DNA as template for ribosomal translation?

Every living cell possesses a genome made up by DNA. For protein synthesis the genes on the DNA must be transcribed into RNA, then the genetic information is handed over to specialized machines in the cell the ribosomes. There the so-called messenger RNA (mRNA) is translated into a protein, which is a sequence of amino acids. The flow of genetic information from DNA to RNA to protein was already described in 1958 by Francis Crick. The aim of this project is to find a shortcut within this central dogma of molecular biology, to allow the ribosomes to translate a DNA instead of an RNA as the carrier of the genetic information. First speculations about the possibility to use DNA as a translation template came from studies in the 1960s. It was observed that under certain conditions, like the presence of antibiotics which induce errors in translation, single stranded DNA was accepted for protein synthesis. The target site of these drugs is the 16S ribosomal RNA, which is responsible for the decoding process of an mRNA during translation. Another hint came from the observation that so-called transfer RNAs (tRNAs) responsible for the delivery of the amino acids are not binding as efficiently to the ribosome mRNA complex when using an mRNA harboring a single DNA codon. This defect in binding could be rescued by the addition of error inducing antibiotics. Since also mutations in ribosomal RNA and ribosomal proteins can lead to miscoding events in translation, this project aims to generate mutated ribosomes that can fulfill the task of reading and correctly translating an mRNA harboring one DNA codon into a protein. The place for inserted mutations within the 16S rRNA will be either completely randomized, or alternatively it will be focused on essential regions, that are directly involved in either decoding or binding of aminoacyl- tRNAs. By applying a distinct selection system, mutated ribosomes that are able to translate the DNA codon within an mRNA, can be separated from those molecules, which cannot and get stuck on the DNA codon during protein synthesis. Within a pool of millions of potential ribosomal mutations this method allows to fish, identify and further characterize those, which were successful in the first step towards shortcutting the central dogma of molecular biology.

Every living cell has a genome made up of DNA. For protein synthesis, the genes on the DNA have to be transcribed into RNA, and then the genetic information is handed off to specialized "machines" in the cell - the ribosomes. There, the messenger RNA (mRNA) is translated into a protein, which is a sequence of amino acids. The flow of genetic information from DNA to RNA to protein was already described by Francis Crick in 1958. The goal of our project is to find a shortcut in this process: instead of going through RNA, can ribosomes read DNA directly to make proteins? First speculations about the possibility of using DNA as a translation template came from studies in the 1960s. It was observed that under certain conditions, such as the presence of antibiotics that induce translation errors, single-stranded DNA is accepted for protein synthesis. Another hint came from the observation that so-called transfer RNAs (tRNAs) responsible for the delivery of the amino acids are not binding as efficiently to the ribosome - mRNA complex when using an mRNA harboring a single DNA-codon. This defect could also be corrected by the addition of error-inducing antibiotics. Since also mutations in ribosomal RNA and ribosomal proteins can lead to miscoding events and potentially alter the binding of tRNA during translation, this project aims to generate mutant ribosomes that can fulfil the task of reading and correctly translating an mRNA harboring a single DNA-codon into a protein. During the course of this project, we explored different strategies to separate ribosomes able to decode DNA from those that are not. Multiple selection protocols based on non-stop mRNAs, stalling sequences or omission of essential proteins were tested and established. At the same time, expression libraries of ribosomes carrying random mutations throughout the entire 16S rRNA were generated. For a more rational approach, we also created targeted mutations in certain areas of the 16S rRNA that are involved in the decoding process, which have been shown to make the decoding step more flexible - potentially allowing these ribosomes to tolerate DNA-codons. To follow the selection process and identify promising mutant ribosomes, we established a sensitive Nanopore sequencing protocol for isolated 16S rRNA. At the end of the project, we attempted a first full selection cycle, but failed to identify any potential candidates. While the funding period of this project has ended, work is continuing to finally answer the question of whether ribosomes can be made to translate DNA. Although we have not yet achieved our ultimate goal, our efforts on this project have led to interesting spin-off projects and publications based on the tools and insights we developed along the way.