In New Study, Scientists Cracking Origins of Life

Chemical reactions driven by the geological conditions on the early Earth might have led to the prebiotic evolution of self-replicating molecules. Scientists at Ludwig-Maximilians Universitaet (LMU) in Munich last August reported on a hydrothermal mechanism that could have promoted the process.

Now, a study done by the university’s physicists is demonstrating that fundamental characteristics of mopolymeric lecules, such as their subunit composition, are sufficient to trigger selection processes in a plausible prebiotic setting.

Before life emerged on Earth, many physicochemical processes on our planet were highly chaotic. A plethora of small compounds, and polymers of varying lengths, made up of subunits (such as the bases found in DNA and RNA), were present in every conceivable combination.

Before life-like chemical processes could emerge, the level of chaos in these systems had to be reduced. In a new study, LMU physicists led by Dieter Braun show that basic features of simple polymers, together with certain aspects of the prebiotic environment, can give rise to selection processes that reduce disorder.

In previous publications, Braun’s research group explored how spatial order could have developed in narrow, water-filled chambers within porous volcanic rocks on the sea bottom. These studies showed that, in the presence of temperature differences and a convective phenomenon known as the Soret effect, RNA strands could locally be accumulated by several orders of magnitude in a length-dependent manner.

“The problem is that the base sequences of the longer molecules that one obtains are totally chaotic”, says Braun.

Evolved ribozymes (RNA-based enzymes) have specific base sequence that enable the molecules to fold into particular shapes, while the vast majority of oligomers formed on the Early Earth most probably had random sequences.

First author of the new report, Patrick Kudella, says “the total number of possible base sequences, known as the ‘sequence space,” is incredibly large. This makes it practically impossible to assemble the complex structures characteristic of functional ribozymes or comparable molecules by a purely random process.”

This led the LMU team to suspect that the extension of molecules to form larger ‘oligomers’ was subject to some sort of preselection mechanism.

At the time of the origin of life, there were only a few, very simple physical and chemical processes compared to the sophisticated replication mechanisms of cells, so the selection of sequences must be based on the environment and the properties of the oligomers.

This is where the research of Braun’s group comes in. For catalytic function and stability of oligomers, it is important that they form double strands like the well-known helical structure of DNA. This is an elementary property of many polymers and enables complexes with both double- and single-stranded parts.

The single-stranded parts can be reconstructed by two processes. First, by so-called polymerization, in which strands are completed by single bases to form complete double strands. The other is by what is known as ligation. In this process, longer oligomers are joined together. Here, both double-stranded and single-stranded parts are formed, which enable further growth of the oligomer.

“Our experiment starts off with a large number of short DNA strands, and in our model system for early oligomers we use only two complementary bases, adenine and thymine”, says Braun, adding, “we assume that ligation of strands with random sequences leads to the formation of longer strands, whose base sequences are less chaotic.”

Braun’s group then analysed the sequence mixtures produced in these experiments using a method that is also used in analysing the human genome. The test confirmed that the sequence entropy, i.e. the degree of disorder or randomness within the sequences recovered, was in fact reduced in these experiments.

The researchers were also able to identify the causes of this “self-generated” order. They found that the majority of sequences obtained fell into two classes—with base compositions of either 70% adenine and 30% thymine, or vice versa.

“With a significantly larger proportion of one of the two bases, the strand cannot fold onto itself and remains as a reaction partner for the ligation”, Braun explains. Thus, hardly any strands with half of each of the two bases are formed in the reaction.

“We also see how small distortions in the composition of the short DNA pool leave distinct position-dependent motif patterns, especially in long product strands”, Braun says.

The result surprised the researchers, because a strand of just two different bases with a specific base ratio has limited ways to differentiate from each other. Co-author of the study, Annalena Salditt, says “only special algorithms can detect such amazing details.”

The experiments show that the simplest and most fundamental characteristics of oligomers and their environment can provide the basis for selective processes. Even in a simplified model system, various selection mechanisms can come into play, which have an impact on strand growth at different length scales, and are the results of different combinations of factors.

According to Braun, these selection mechanisms were a prerequisite for the formation of catalytically active complexes such as ribozymes, and therefore played an important role in the emergence of life from chaos.

Last August, they state that life is a product of evolution by natural selection, an obvious take-home lesson from Charles Darwin’s book The Origin of Species, published over 150 years ago. But how did the history of life on our planet begin? What kind of process could have led to the formation of the earliest forms of the bio-molecules we now know, which subsequently gave rise to the first cell?

