World’s first successful in-vitro synthesis of ribosomes

A research group led by Professor Wataru Aoki of the Graduate School of Engineering, The University of Osaka, has succeeded for the first time in the world in synthesizing ribosomes—the protein factories of cells—from genes in vitro. Ribosomes are complex structures assembled through the coordinated action of approximately 200 types of molecules, and reproducing them artificially in vitro has long been considered difficult.

Guided by the principle of “reproducing the intracellular environment in vitro,” the research group successfully synthesized ribosomes from genes by combining the required factors under appropriate conditions.

Why creating artificial ribosomes is important

Ribosomes exist within the cells of living organisms and synthesize proteins by linking amino acids according to genetic information. If ribosomes can be artificially modified to incorporate non-natural monomers that do not exist in nature, it may become possible to create pharmaceuticals and useful enzymes with functions that have not been achievable to date.

However, it is known that attempts to modify ribosomes within living cells result in toxicity, causing the cells to no longer function normally. On the other hand, research has been conducted to modify molecules other than ribosomes so that ribosomes can use non-natural monomers^*1 as materials To date, it has been possible to use over 200 non-natural monomers for protein synthesis. However, to efficiently polymerize non-natural monomers, it ultimately proved necessary to modify the ribosome itself.

Therefore, the research group reasoned that if ribosomes could be artificially synthesized in vitro without using cells, it would be possible to modify ribosomes freely without concern about cytotoxicity.

*1 Non-natural monomer
Molecules with chemical structures that living organisms do not normally utilize and that are expected to serve as materials for creating molecules with new properties.

Reproducing the intracellular environment in vitro

Ribosomes are synthesized through a complex process

Ribosomes in living organisms are formed through highly complex processes. For example, the ribosome of Escherichia coli consists of three types of ribosomal RNA^*2 and 54 types of proteins, and its synthesis requires the action of nearly 100 auxiliary proteins. Ribosomes are not produced simply by the presence of their constituent materials. Each component binds in a prescribed order, undergoing stepwise structural changes as assembly progresses.

Because of the complexity of this process, it has been difficult to artificially generate molecules that function in the same way as ribosomes in living systems.

*2 Ribosomal RNA
RNA that constitutes the ribosome and is involved in the reactions that synthesize proteins.

Creating the intracellular environment

The research group reasoned that ribosomes could be synthesized if an environment identical to that inside cells was reproduced in vitro.

First, Escherichia coli cells were disrupted to obtain an S150 lysate. This solution was considered to contain many auxiliary proteins required for ribosome assembly. Furthermore, low-molecular-weight compounds abundant in living organisms were added to reproduce an environment similar to those inside biological cells. Next, native ribosomes, RNA polymerase (RNA-synthesizing enzyme), ribosomal RNA genes, and genes encoding 54 ribosomal proteins were added (Fig. 1).

After repeatedly optimizing the reaction conditions, the research group succeeded in synthesizing ribosomes from genes in vitro. This achievement represents the world's first example of ribosome synthesis in a completely artificial environment.

Toward understanding ribosomes and the synthesis of useful substances

Ribosomes are known to use amino acids present in the body for synthesis, while non-natural monomers are rarely used as synthetic materials. The fundamental question of why such a difference exists has not been clarified. The successful construction of artificial ribosomes in this study makes it possible to modify and evaluate ribosomes, tasks that were previously difficult, and may greatly contribute to a deeper understanding of ribosomal mechanisms (Fig. 2).

If artificial ribosomes can be designed to efficiently polymerize non-natural monomers, this could lead to the development of superior pharmaceuticals that are easily absorbed by the body and suitable for oral administration, highly stable industrial enzymes that are resistant to degradation, and bioplastics with low environmental impact. In particular, the ability to efficiently handle D-amino acids would bring us one step closer to creating enzymes capable of unprecedented reactions and pharmaceuticals with high resistance to degradation.