Scientists at the Federal University of São Paulo (Unifesp) managed, for the first time in Brazil, to directly sequence the RNA of Sars-CoV-2, the virus that causes Covid-19. The results of the research, supported by FAPESP, were published in an article that has not yet been peer reviewed on the bioRxiv platform.
According to the authors, the technique allows mapping the viral genome with approximately 25 times more resolution than conventional sequencing methods. In this way, it is possible to have a more accurate sense of the pathogen’s biology and how its genome is evolving.
“It is very promising, as it allows us to understand, for example, why there are strains that are more virulent or more capable of escaping from our immune system”, says Marcelo Briones, a researcher at the Medical Bioinformatics Center of Escola Paulista de Medicina (EPM) to the Agency. -Unifesp) and research coordinator.
As Briones explains, Sars-CoV-2 is a single-stranded RNA virus, that is, its genetic material is composed of a single strand of nucleotides, whose bases are guanine, adenosine, cytosine and uracil.
To sequence it by the conventional method, a technique known as RT-PCR (reverse transcriptase polymerase) is used to convert RNA molecules into complementary DNA (cDNA)—remembering that the DNA molecule is formed by two strands of nucleotides.
That is, a complementary copy of the RNA strand of the virus is made. These cDNA molecules are then amplified (billions of clones are generated) and sequenced. Among the strategy’s advantages are the speed and the possibility of sequencing even in samples with very little genetic material.
“Conventional sequencing of this virus is like trying to identify a person by looking only at their shadow. With the method used in our study, we can look directly at the viral RNA as it is found in vivo. It’s much more trustworthy”, says the researcher.
Carla Braconi, professor at the Department of Microbiology, Immunology and Parasitology at EPM-Unifesp and co-author of the article, says that the research was carried out with one of the first strains of Sars-CoV-2 isolated in Brazil, in early 2020.
“We received the viral isolate from Professor José Luiz Proença-Módena [da Universidade Estadual de Campinas] and we cultivate the pathogen in vero cells [linhagem celular de rim de macaco altamente suscetível ao Sars-CoV-2]. Then we extracted the viral RNA and sequenced it with a portable technology called MinION, from Oxford Nanopore Technologies”, he says.
According to Briones, RNA is sequenced exactly as it leaves the vero cell, without going through RT-PCR or amplification. “We just ‘hang’ an adapter on the end of the molecule and a cDNA strand so the RNA strand is stretched out. And then only the RNA goes, base by base, in the sequencer. And each type of base [citosina, guanina, adenosina ou uracila] and its modifications, such as methylation, interrupt the electrical flow of the device with a different pattern and that’s how we identify which is which.”
The process produces a graph that resembles an electroencephalogram, which is then interpreted with bioinformatics tools. The final generated sequence can then be compared to reference models.
“Initially, one gets the impression that the obtained sequence has a lot of errors. But actually, they are the modified RNA bases. And part of these modifications go unnoticed by conventional sequencing”, says the researcher.
The analysis, carried out by postdoctoral researcher João Campos, focused on the methylation pattern of the viral RNA. In other words, we sought to look —among the nearly 30,000 bases that make up single-stranded RNA— which received the addition of a methyl radical (CH3).
“This type of biochemical modification in RNA is very important for the proper functioning of viruses such as Sars-CoV-2, as well as some arboviruses. [entre eles dengue e zika] that integrate group 4 in the Baltimore classification system, composed of viral genomes with single-stranded RNA and positive polarity”, says Braconi.
The authors explain that RNAs in general have about a hundred modified bases that are essential for their biological functions. “After Sars-CoV-2 enters the cell and ‘forces’ it to make copies of its genetic material, an enzyme comes along that methylates these RNAs and these modifications start to play a role. They are part of the information the virus needs to survive. Without analyzing this methylation pattern, therefore, it is not possible to know the real genetic richness of Sars-CoV-2”, says Briones.
A frequently modified base in the RNA of Sars-CoV-2 is N6-methyladenosine (m6A), which is implicated in the evasion of the immune response. “This modification allows the virus to escape the interferon activation system [proteínas produzidas por células de defesa com ação antiviral]. It is, therefore, a potential target for drugs and there are already studies in this regard”, says Briones.
If it were possible to create a drug capable of totally blocking the viral RNA methylation process, says the researcher, the new coronavirus would disappear from the cells and would be the end of Covid-19. “The problem is that, if we block methylation too much, the host cells also end up dying, because the enzymes that methylate the viral RNA are the same ones that methylate the RNAs in the cells. So it needs to be something with very specific action.”
The Unifesp group pioneered the direct RNA sequencing of Sars-CoV-2 coupled with the identification of m6A bases. The work was carried out within the scope of the Thematic Project “Investigation of elements induced by the vaccine response in individuals submitted to clinical tests with the ChAdOx1 nCOV-19 vaccine”, coordinated by Professor Luiz Mário Janini. Researchers Juliana Maricato and Fernando Antoneli also participated.
“In two of the previously published works [por grupos do exterior] only RNA sequencing was done. A third party also did direct sequencing, but identified the 5mC base. There is also a fourth work that identified the same m6A base, but using other techniques that do not involve direct RNA sequencing”, informs Briones.
According to the researcher, this detailed understanding of how the genome of the new coronavirus works allows scientists to have a clearer idea of how the pathogen is evolving.
“People who talk about mutations suffered by the virus —which in the strict sense correspond to changes from one base to another in the RNA sequence— are, in my view, stuck in the DNA paradigm. This makes no sense, as this virus works under another logic. It never has DNA in its replicative cycle and therefore to speak of ‘transcripts’ of this virus is absurd. Sars-CoV-2 lives completely in the RNA world”, says Briones, referring to the RNA world hypothesis, according to which the current world, with life based mainly on DNA and proteins, was preceded by a world in which life was based on RNA.
“The level of complexity of the RNA molecule is extraordinary and, with the new technologies that make direct sequencing possible, a new universe of research opens up. We are taking this train at the beginning. There is still going to be a great deal of development, but it is the way forward”, he says.
The next step at Unifesp will be to sequence the genomic RNA of recently identified Sars-CoV-2 variants and investigate whether there are significant differences in the methylation pattern.
The article “Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants” can be read on the bioRxiv platform.