For the first time in history, the influenza virus gene can finally be detected directly in its original RNA form.

The latest technology allows the flu virus to be sequenced for the first time in the form of its original RNA. The genetic code of the influenza virus, like other viruses, is stored in RNA, so if you want to measure its gene sequence, it can only be obtained by sequencing the reverse-transcribed DNA under the previous limited technology. However, this new invention uses nanopore sequencing technology to directly read out its RNA sequence as it passes through a tiny molecular pump.

For the first time in history, the influenza virus gene can finally be detected directly in its original RNA form.

“This is the first time in history that we can look at the original form of the gene,” says microbiologist John Barnes of the Center for Disease Control and Prevention (CDC) in Atlanta. Barnes led the study and published a pre-release of the paper on BioRxiv on April 12. He said: "This will bring a lot of possibilities for subsequent research."

Barnes and his team are most interested in studying the genes of viruses. Other studies involve RNA in various tissues and organs, as well as RNA in humans. Researchers have long wanted to elucidate their role in cellular function by measuring molecular modifications on RNA, but it has been difficult to conduct such experiments.

“The biggest breakthrough this invention will bring is the ability to discover RNA modifications that are transformative,” said Ewan Birney, co-director of the European Bioinformatics Institute (EMBL-EBI).

Ewan Birney

RNA is very similar to its cousin DNA at the chemical level. In cellular organisms, RNA is a bridge between DNA-encoding genes and proteins and plays other roles in cells. But many viruses, including those that cause Ebola, polio, and the common cold, store their genes in RNA instead of DNA.

Barnes is also the leader of the CDC Influenza Genomics team, and he said that no one has ever done RNA sequencing because it feels almost impossible. In the past, methods for sequencing raw RNA required disruption of the original chemical structure of RNA, or isolated bases one after another, and these methods have changed little since their inception in the late 1970s. However, DNA sequencing has been greatly developed, and accordingly, almost all current "RNA sequencing" uses a reverse transcriptase to reverse transcribe RNA into DNA and then sequence it.

Nanomaterials provide an easier way to sequence RNA. This technique allows the current to pass through a nanometer-scale molecular hole. When the genetic material passes through the aperture, by measuring the amplitude of the current fluctuation, it is possible to know which nucleotide is passed.

In January of this year, researchers at Oxford Nano-holes used a device called MinION to directly measure RNA sequences. Their attempt at this time was aimed at messenger RNA, whose role in the RNA family is to transmit DNA information and translate proteins.

Figure Minion

Barnes' team used the technology in the genome of influenza A, which contains approximately 13500 RNA bases and consists of eight fragments. Barnes said that because the job requires a lot of flu virus and the inevitable sequencing error has to be removed, the original data has to be processed many times, so his team made several attempts to debug the device and the error. The result of the RNA sequence is obtained. But nanotechnology is indeed growing rapidly, and Barnes hopes that with further improvements, direct sequencing of influenza and other RNA viruses can become routine.

On the wish list of Barnes and other scientists, the first is to identify the molecular modifications of RNA. At present, more than 100 molecular modifications have been discovered, but researchers have little knowledge of their effects, in large part because scientists cannot systematically study their molecular transcriptional translation, cellular function, and individual physiology. The role of the level, and the emergence of new nanotechnology is expected to solve this problem.

At present, the technology of the Oxford Nanopore team has been able to directly measure two common RNA modifications and markers. The company's consultant, Birney, analyzed that perhaps the addition of machine learning algorithms to crack the meaning of the markup and find more modifications would make the technology even more useful.

Bryan Cullen, a virologist at Duke University, says sequencing of modified RNA has been a big problem in the field. Last year, his team discovered a marker called m6A that could cause changes in viral gene expression when the virus infects mice, eventually allowing the virus to multiply. However, behind this discovery, there is a lot of time and resource consumption, which is the current status of RNA modification detection.

The advantages of nanotechnology sequencing, in addition to the more convenient measurement of RNA modification, can also reveal the hidden diversity of RNA viral sequences. Stacy Horner, co-director of the Duke University RNA Biology Center, said that compared to other existing technologies, the original result is a rough splicing of a large number of short RNA sequences, so the diversity of the sequence will Lost in the process.

Birney said, "Although this technology is not yet perfect, biologists are still looking forward to the direct measurement of the entire virus gene and other RNA molecules in normal organisms in the future." The smaller the more important, when we use The smaller the material scale, the more direct observation of its morphological function at a level closer to the molecule, the method is simpler and the accuracy is greatly improved. The application of nanomaterials to the direct sequencing of RNA is undoubtedly a major breakthrough in technology.