Life on Earth is likely to have arisen by an RNA molecule that started to make copies of itself. RNA researchers have succeeded in recreating an RNA copy machine and determining its structure through single-particle cryogenic electron microscopy. The research elucidates RNA’s role in self-replication and contributes to deeper understanding of the origin and evolution of life. The research also paves the way for using advanced RNA machines in medicine and biotechnology.
Before DNA and proteins evolved, RNA was the only molecule that defined life – by both storing the genetic information and carrying out the processes of life through RNA catalysts (ribozymes). This RNA world is believed to have been the basis for the emergence of more complex life forms with DNA and proteins – the world we know today.
Scientists hunting ancient DNA have uncovered worlds that are 1 to 1.5 million years old, but RNA researchers are now seeking to uncover an RNA world that presumably existed around 4 billion years ago, of which all physical traces have disappeared.
“To decode the secrets from this early era, we have recreated an analogue of the original replication machine – a replicase – in the laboratory and have now determined its molecular anatomy with single-particle cryogenic electron microscopy to better understand its working mechanism,” explains co-author Ebbe Sloth Andersen, Associate Professor, Interdisciplinary Nanoscience Center, Aarhus University, Denmark.
The new study was carried out in collaboration with Philipp Holliger’s research group at the Medical Research Council Laboratory of Molecular Biology in Cambridge, United Kingdom, which developed the RNA replicase, and Ebbe Sloth Andersen’s research group from Aarhus University, which determined its structure by using advanced microscopy technology.
Recreating the mythical RNA replication machine
The RNA replicase has been in development in research laboratories around the world for the past 30 years. It was originally discovered like a needle in a haystack between random sequences in selection experiments. The first version could only attach two RNA strands together, but through later development it was improved to function as an RNA polymerase. Philipp Holliger’s laboratory has developed the polymerase for years to enable the replicase to become increasingly efficient.
“The RNA replicase developed in Holliger’s laboratory can copy long and structurally complex RNA strands. The RNA copying process takes place in a special eutectic ice phase, a slush-ice-like state, in which the RNA is upconcentrated in the water between the ice crystals,” says assistant professor Emil L. Kristoffersen, who worked in Philipp Holliger’s laboratory.
On returning to Aarhus, Emil L. Kristoffersen established a collaboration with Ebbe Sloth Andersen’s research team at Aarhus University to analyse the structure and functional landscape of the replicase.
Cryogenic electron microscopy reveals kissing molecules
In Aarhus, the researchers analysed the RNA replicase using cryogenic electron microscopy, a technique that uses electron beams to examine samples that are cooled to cryogenic temperatures. This technique has proven particularly effective in probing complex and dynamic RNA structures in detail that were previously inaccessible using older methods.
“The structure of the RNA replicase was full of surprises. It generally resembles an open hand and thus has the same structure as certain protein-based polymerases. The machine comprises two domains connected by kissing-loop interactions that improve the replication. In addition, it has developed a domain for correcting errors,” explains Ebbe Sloth Andersen.
To obtain deeper understanding of the RNA replicase mechanism, the researchers performed a detailed mutational analysis to identify key components of the RNA structure. This emphasised the importance of the catalytic core and of several new RNA motifs with various supporting roles in the mechanism. Mutations disrupting the kissing-loop interactions had a negative effect on the copying efficiency.
“The experimental data provides valuable knowledge about elements that are important for efficient self-replication of RNA molecules and points towards experiments and optimisation of the copy machine,” elaborates Emil L. Kristoffersen.
Further exploration of the RNA world and its applications
The structure and mutational analysis of the RNA replicase opens new research opportunities to study the RNA world. This structural insight enables more efficient replication methods to be rationally developed and thus to recreate the RNA world in a laboratory environment. But nature can also show the way:
“The simplest evolutionary systems that exist today are the viroids, which infect plants and destroy millions of Euros worth of crops each year. Viroids practically still exist in an RNA world, since they only consist of small circular RNA molecules and use ribozymes as part of their replication mechanism. Clearly these should be studied further,” explains Emil L. Kristoffersen.
This new study also highlights how research into the origin of life can lead to the discovery of RNA core structural motifs, which can also potentially be used for future RNA nanotechnology and medicine.
“RNA structures can be used for precisely delivering drugs, since they can be tailored to interact directly with selected cells or proteins. In addition, they can be used in diagnostic tools or as an integral part of innovative treatment methods, such as in gene therapy or personalized medicine,” concludes Ebbe Sloth Andersen.
