Normal gene activity in the afflicted cells is changed during pathological events. Therefore, obtaining a precise picture of gene activity could hold the key to the creation of new, targeted therapies. Examining genes and the processes they trigger can also be used to determine if these therapies function as intended.
It is understandable why research is concentrated on strategies and tactics that provide specifics about the genetic activity of particular cells. A technique that has just been created by a research team at the University of Würzburg (JMU) represents a substantial advancement over the techniques already in use.
Participants included researchers from the Helmholtz Institute for RNA-based Infection Research (HIRI) and the Institute for Molecular Infection Biology (IMIB). The current issue of the journal Nucleic Acids Research has an overview of the results from their research.
Analysis of a synthetic transcriptome
We have developed a technique that can be used to analyze the translational landscape of a fully customizable synthetic transcriptome, in other words one outside the cell.”
Jörg Vogel, Study Principal Author and Director, Helmholtz Institute for RNA-based Infection Research
Vogel also heads the Institute for Molecular Infection Biology at the Julius Maximilian University of Würzburg.
The abbreviated form of the scientific name for the new method is INRI-seq, or in vitro Ribo-seq.
All of the genes that are active in a cell at any one time are compiled into a transcriptome. It is made up of the total amount of mRNA, which are the carriers of protein blueprints from the cell nucleus to the ribosomes.
The “protein factories” of the cell, or ribosomes, are responsible for translating the nucleotide sequence of mRNA into the amino acid sequence of a protein.
Refinement of comparable methods
INRI-seq is, in theory, an improvement over comparable approaches that aim to do the same thing but yield findings that are less precise or have other drawbacks. For instance, RNA sequencing (RNA-seq) establishes the mRNA concentration in cells, enabling inferences about their active genes.
The final protein abundance, however, does not necessarily relate to the corresponding mRNA concentrations.
Ribosome profiling is a technique that is more precise (Ribo-seq). This has evolved into one of the key techniques for directly assessing protein synthesis on a transcriptome-wide scale over the last ten years.
Vogel stated, “While Ribo-seq has greatly advanced the study of translation-related processes, the method has not been without limitations.”
Numerous limitations of Ribo-seq
Since it is difficult to use Ribo-seq to find poorly expressed genes, many genes cannot be documented in conventional study designs. Similar to this, it is challenging to conduct ribo-seq research on microorganisms from significant ecological environments like the human gut since many of them cannot be cultivated in a lab.
The fact that “on the mechanistic level, Ribo-seq-based studies of molecules affecting translation, such as special antibiotics, can be hampered by cellular responses,” as stated by Vogel, is another drawback. It can be challenging to distinguish between direct and indirect impacts on translation because ribo-seq is done on living cells.
The researchers from Würzburg have created INRI-seq for the global investigation of translation in a cell-free environment to bypass some of these restrictions. To better manage the amounts of each individual mRNA, INRI-seq combines a commercially available in vitro translation system with an in vitro-synthesized, fully customizable transcriptome.
“With INRI-seq, for example, it is no longer necessary for translation-modulating substances to traverse cellular membranes or to extract ribosomes from a large number of living cells,” Vogel added.
He further stated, “You also need a lot less of the often-expensive substance that you want to study, such as a new antibiotic that can only be produced on a small scale. INRI-seq therefore also saves time and money.”
Higher success rate in the experiment
Using a transcriptome of the bacteria Escherichia coli that was artificially created, the research team showed how well the system functions. In a study on living cells, which was technically similar, INRI-seq found nearly four times as many sites where translation processes are started, proving its high sensitivity.
The study team stated, “INRI-seq bears great potential as an alternative method for studying translation process and thus also substances that can influence these processes.”
Hör, J., et al. (2022). INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors. Nucleic Acids Research. doi.org/10.1093/nar/gkac838