Periodic Reporting for period 5 - RiboLife (Resurrecting LUCA - Engineering of RNA-encoded Cellular Life Using Dual Evolution and Intergenomic Transplantation)
Berichtszeitraum: 2023-11-01 bis 2024-12-31
In RiboLife, we set out to investigate whether RNA-based genetic storage and replication can be reintroduced into modern cellular systems. While all known cellular life relies on DNA for storage of genetic information, evolutionary evidence suggests that primitive life forms may have used RNA-based genomes. To date, hardly any studies have investigated whether modern cells could, in part, also use RNA as a persistent genome for essential genes. A demonstration of this would be a significant contribution to the understanding of the origin of life on Earth but would also provide important new tools for biotechnology and synthetic biology.
To address this challenge, we aimed to develop synthetic, self-replicating RNAs (srRNAs) that could function in both cell-free and intracellular environments. During the project, we successfully developed srRNA scaffolds capable of supporting the replication of large RNA molecules, including large mRNAs and even ribosomal RNAs (rRNAs), without DNA intermediates. This achievement demonstrated for the first time that long, functional non-coding RNAs that are critical for cellular survival can be replicated by RNA replicases in vitro. We also identified essential host factors that modulate RNA replication in a fully reconstituted system. These findings pave the way for synthetic RNA-based gene expression systems that function independently of DNA.
We also established a novel approach for intracellular RNA replication using bacterial host systems. This allowed us to transplant single essential genes onto synthetic RNA constructs, a critical step towards the creation of RNA-DNA hybrid cells. This proof of concept lays the foundation for new gene evolution strategies, including orthogonal translation systems based on RNA genomes.
To address this challenge, we aimed to develop synthetic, self-replicating RNAs (srRNAs) that could function in both cell-free and intracellular environments. During the project, we successfully developed srRNA scaffolds capable of supporting the replication of large RNA molecules, including large mRNAs and even ribosomal RNAs (rRNAs), without DNA intermediates. This achievement demonstrated for the first time that long, functional non-coding RNAs that are critical for cellular survival can be replicated by RNA replicases in vitro. We also identified essential host factors that modulate RNA replication in a fully reconstituted system. These findings pave the way for synthetic RNA-based gene expression systems that function independently of DNA.
We also established a novel approach for intracellular RNA replication using bacterial host systems. This allowed us to transplant single essential genes onto synthetic RNA constructs, a critical step towards the creation of RNA-DNA hybrid cells. This proof of concept lays the foundation for new gene evolution strategies, including orthogonal translation systems based on RNA genomes.
RiboLife aimed to explore RNA-based genetic storage and replication, leading to significant advancements in synthetic RNA biology and RNA-DNA hybrid systems. Throughout the project, research was structured around three main objectives:
1. Development of artificial self-replicating RNA scaffolds
2. Engineering of self-replicating RNA nanostructures
3. Transplantation of essential cellular functions onto synthetic RNA genomes
A breakthrough of the project was the first successful replication of ribosomal RNAs (23S, 16S, and 5S, including tethered constructs) in a DNA-free system. This result demonstrates that RNA-based genetic replication can sustain core cellular functions, opening new avenues in synthetic ribosome engineering. Additionally, we identified critical bacterial host factors involved in regulating phage RNA replicases, which enabled the first complete in vitro reconstitution of the MS2 replicase complex.
Another major achievement was the development of novel self-replicating RNA scaffolds derived from bacteriophage MS2. These engineered RNAs were designed to carry functional genetic elements, including ribozymes and protein-coding genes, demonstrating their potential as synthetic gene expression platforms. Towards the later stages of the project, we successfully achieved partial intracellular RNA replication, showing that essential genes can be transplanted onto RNA-based genetic carriers, which represents an important step toward RNA-based genome engineering.
An unexpected yet highly promising result emerged when we explored other strategies to create RNA-DNA hybrid systems. We discovered that bacterial retrons can be harnessed to synthesize functional single-stranded DNA aptamers inside cells from RNA templates, a finding that holds significant potential for DNA-based therapeutics, gene silencing, and programmable nucleic acid synthesis.
