Prof. Clemens Richert and his team of researchers from the University of Stuttgart are investigating the role of ribonucleic acid (RNA) in the development of life on earth. An important issue that has not yet been solved is how double-strand RNA can be created without the involvement of enzymes. Richert’s experiments have led to a breakthrough discovery as well as some mechanistic explanations.
The RNA world hypothesis proposes that life based on RNA predates the current world of life based on DNA. Did life on earth start with the emergence of RNA that was able to replicate other RNAs, including itself? “Only a handful of serious scientists now doubt the RNA world hypothesis. RNA can store genetic information and catalyse biochemical reactions,” said Prof. Dr. Dr. Clemens Richert, head of the Department of Biological Chemistry at the University of Stuttgart. One of Richter’s major research priorities is the enzyme-free extension of primers, a process that is also believed to have taken place in the enzyme-free world at the time when RNA double-strands first appeared.Richert refers to this as a chicken and egg causality dilemma: enzymes normally catalyse the biochemical steps involved in the creation of double-strand RNAs by adding complementary nucleotides to single-strand RNA templates. However, the enzymes also have to be encoded in genes. An RNA molecule that can catalyse a biochemical reaction is referred to as a ribozyme. But how did the first double-strand RNA molecules come into being? The RNA world hypothesis proposes that double-strand RNA occurred spontaneously under specific physical and chemical environmental conditions. Experiments have since shown that a mineral-containing aqueous solution, associated with temperatures close to or below freezing point enable nucleotides to bind to single-strand RNA fragments. The formation of ice in the water leads to the formation of channels between the ice crystals, which promotes the spatial association of the individual building blocks. Hauke Trinks, a physicist from the Max Planck Institute for Biophysical Chemistry, and Christof Biebricher from the Technical University of Hamburg-Harburg have previously shown that sea ice has many features that are important for catalytical actions to occur, including those that enable the generation of long chains of RNA.
The biggest problem associated with all the approaches so far is that the reaction has come to a standstill relatively quickly. This is due to chemical reactions – so-called hydrolyses – that occur at the activated nucleotide monomers contained in the liquid. The hydrolised products can bind to the reaction site and inhibit the formation of double strands,” said Richert. Richert and his team have developed a method to get rid of the inhibitors – they simply wash them away. The method works as follows: short single-strand RNA molecules are immobilised by attaching them to iron oxide particles to which a nucleotide mixture is added. These nucleotides then form a complementary RNA strand on the basis of the RNA template. The solution is repeatedly replaced with a fresh one in order to keep the concentration of inhibitory monomers at a tolerable limit. Using this method, the researchers were able to successfully generate double-strand RNA.
“We used iron oxide particles for the simple reason that they are magnetic. The particles can thus be contained within the vial and the supernatant is easily removed with a pipette,” said Richert. The researchers showed that any of the four nucleobases (adenine, cytosine, guanine and uracil) was successfully copied in the absence of enzymes. The experiment thus confirmed that double-strand RNA could form without the involvement of enzymes. Richert and his team thus closed an important gap in the RNA world hypothesis. The researchers’ next goal is to induce the formation of complimentary strands that are as long as possible and move one step closer to being able to produce functional RNA units. This is a process that requires many individual reactions that Richert hopes to be able to bring together in one single experimental approach. “Our ultimate goal is to find out how we can turn individual nucleotides into a catalytical ribozyme in a kind of protocell, which would then have an evolutionary advantage,” said Richert.
Richert’s future plans are to couple RNA to clay surfaces. Although this might make the experiments more complex, Richert believes that clay minerals are excellently suited to forming longer RNA strands. “We have already been able to solve a fundamental problem. Our next goal is to simulate a scenario that comes closer to what might have happened during evolution,” said Richert. He also wants to do something more than just washing off inhibitory molecules. He believes a smarter way of doing it would be to reactivate the deactivated monomers and he is hoping to find a way to do this in a reliable and controllable manner.
Enzyme-free reactions involving nucleic acids are one of Richert’s principal research priorities focusing on both RNA and DNA, which is far more stable than RNA. “Our main concern is how we can find cheaper and more robust ways to turn processes that are normally catalysed by enzymes into enzyme-free processes. In a DFG-funded project, we are currently investigating the enzyme-free peptide chain extension at the N-terminus of DNA strands,” said Richert who also has in mind the possible commercial exploitation of the new methods. “I believe that the enzyme-free primer extension would be of great interest in applications that could be used to sequence small RNA or DNA fragments both reliably and inexpensively. I am sure that any researcher who is seeking to develop unusual forms of nucleic acid structures can also benefit from using this method,” said Richert who is open to working with companies that are involved in this particular field.
Further information:University of StuttgartInstitute of Organic ChemistryProf. Clemens RichertPfaffenwaldring 5570569 StuttgartTel.: +49 (0)711/ 685 - 64311E-mail: lehrstuhl-2(at)oc.uni-stuttgart.de