Many important research news in the past few months relates to the investigation of the origin of life, the possibility to generate artificial chromosomes and to create a DNA containing synthetic base pairs. A brief summary of latest discoveries
We do all remember about school books describing the origin of life in the ancient oceans and the genetic code made up of only four letters – A, T, G, C – the four DNA’s bases those combinations in triplets encode for the transmission of the hereditary information.
Several studies have been recently published in the scientific literature that may contribute to rewrite these biological paradigms about the origin of life, on one hand, and on the possibility to create artificial DNA or chromosomes, on the other. These last two discoveries are particularly significant as they may open the door to the creation of artificial forms of life, in which DNA and chromosomes might be ‘tailor-made designed’ and synthetized in the lab.
Artificial DNA’s bases
The possibility of insert two synthetic hydrophobic bases within the DNA sequence is by far the most important discovery recently published in the literature. The research group led by Floyd Romesberg of the Scripps Research Institute in La Jolla, California, has been working since 15 years to demonstrate the possibility to expand the genetic code by the identification of artificial bases that may be inserted in the DNA strands. Their recent results, describing the in-vivo use of a six-bases genetic code, have been published in Nature1.
The two new artificial hydrophobic bases – named d5SICS and dNaM, respectively – were identified in 20082 upon the screening of 60 candidates and of the 3600 resulting combinations. The researchers have now demonstrated the possibility to insert such bases within the DNA sequence and to replicate it in-vivo.
A modified DNA plasmid containing a single pair of the artificial bases has been inserted in the Escherichia coli bacterium and externally supplemented with the new bases. The bacterial proliferation has occurred normally and the duplicated DNA of the new born colonies do contain the artificial bases (Figure 1).
A main challenge for the researchers has been how to make available within the bacterial cells the artificial nucleotide phosphates needed for DNA’s duplication: they engineered the E. coli cells in order to express a gene from a diatom alga codifying for a protein that allows the nucleotides molecules to pass through the bacterium’s membrane.
The fact that the artificial nucleotides must be externally supplemented should also, according to Romesberg’s group, protect from the possibility that the engineered bacteria might escape the lab: as they won’t find any source of synthetic nucleotides in the external world, they would switch back and use the four standard bases for replication, says the scientists (Figure 2).
The possibility to insert two new, and structurally completely different as they are hydrophobic, bases within the DNA sequence might hugely expand its coding capability. The standard biological alphabet uses four bases to code for 20 different aminoacids; with the insertion of two more bases the number of possible combination would exponentially increase, as the resulting number of aminoacids and, thus, of resulting proteins.
The technique would allow for the production of completely new proteins and enzymes, for example to be used as drugs, diagnostics or vaccines, or to engineer cells to produce specific substances. A new company, Synthorx, has been already founded by Avalon Ventures in order to exploit the economical potential of the discovery. Romesberg’s group is now working to demonstrate that the unnatural base pair might be transcribed into RNA and finally transduced to a protein. «A lot of times people will say you’ll make and organism completely out of your unnatural DNA. That’s just not going to happen, because there are too many things that recognize DNA. It’s too integrated into every facet a cell’s life» – says Romesberg about the possibility that the new findings may open the door to artificial forms of life.
The first synthetic chromosome
Syn-III is the name of the first completely synthetic chromosome. It has been assembled at the New York University Langone Medical Center by the group led by Jef Boeke and the results have been published in Science3.
The artificial chromosome has been inserted into Saccharomyces cerevisiae cells, and the yeast has maintained unaltered its proliferation ability.
The international group has worked seven years to reach the goal: syn-III is made up of 273.871 base pairs and is shorter than its natural counterpart (316.667 base pairs). The artificial chromosome has been produced after a careful process of genetic planning: 47.841 base pairs have been removed as considered not necessary for replication and cellular growth. Junk DNA sequences have also been eliminated as well as ‘jumping DNA’, portions of the genome that might migrate and cause mutations. As prof. Boeke comments: “When you change the genome you’re gambling. One wrong change can kill the cell. We have made over 50.000 changes to the DNA code in the chromosome and our yeast still live. That is remarkable. It shows that our synthetic chromosome is hardy, and it endows the yeast with new properties”.
