Thursday, 6 October 2016

Replication, Replication, Replication I

This post and the one below it are linked. Here, I discuss a topic that interests us as a group, and below I look at some recent related papers. This post should make reasonable sense in isolation, the second perhaps less so.

Replication is at the heart of biology; whole organisms, cells and molecules all produce copies of themselves. Understanding natural self-replicating systems, and designing our own artificial analogues, is an obvious goal for scientists - many of whom share dreams of explaining the origin of life, or creating new, synthetic living systems.

Molecular-level replication is a natural place to start, since it is (in principle) the simplest, and also a necessary component of larger-scale self-replicating systems. The most obvious example in nature is the copying of DNA; prior to cell division, a single copy of the entire sequence of base pairs in the genome must be produced. But the processes of transcription (in which the information in DNA sequence is copied into an RNA sequence) and translation (in which the information in RNA sequence is copied into protein sequence) are closely related to replication. The information initially present in the DNA sequence is simply written out in a new medium, like printing off a copy of an electronic document. This process is illustrated in the figure above (which I stole from here). This figure nicely emphasies the polymer sequences (shown as letters) that are being copied into a new medium (note: three RNA bases get copied into one amino acid in a protein: AUG into M, for example). An absolutely fundamental feature of both replication and copying processes is that the copy, once produced, is physically separated from the template from which it was produced. This is important, otherwise the copies couldn't fulfill their function, and more copies could not be made from the same template.

This single fact - that useful copies must separate from their template yet retain the copied information - makes the whole engineering challenge far harder. It's (reasonably) straight-forward to design a complex (bio)chemical system that assembles on top of a template, guided by that template. All you need are sufficiently selective attractive interactions between copy components and the template. But if you then want to separate your copy from the template, these very same attractive interactions work against you, holding the copy in place - and more accurate copies hold on to the template more tightly. My collaborators and I formalise this idea, and explore some of the other consequences of needing to separate copies from templates, in this recent paper.

Largely because of this problem, no-one has yet constructed a purely chemically driven, artificial system that produces copies of long polymers, as nature does. Instead, it has proved necessary to perform external operations such as successively heating and cooling the system. Copies can then grow on the template at low temperature, and then fall off at high temperature, allowing a new copy to be made when the system is cooled down. This is exactly what is done in the PCR, an incredibly important process for amplifying a small amount of DNA in areas ranging from forensics to medicine.

As a group, we're very interested in how copying/replication can be achieved without this external intervention. Two recent papers, discussed in the blog entry below, highlight the questions at hand.

Replication, Replication, Replication II

Here I discuss two recent experimental papers that are related to the challenge of replication or copying, following on from the discussion in "Replication, Replication, Replication I". My take on these papers is heavily couched in terms of that discussion.

Semenov et al.: Autocatalytic, bistable oscillatory networks of biologically relevant reactions
Nature 537, 656–660 (2016)
A catalyst accelerates chemical involving a substrate. For example, amylase accelerates the interconversion of starch and sugars, helping us to digest food. As we learnt at school, a key feature of catalysts is that they are not consumed by the reaction - a single amylase can digest many starch molecules. This fact should remind us of the replication/copy process discussed above, in which it is important that a new copy separates from its template so that the template is not be consumed by the copy process, and can go on to produce many more copies. Indeed, templates for copying/replication must be catalysts. In the specific case of replication, the process is autocatalytic, meaning that a molecule is a catalyst for the production of identical molecules. Simple autocatalytic systems are thus often seen as a bridging point to the full complexity of life.

Semenov et. al. show that a particularly simple set of molecules can exhibit autocatalytic behaviour. Although autocatalysis has been previously demonstrated, the novelty of their approach is the use of such simple organic molecules (which could plausibly have been present on Earth prior to living organisms). Additionally, they are able to show relatively sophisticated behaviour from their system - not just exponential growth of the output molecule (the natural behaviour of autocatalytic systems). When molecules that cause inhibition of autocatalysis and degradation of components are added, for example, the output concentration can be made to oscillate.

Although fascinating, the work of Semenov et al. does not solve the question raised in the blog post above. There are no long polymers in this system, and so the difficulty of separating strongly-interacting copies and templates does not arise. But as a consequence, this autocatalytic mechanism passes on very little information (arguably, none) to the new molecules produced. Autocatalysis alone is not enough  - we are still a long way from processes such as DNA replication, transcription and translation.

Meng et al.: An autonomous molecular assembler for programmable chemical synthesis 
Nature Chemistry 8, 542–548 (2016)
This paper, co-authored by my collaborators in the Turberfield group, takes a completely different approach. The idea is to specify the sequence of a molecular polymer using a DNA-based programme. As I have talked about before, the exquisite selectivity of base-pairing in DNA allows reactions to be programmed into carefully designed single strands, allowing them to self-assemble into a complex patterns when mixed. In this case, the authors mix sets of short DNA strands that are designed to assemble into a long double-stranded structure in a specific order. The selectivity of interactions allows the strands to be programmed to bind one-by one to the end of the structure in the desired sequence.

This process (the hybridisation chain reaction) is not new. The advance is using it to template the sequence of a second (chemically quite different) polymer that can't assemble with a specific sequence on its own - for simplicity, lets call this polymer X (its details aren't important). The authors ingenuously attach building blocks of X to the DNA strands - with each distinct DNA sequence paired with a distinct building block. When a new strand is incorporated via the hybridisation chain reaction, it brings with it the associated building block and adds it to X, which grows simultaneously with the double-stranded DNA construct. The details of this process are a bit fiddly, and due to a technicality a new building block is only added for every second strand incorporated, but the process as a whole allows them to assemble a specific polymer X using DNA-based instructions set by the sequences of the original strands. The authors call this programmed chemical synthesis.

The authors are inspired by the ribosome (see fig, stolen from here), the biological machine that translates an RNA sequence (the red polymer) into a polypeptide sequence (green), which eventually folds into a protein. The ribosome uses RNA base pairing to bring a set of peptide building blocks together in the right order, like the device of Meng et al. uses DNA base-pairing to form polymer X. However, there is a key difference. The information-carrying RNA strand in the figure is not consumed by the process; it acts as a catalyst, as discussed, and the ribosome walks along it until the end and then releases it. The information-carrying components of the system of Meng et al. (the strands that carry the molecular programme) are consumed, being incorporated into a long double-stranded DNA molecule that the authors actually use to analyse the success of the process. Thus although the system allows programmable self-assembly, it doesn't implement catalysis and hence can't perform copying/replication.

Both papers are great pieces of work, but one demonstrates autocatalysis without information transfer, and the other demonstrates the ability to programme polymer assembly without autocatalysis. The challenge to produce chemical systems that copy or replicate is still on.