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Austrian Researchers, Lexogen Develop and Commercialize Technique for Sequencing Metabolic RNA

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SAN FRANCISCO (GenomeWeb) – Standard RNA sequencing provides information about the relative abundance of transcripts but doesn't provide information about the dynamics of how quickly RNA molecules are made and turned over. But now, researches at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) in Vienna, Austria have developed a sequencing technique that enables these dynamics to be seen.

Vienna-based transcriptomics company Lexogen has licensed the method and launched a sample prep kit based on the technique, which was described this week in Nature Methods.

Stefan Ameres, senior author of the study and group leader in the mechanism and biology of RNA silencing lab at IMBA, said that although RNA-seq is great for "generating a snapshot of the RNA molecules present inside cells and tissue at a given point of time," it has limitations. "It doesn't provide information about how this expression profile is actually generated — how each of these RNAs are made inside the cells and how rapidly they're turned over, which you need to understand gene regulation."

He said that his group made use of a technique known as metabolic RNA labeling and coupled that with next-generation sequencing. Metabolic RNA labeling relies on adding synthetic nucleotides to cells in culture, which then become incorporated into newly transcribed RNAs. However, he said, it can be difficult to identify the synthetic nucleotides.

So, the researchers chemically modified the nucleotide analog in such a way that when they performed the library prep for sequencing, that modified nucleotide would become misincorporated during the reverse transcription step. After sequencing, at every location where that modified nucleotide was incorporated, there would be a specific mismatch.

"That lets you bioinformatically separate out the labeled from unlabeled RNA," Ameres said. Previous methods of metabolic RNA labeling relied on biochemically separating out the labeled from unlabeled RNA, which is "tedious," Ameres said.

The technique — thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) — involves incorporating 4-Thiouridine (S4U) nucleotides into newly synthesized RNA, by growing cultured cells in S4U. This labels the newly made RNA. Then, when RNA is extracted, it includes both the newly made and labeled RNA as well as the previously existing RNA, which is unlabeled. The RNA is then mixed with iodoacetamide, which modifies the labeled RNA nucleotides. During the reverse transcription step, at positions where there is a labeled RNA nucleotide, guanine is incorporated instead of adenine.

Lexogen CEO Alexander Seitz said that the firm has now developed the technology into a kit and it is currently available for customers to order. He said initial interest will likely be in the basic research space from labs studying RNA dynamics and gene expression. But, ultimately, he said it would have significant value in drug and diagnostic discovery.

Being able to "see the gene expression and how it's controlled" is important for drug discovery, he said. And it will enable scientists to better understand gene networks and how genes respond to stimuli. Currently, researchers can evaluate total gene expression in response to a stimuli, but can't distinguish which genes are being expressed as a direct response to that stimuli versus which genes are already being expressed. With SLAM-seq, "you can see the response in a timely manner and assess what's new."

Ameres described the approach as taking a movie of RNA as opposed to a photo of a single point in time. It will "provide temporal resolution to gene expression profiling," he said. Applications in the research field will include studying specific pathways, like the microRNA pathway or epitranscriptome pathway. "You can interfere with the processes and then understand their impact," he said.

In his lab, he said he is interested in using it to study small RNAs, like microRNAs. "We know very little about how rapidly microRNAs are made in cells, such as the key kinetic bottlenecks of forming complexes," he said. His lab is interested in applying SLAM-seq in a targeted fashion to "get a dynamic picture to understand how those processes are regulated inside the cells and coordinated to function efficiently."

He added that the method is a very simple protocol that won't add a lot of extra labor, equipment, or cost. "The most expensive part is still the sequencing," he said. In addition, he said it should be compatible with any RNA sequencing protocol that has a reverse transcription step.

Seitz added that the new product "fits perfectly" with Lexogen's existing array of transcriptomics products. For instance, the SLAM-seq kit can be combined with the company's QuantSeq 3' mRNA-seq library prep kits, which enable as little as 100 picograms of total RNA input.

In fact, Seitz said, the researchers at the Institute of Molecular Biotechnology had been working with Lexogen's QuantSeq kits to develop the metabolic RNA sequencing protocol and reached out to the company. Seitz said such a protocol was "missing from our portfolio and everyone else's portfolio."

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