Taken together, these findings significantly advance the field of synthetic RNA biology by establishing a new platform for RNA-based information storage, gene evolution, and synthetic gene expression. The ability to replicate ribosomal RNA without DNA intermediates provides a framework for designing artificial translation systems, while the retron-based RNA-DNA hybrid approach offers novel tools for intracellular genetic programming. We have published our main findings in peer-reviewed publications in high-impact journals. This includes a recent systematic review on synthetic self-replicating RNAs and their applications. Remaining key challenges that require further research concerns in particular the long-term stability of srRNAs inside living cells. Ongoing experiments aim to build on our discoveries, with potential applications in biotechnology, synthetic biology, and molecular genetics. The results of RiboLife not only provide new insights into the potential of RNA-based genetic systems but also lay the groundwork for future applications in biotechnology, synthetic biology, and molecular therapeutics.
1. Development of artificial self-replicating RNA scaffolds
2. Engineering of self-replicating RNA nanostructures
3. Transplantation of essential cellular functions onto synthetic RNA genomes
A breakthrough of the project was the first successful replication of ribosomal RNAs (23S, 16S, and 5S, including tethered constructs) in a DNA-free system. This result demonstrates that RNA-based genetic replication can sustain core cellular functions, opening new avenues in synthetic ribosome engineering. Additionally, we identified critical bacterial host factors involved in regulating phage RNA replicases, which enabled the first complete in vitro reconstitution of the MS2 replicase complex.
Another major achievement was the development of novel self-replicating RNA scaffolds derived from bacteriophage MS2. These engineered RNAs were designed to carry functional genetic elements, including ribozymes and protein-coding genes, demonstrating their potential as synthetic gene expression platforms. Towards the later stages of the project, we successfully achieved partial intracellular RNA replication, showing that essential genes can be transplanted onto RNA-based genetic carriers, which represents an important step toward RNA-based genome engineering.
An unexpected yet highly promising result emerged when we explored other strategies to create RNA-DNA hybrid systems. We discovered that bacterial retrons can be harnessed to synthesize functional single-stranded DNA aptamers inside cells from RNA templates, a finding that holds significant potential for DNA-based therapeutics, gene silencing, and programmable nucleic acid synthesis.
Taken together, these findings significantly advance the field of synthetic RNA biology by establishing a new platform for RNA-based information storage, gene evolution, and synthetic gene expression. The ability to replicate ribosomal RNA without DNA intermediates provides a framework for designing artificial translation systems, while the retron-based RNA-DNA hybrid approach offers novel tools for intracellular genetic programming. We have published our main findings in peer-reviewed publications in high-impact journals. This includes a recent systematic review on synthetic self-replicating RNAs and their applications. Remaining key challenges that require further research concerns in particular the long-term stability of srRNAs inside living cells. Ongoing experiments aim to build on our discoveries, with potential applications in biotechnology, synthetic biology, and molecular genetics. The results of RiboLife not only provide new insights into the potential of RNA-based genetic systems but also lay the groundwork for future applications in biotechnology, synthetic biology, and molecular therapeutics.
RiboLife has greatly advanced our ability to develop self-replicating RNA systems by capitalizing on the replication machinery of RNA bacteriophages. Using our cell-free prototyping approach, we have been able to extend the coding capacity of synthetic srRNAs beyond the state of the art. This has direct implications for RNA-based gene expression technologies, synthetic biology and vaccine development.
Key breakthroughs include:
-The first demonstration that large messenger RNAs, but also ribosomal RNAs, can be replicated by an RNA-only system, enabling potential applications in ribosome engineering and artificial translation systems.
-The ability to test these RNA genomes for intracellular replication, paving the way for bacteria with hybrid RNA-DNA genomes.
-A new approach for the intracellular synthesis of ssDNA aptamers from RNA that could significantly advance the development of DNA-based therapeutics and sensors.
Our findings provide a new framework for the design of programmable RNA-based genetic systems, opening new avenues for biotechnology, synthetic gene evolution and biomedical applications.
Key breakthroughs include:
-The first demonstration that large messenger RNAs, but also ribosomal RNAs, can be replicated by an RNA-only system, enabling potential applications in ribosome engineering and artificial translation systems.
-The ability to test these RNA genomes for intracellular replication, paving the way for bacteria with hybrid RNA-DNA genomes.
-A new approach for the intracellular synthesis of ssDNA aptamers from RNA that could significantly advance the development of DNA-based therapeutics and sensors.
Our findings provide a new framework for the design of programmable RNA-based genetic systems, opening new avenues for biotechnology, synthetic gene evolution and biomedical applications.