10 June 2008

Artificial protocells synthesize DNA without the help of enzymes

Alexander Markov, "Elements"

American biologists have taken an important step towards understanding the initial stages of the origin of life. They managed to create a "protocell" with a shell of simple lipids and fatty acids, capable of drawing activated nucleotides from the environment – the "bricks" necessary for DNA synthesis. The protocell cannot independently carry out matrix synthesis (replication) of DNA from beginning to end, but successfully copes with the most important stages of this process, and all reactions go without the participation of any proteins or other complex biological molecules-catalysts.

One of the key aspects of the problem of the origin of life is the question of what type of metabolism the first living organisms had. Some scientists, following academician A. I. Oparin, believe that the first "protocells" were heterotrophs, that is, consumers of finished organic matter dissolved in the waters of ancient reservoirs (the theory of "primary broth"). Or maybe life originated in cracks and cavities of rocks or in hydrothermal vents, where organic matter formed in the bowels of the Earth served as food for the first organisms.

Other experts consider it more likely that the first organisms were autotrophs, that is, they did not need ready-made organic matter and synthesized it themselves from carbon dioxide and other simple substances, using the energy of redox reactions (chemoautotrophs) or light (photoautotrophs). However, the idea of the primacy of photoautotrophs seems doubtful, since comparative genomics data convincingly indicate the later appearance of photosynthesis compared to some types of chemoautotrophic metabolism, such as methanogenesis and anaerobic oxidation of methane.

Molecular data, however, do not yet give a clear answer to the question of who appeared earlier – heterotrophs or chemoautotrophs. In favor of the primacy of heterotrophs is evidenced, first of all, by the obvious fact that their metabolism as a whole is simpler. All living organisms should be able to use ready-made organic matter to build their own cells, but autotrophs also need to synthesize this organic matter themselves from simple molecules. It is logical to assume that the ability to bind CO2 and synthesize organic matter developed later as a "superstructure" over heterotrophic metabolism.

However, serious arguments are also put forward against the idea of the primacy of heterotrophs. One of them is that since all living organisms multiply exponentially, the very first heterotrophic form of life that appeared on the planet would have eaten all the primary broth, no matter how much it was, for an insignificant period of time by geological standards. She simply would not have had time to go through the entire path of evolutionary development necessary for the transformation of a heterotrophic organism into an autotrophic one. It can be argued that the "broth" was gradually fed by organic matter, formed, for example, during geochemical processes in the bowels of the planet.

The other argument is harder to deflect. The membranes (shells) of modern cells consist of phospholipids, and these membranes are practically impervious to polar and charged molecules, including complex organic compounds such as sugars or nucleotides. To transport these molecules across the membrane, modern cells have a set of special transport proteins. At the dawn of life, of course, there could not be such proteins. Consequently, the protocell simply could not receive complex organics from the external environment. She had to be content with those simple inorganic molecules that are able to pass through the phospholipid membrane without assistance. Conclusion: the first living cells were autotrophs.

An article by American biologists published on June 4 on the website of the journal Nature is a very successful attempt to deflect this argument from the opponents of the "heterotrophic theory". The authors assumed that the protocell membrane did not necessarily have to consist of the same lipids as the membranes of modern cells. By the way, the primary "substance of heredity" also did not necessarily have to be DNA or RNA in their current form. Stable two-layer membranes (and bubbles surrounded by such membranes) are obtained from a variety of different lipids, fatty acids, alcohols and other amphiphilic compounds (that is, having a polar hydrophilic "head" and a hydrophobic hydrocarbon "tail"). Such molecules in water can assemble themselves into two-layer membrane films: hydrophobic tails turn inward, away from the water, and hydrophilic "heads" stick out, forming both surface layers of the membrane.

A hypothetical protozoan protocell feeding on ready-made organic matter.The protocell membrane grows due to the inclusion of suitable molecules from the external environment.

The protocell is divided by a simple "falling apart in half" under the action of internal or external physical forces. Its main "food" consists of activated nucleotides. They seep through the membrane and are used for spontaneous (non-enzymatic) reproduction of nucleic acid molecules (RNA, DNA or some of their early modifications). Fig. from the discussed article in Nature.

Phospholipids are quite complex molecules. Rather, the membranes of the protocells had to be assembled from simpler amphiphilic compounds that could be formed abiogenically.

The authors studied the properties of small bubbles (hundreds of nanometers in size, which is comparable to the smallest living cells) surrounded by membranes of various fatty acids. At first, they tried to figure out what determines the permeability of membranes for simple organic compounds, such as ribose sugar (this sugar is one of the necessary components of nucleotides, from which, in turn, RNA and DNA molecules are assembled). It turned out that membranes made of simple fatty acids pass ribose a little better than phospholipid membranes, but still bad.

However, the permeability increases dramatically if you use a mixture of fatty acid with a monoester of the same acid and glycerin. Numerous experiments have shown that the permeability of the membrane depends primarily on the shape of the molecules from which it is made: the larger the "head" of the molecule in relation to the length of the "tail", the higher the permeability. For example, in fatty acids, the role of the "head" is played by the carboxyl group (–COOH), which is small in size. Long hydrophobic "tails" in the thickness of the membrane are located closely and tightly stick together. In the glycerin ester of the same fatty acid, the role of the "head" is played by a glycerin molecule, much larger. Because of this, the hydrophobic "tails" in the thickness of the membrane are placed more freely, and the whole structure as a whole turns out to be more loose, fluid and mobile. Based on the experiments carried out, the authors proposed a theoretical model for the passage of charged molecules through membranes (see Figure).