In the case too, as for the unnatural base pair inserted into DNA, the new method might be used to engineer cells with synthetic chromosomes in order to produces specific substances – i.e. drugs (artemisin), vaccines (hepatitis B), biofuels (ethanol, buthanol, biodiesel).
The yeast modified cells have been tested under a wide set (19) of different conditions, including pH, temperature, hydrogen peroxide content: just in one case the proliferation response was altered. The genetic structure remained unchanged after 125 cell’s divisions, thus demonstrating the stability of the artificial chromosome.
Syn-III chromosome is the shorter among the S. cerevisiae genetic makeup. Boeke and his group are now working to synthetize longer artificial chromosomes, making use of up to 10.000 base pairs.
The origin of metabolism
On the over side of evolution, new insights are emerging on the sequence of events that generated the first organised forms of life in the ancient oceans. The classical Urey-Miller hypothesis talks about RNA as the first biomolecule emerging from the Archean seas.
The recent findings, published in Molecular Systems Biology4,5 by the research group of the University of Cambridge led by Markus Ralser, may change the order of events as they suggest that metabolism would have originated first as the result of a complex network of reactions catalised by the inorganic ions present in the Archean oceans. RNA, according to the new theory, would have been selected at a later stage as the molecule useful to code for genetic information. The two alternative pathways are schematically represented in figure 3.
Under the classical hypothesis, metabolism would be the result of the evolutionary pressure on prebiotic enzymes codified by early RNA molecules. In the new one, the metabolic network would have been the first to appear, as explained by Markus Ralser: “Our results demonstrate that the conditions and molecules found in the Earth’s ancient oceans assisted and accelerated the interconversion of metabolites that in modern organisms made up glycolysis and the pentose-phosphate pathways, two of the essential and most centrally placed reaction cascades of metabolism”. According to the results from University of Cambridge, these metabolic reactions were particularly sensitive to the presence of ferrous ions (Fe2+) that helped catalyse many of the observed chemical reactions.
The ferrous ions would have originated – according to the geoscientist Alexandra V. Turchyn – from early oceanic sediments, as these soluble forms of iron were one of the most frequently found molecules in the ancient oceans. Researchers have replicated in the lab the conditions of the prebiotic oceans, including compositions of the early sediments, anorexic environment and a temperature (50-90°C) similar to a hydrothermal vent of an oceanic volcano. Upon incubation of different metabolites and in the presence of iron and other compounds found in oceanic sediments, the group observed 29 metabolic-like chemical reactions, some of which producing precursors of the building blocks of proteins or RNA, such as ribose 5-phosphate. “These results indicate that the basic architecture of the modern metabolic network could have originated from the chemical and physical constraints that existed in the prebiotic Earth”, commented Markus Ralser.
These recent finding are just the first drops of a possibly completely new vision of life on Earth, both as its appearing as well as future evolution. It will be fundamental to carefully evaluate the safety for the mankind and the entire ecosystem of such new techniques of genetic engineering: aliens might originate directly from man’s hands, we could argue.
1. D.A. Malyshev et al., A semi-synthetic organism with an expanded genetic alphabet, Nature, 509, 385-388, 2014, doi: 10.1038/nature13314
2. A. Leconte et al., Discovery, characterisation and optimisation of an unnatural base pair for expansion of the genetic alphabet, JACS, 130, 2336-2343, 2008, doi: 10.1021/ja078223d
3. N. Annaluru et al., Total synthesis of a functional designer eukaryotic chromosome, Science, Vol. 344, pp. 55-58 , doi: 10.1126/science.1249252
4. M.A. Keller et al., Non-enzymatic glycolysis and pentose phosphatepathway-like reactions in a plausible Archean ocean, Molecular Systems Biology, 10, 725, 2014, doi: 10.1002/msb.20145228
5. P.L. Luisi, Prebiotic metabolic networks?, Molecular System Biology, 10, 729, 2014, doi: 10.1002/msb.20145351