural selection experiments on these proteins, XNA or ribozymes using dynamic combinatorial libraries. Just imagine what this could teach us about abiogenesis, in both terrestrial and extraterrestrial settings, given the discovery of stable self replicating structures.

I am seeing this as an engineering achievement, not something that enables fundamentally new science. Its not a discovery of stable self replicating structures, those have already been discovered and understood, its a prototyping of a different stable self replicating structure. An amazing achievement, but still it seems more like engineering than science. What am I missing?

 

[Auto-Didact]

I am seeing this as an engineering achievement, not something that enables fundamentally new science. Its not a discovery of stable self replicating structures, those have already been discovered and understood, its a prototyping of a different stable self replicating structure. An amazing achievement, but still it seems more like engineering than science. What am I missing?

The substrate independent mathematically modeled abstraction of this phenomena gained by adopting a nonlinear science/dynamical systems perspective. Taking this perspective, directly enables the changing of the subject matter from engineering into biophysics/mathematical biology.
Here is another similar "engineering achievement" which, despite predominantly being focused on engineering, clearly is capable of providing answers to particular fundamental questions in biological theory.

I should also make clear that abiogenetic stable self-replicators have not yet actually been found. The hope is precisely that such engineering approaches will give different stable self replicating structures and so enable a mapping out of the state space of possibilities. This naturally enables the creation of a dynamical systems theory of self-replicators, a strategy which might lead the way to an actual theoretical prediction and experimental discovery of the long sought after abiogenetic self-replicator.