Scientists believe that, on the (relatively) young Earth, environments must have existed, which were conducive to prebiotic, molecular evolution.

A dedicated group of researchers is engaged in attempts to define the conditions under which the first tentative steps in the evolution of complex polymeric molecules from simple chemical precursors could have been feasible.

LMU biophysicist, Dieter Braun, explains “to get the whole process started, prebiotic chemistry must be embedded in a setting in which an appropriate combination of physical parameters causes a non-equilibrium state to prevail.”

Together with colleagues based at the Salk Institute in San Diego, he and his team have now taken a big step toward the definition of such a state. Their latest experiments have shown the circulation of warm water (provided by a microscopic version of the Gulf Stream) through pores in volcanic rock can stimulate the replication of RNA strands.

The new findings appear in the journal Physical Review Letters.

As the carriers of hereditary information in all known life forms, RNA and DNA are at the heart of research into the origins of life. Both are linear molecules made up of four types of subunits called bases, and both can be replicated—and therefore transmitted. The sequence of bases encodes the genetic information.

However, the chemical properties of RNA strands differ subtly from those of DNA. While DNA strands pair to form the famous double helix, RNA molecules can fold into three-dimensional structures that are much more varied and functionally versatile.

Indeed, specifically folded RNA molecules have been shown to catalyze chemical reactions both in the test-tube and in cells, just as proteins do. These RNAs therefore act like enzymes, and are referred to as ‘ribozymes.”

 The ability to replicate and accelerate chemical transformations motivated the formulation of the “RNA world’ hypothesis. This idea postulates that, during early molecular evolution, RNA molecules served both as stores of information like DNA, and as chemical catalysts. The latter role is performed by proteins in today’s organisms, where RNAs are synthesized by enzymes called RNA polymerases.

Ribozymes that can link short RNA strands together—and some that can replicate short RNA templates—have been created by mutation and Darwinian selection in the laboratory. One of these “RNA polymerase’ ribozymes was used in the new study.

Acquisition of the capacity for self-replication of RNA is viewed as the crucial process in prebiotic molecular evolution. In order to simulate conditions under which the process could have become established, Braun and his colleagues set up an experiment in which a 5-mm cylindrical chamber serves as the equivalent of a pore in a volcanic rock.

On the early Earth, porous rocks would have been exposed to natural temperature gradients. Hot fluids percolating through rocks below the seafloor would have encountered cooler waters at the sea-bottom, for instance.

This explains why submarine hydrothermal vents are the environmental setting for the origin of life most favored by many researchers. In tiny pores, temperature fluctuations can be very considerable, and give rise to heat transfer and convection currents.

These conditions can be readily reproduced in the laboratory. In the new study, the LMU team verified that such gradients can greatly stimulate the replication of RNA sequences.

One major problem with ribozyme-driven scenario for replication of RNA is that the initial result of the process is a double-stranded RNA. To achieve cyclic replication, the strands must be separated (‘melted’), and this requires higher temperatures, which are likely to unfold—and inactivate—the ribozyme. Braun and colleagues have now demonstrated how this can be avoided.

“In our experiment, local heating of the reaction chamber creates a steep temperature gradient, which sets up a combination of convection, thermophoresis and Brownian motion”, says Braun.

Convection stirs the system, while thermophoresis transports molecules along the gradient in a size-dependent manner. The result is a microscopic version of an ocean current like the Gulf Stream. This is essential, as it transports short RNA molecules into warmer regions, while the larger, heat-sensitive ribozyme accumulates in the cooler regions, and is protected from melting.

The researchers were indeed, astonished to discover that the ribozyme molecules aggregated to form larger complexes, which further enhances their concentration in the colder region. In this way, the lifetimes of the labile ribozymes could be significantly extended, in spite of the relatively high temperatures. “That was a complete surprise”, says Braun.

The lengths of the replicated strands obtained are still comparatively limited. The shortest RNA sequences are more efficiently duplicated than the longer, such that the dominant products of replication are reduced to a minimal length. Hence, true Darwinian evolution, which favors synthesis of progressively longer RNA strands, does not occur under these conditions.

“However, based on our theoretical calculations, we are confident that further optimisation of our temperature traps is feasible”, says Braun. A system in which the ribozyme is assembled from shorter RNA strands, which it can replicate separately, is also a possible way forward.

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