The worldwide consideration on the SARS-CoV-2 virus has emphasised the dearth of analytical methodologies for fast and cost-effective identification and characterization of viral RNA. Many virus households, together with pathogenic varieties akin to coronaviruses, human retroviruses, and influenza, are RNA viruses, that means their genetic materials happens within the type of RNA as a substitute of DNA. When these viruses infect host cells, the viral RNA bundle hijacks the contaminated cell’s molecular equipment and begins producing its personal viral molecules, which ultimately overwhelm the cell, releasing new viral molecules to contaminate different cells within the system.
Nearly 20 years in the past, an RNA virus now generally known as SARS-CoV-1 unfold by way of the human inhabitants killing almost 775 individuals who suffered from the extreme acute respiratory syndrome (SARS) that the virus precipitated. Since 2012, Center East respiratory syndrome (MERS), one other illness attributable to an RNA coronavirus, has killed greater than 850 individuals, roughly 35 p.c of these it has contaminated. Scientists had been alarmed by the severity of those viral infections and thru genetic evaluation had been in a position to get an concept of how these viruses infect cells. However the inferiority of RNA-characterizing methods with respect to DNA-focused strategies signifies that researchers’ understanding of the biology and processes related to these infections continues to be arguably nascent.
RNA differs from DNA in that it’s modified post-transcriptionally with an assortment of chemical moieties on its nucleobase and ribose sugar. Up to now, there have been greater than 140 modifications detected in RNA, and whereas the best focus is discovered on switch RNA, which delivers amino acids to the ribosome throughout translation, each RNA studied has contained some stage of modification. Greater than 70 years of research has yielded some understanding of the organic function RNA modifications play. Data of the roles they play in viral RNA, nevertheless, is scant. Analytically, investigation of viral RNA is hindered by various technical challenges, akin to an lack of ability to generate the precise sequence of the viral RNA with the modifications of their correct places.
For instance, the genetic sequence of SARS-CoV-2 was revealed in 2020. The authors of that paper reported the presence of 41 modification websites discovered within the viral RNA sequence. These modifications had been listed as unknown as a result of the truth that RNA sequencing (RNA-seq) can’t immediately establish modifications. RNA-seq enzymatically produces a complementary DNA strand (cDNA). Within the course of of making the cDNA, the enzyme “studying” the RNA stops when it encounters a modification. These arduous stops are exploited to establish the possible presence of a modification, however not its id (methylation, acetylation, and so on.).
This lacking info is essential, particularly within the context of viral RNA, as modifications have been proven in nonviral RNAs to help in structural stability or act as determinants for RNA protein binding, two key attributes for viral RNA an infection and replication. Within the context of SARS-CoV-2 two rapid questions are raised: What are the chemical buildings of the unknown modifications, and the way are they contributing to the virus’s biology?
Since 1985, the gold commonplace for finding out RNA modification has been mass spectrometry. The strategy, developed within the lab of James McCloskey on the College of Utah, can inform not provided that there are modifications current but in addition what the modifications are and the place they exist within the sequence. The 2 largest limitations of mass spectrometry for transcriptomics are, firstly, the necessity for big samples and secondly, ionization inefficiencies. Viral RNA can’t be purified, economically, on the concentrations crucial for full characterization. Moreover, giant molecules akin to intact mRNA can’t simply be analyzed because of the nature of the electrospray ionization; The bigger the molecule the tougher it’s to ionize totally and to take care of the cost throughout the fragmentation course of used to map the modification to its respective place inside the sequence. Nonetheless, mass spectrometry continues to be at the moment the one technique to immediately sequence RNA.
Newer applied sciences akin to nanopore sequencing supply the promise of having the ability to deal with small samples and in addition to learn and find RNA modifications. Nanopore sequencing differs from commonplace RNA-seq in that the RNA transcript is fed by way of a round protein advanced. Because the nucleobases go by way of the pore, electrical indicators will be detected and transformed to sequence ID. The RNA is learn immediately, with out the necessity to generate cDNA. The limitation of nanopore sequencing is the necessity to prepare the system to acknowledge every RNA modification’s signature. This turns into a Herculean job, as every nucleotide’s electrical signature is affected by their neighboring nucleotides. The time and prices related to producing a coaching library for all of the modifications with all of the neighboring nucleotides is prohibitive.
We’re informed the following pandemic is correct across the nook. If so, one other Area Race–kind effort is required, this time with a aim of creating strategies that may extra totally characterize RNA as a substitute of flying to the moon.
Robert Ross is Senior Product Functions Specialist at Thermo Fisher Scientific, engaged on improvement of liquid chromatography tandem mass spectrometry (LC-MS/MS) characterization of nucleic acids. E-mail him at robert.ross2@thermofisher.com.