A diagram of the passage of polar or weakly charged molecules through a two-layer lipid membrane.The molecule first adheres to the hydrophilic "heads" of lipids (highlighted in red).

This leads to a change in the orientation of lipid molecules. Under certain conditions, lipids can "flip" their heads to the other side of the membrane, dragging the captured molecule with them. Fig. from additional materials to the article under discussion in Nature.

The authors found several variants of the membrane composition, in which its permeability to ribose is high. Further experiments were carried out with two of these variants. The first of them is a mixture of myristoleic acid with its glycerol monoester (glycerol monoester of myristoleic acid). This mixture gives stable bubbles with good permeability, but it has one drawback: myristoleic acid contains 14 carbon atoms and one double bond, and its presence in the "primary broth" in sufficiently high concentrations is considered unlikely. The second option is a mixture of decanoic acid with the corresponding glycerin monoester and decanoic alcohol. This mixture is closer to reality (that is, to what could be in the primary broth), because there are only 10 carbon atoms in decanoic acid and there are no double bonds.

Then the authors began to study the permeability of these bubbles in relation to activated nucleotides – those "bricks" from which the cell collects RNA and DNA molecules. If the real protocells were heterotrophs, such nucleotides should have been their main "food". Modern cells use nucleotides with three phosphoric acid residues attached to them (nucleotide-triphosphates). However, nucleotide triphosphates, as it turned out, flatly refuse to pass through any lipid membranes. The reason is that they carry too strong a negative charge. Nucleotide diphosphates and nucleotide monophosphates have less charge, and they manage to pass through myristolein and decane membranes, but DNA is not synthesized by itself from such "bricks".

However, there was a workaround here too. Nucleotides can be activated in a different way – by attaching to them, instead of three phosphates, one phosphate and an imidazole molecule (imidazole is a simple organic compound widely distributed in wildlife and representing a ring of three carbon atoms and two nitrogen atoms; imidazole is an integral part of one of the 20 "canonical" amino acids – histidine). Imidazole-activated nucleotides are suitable for DNA and RNA synthesis, but they have only one negative charge, not four, like nucleotide triphosphates. Such nucleotides have already been used previously in experiments on the synthesis of nucleic acids without the participation of enzymes.

Many researchers admit that at the dawn of life, not nucleotide triphosphates could be used for the synthesis of nucleic acids, as now, but nucleotides activated by imidazole. Such nucleotides are even better at this job than nucleotide triphosphates, especially in the absence of catalyst proteins. The authors of the article under discussion add another consideration to this: they suggest that the transition from nucleotides activated by imidazole to less effective nucleotide triphosphates was due to the need to prevent the leakage of nucleotides from the cell (nucleotide triphosphates, as we remember, do not pass through membranes). This, of course, happened already when the cells themselves learned to synthesize the building blocks for the synthesis of nucleic acids and stopped "sucking" them from the outside.

As expected, the nucleotides activated by imidazole passed quite freely through the myristolein and decane membranes. This success inspired the authors to attempt to create an artificial protocell that would "feed" on activated nucleotides and carry out matrix synthesis (replication, copying, reproduction) of DNA or RNA molecules without the help of enzymes.

To date, chemists have already made some progress in the study of non-enzymatic replication of nucleic acids. However, the conditions necessary to complete the replication cycle without the help of proteins have not yet been found. There are two main unresolved problems. Firstly, conditions have not yet been found in which the matrix synthesis of any DNA or RNA molecule would go by itself, regardless of the sequence of nucleotides in the matrix. Some sequences can be replicated, others cannot. Secondly, in order for the process of spontaneous replication to begin, a "seed" is needed - a primer. This means that if you take a simple single-stranded DNA or RNA molecule, then replication does not begin on such a matrix without the help of enzymes. It still has to start with the use of enzymes. But if part of the nucleotides of the second (complementary) chain is already in place, then the replication process can continue under certain conditions without the help of enzymes. And this is already a lot.

If the full cycle of non-enzymatic replication of NK had already been discovered, then the authors of the article under discussion would probably have come close to creating a real living organism. In the meantime, they had to be content with what they had. They took short DNA molecules with a seed and with an under-replicated "tail" consisting of 15 C nucleotides (cytidines). The molecules were placed inside membrane bubbles.

These filled bubbles – model protocells – were placed in an environment optimal for non-enzymatic DNA synthesis (pH 8.5, temperature 4 ° C, plus two more simple organic compounds theoretically compatible with the ideas of the primary broth). After that, the protocells began to receive "food" – activated nucleotides. The official name of "feed": 2'-amino-2',3'-dideoxyguanosine-5'-phosphorimidazole. From time to time, some of the protocells were extracted from the solution to see how replication was going.

She walked well, albeit slowly. In the end, all the protocells coped with the task, that is, they completed the replication of under-replicated DNA molecules, attaching a complementary guanosine (G) to each of the 15 cytidines (C). It took them 24 hours, 96 minutes per nucleotide. In real living cells, DNA replication is carried out tens of millions of times faster, but there are super–efficient catalysts - enzymes.

The results obtained show that the first living cells could still be heterotrophs. And they also show that in the very near future scientists, apparently, will be able to reproduce in the laboratory all the key stages of the origin of life from inanimate matter.

Source: Mansy et al., Template-directed synthesis of a genetic polymer in a model protocell // Nature. Advance online publication 4 June 2008 (doi:10.1038/nature07018).

Portal "Eternal youth" www.vechnayamolodost.ru10.06.2008

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