Scientists have solved a long-standing mystery of how living cells distinguish the two types of nucleic acids, RNA and DNA, that make up the genetic information in all life forms. This key insight into the ancient mystery of how cells can tell apart the nearly identical building blocks used in the synthesis of the two nucleic acids can help design more potent and selective inhibitors against viral RNA polymerases (RNAPs)—enzymes that synthesize RNA from a DNA template.

These findings are reported in the article, “The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases,” published in the journal Nature Communications by an international team of scientists from the University of Turku, Finland, and Penn State University.

The synthesis of DNA is carried out by enzymes called DNA polymerases (DNAP) and is needed to accurately transfer the genetic information from generation to generation while the synthesis of RNA is carried out by RNAPs, needed to decode the genetic information to produce proteins that carry out the structural and catalytic functions in the living cell.

The ancient problem faced by RNA and DNA polymerases is that the DNA and RNA building blocks are very hard to tell apart. These building blocks are identical except for a small part of the molecule, a hydroxyl group at the second position of a ring structure (2’OH group) that is present in the RNA building blocks but is absent in the DNA building blocks.

DNAPs avoid using the RNA building blocks through a structurally restrictive inner sanctum or active site (where the enzymatic action happens) of the enzyme, that is just big enough to allow entry of the leaner DNA building blocks but is too small to accommodate the slightly bigger RNA building blocks that have additional 2’OH groups that add to their bulk. Only DNA building blocks can therefore bind to the active site cavity and get incorporated into the growing DNA polymer chain.

“RNA polymerases cannot use the same strategy because the smaller DNA building blocks will always fit into the same active site cavity as the RNA building blocks,” said Georgiy Belogurov, PhD, senior researcher at the University of Turku and senior author on the paper.

The question that had been unanswered until now is: how do RNAPs avoid using the smaller DNA building blocks to make RNA chains?

To solve this ancient mystery, the research team headed by Belogurov performed complex biochemical measurements using RNAPs that were altered by carefully engineered mutations. The research team at Penn State University, led by Katsuhiko Murakami, PhD, obtained a detailed three-dimensional structure of RNAP with the DNA building block.

By the combined analysis of the biochemical and structural data Janne Mäkinen, a doctoral candidate and the first author of the study, and his colleagues discovered that RNAPs evolved an active site cavity that warps the DNA building blocks so that they are no longer suitable for incorporation into the RNA chain.

“The deformed DNA building blocks then dissociate from the RNAPs instead of being attached to the growing RNA polymer,” said Mäkinen.

This study was funded by the Academy of Finland, Sigrid Juselius Foundation (Finland), and the National Institute of Health (USA) and has long-reaching implications for translational research.

“RNA viruses such as SARS-Cov-2, the causative agent of COVID-19, also synthesize RNA as a part of their infectious cycle. Viruses use their own RNA polymerases that are very different from RNA polymerases of the human cell but also need to select the RNA building blocks and reject the DNA building blocks,” said Belogurov.

The study concluded that viral and human RNAPs use different mechanisms to reject the DNA building blocks. This indicates it may be possible to design a synthetic molecule similar to a DNA building block that would selectively bind and inhibit viral RNAPs but will be rejected by the human RNAPs and therefore will not interfere with the synthesis of RNAs needed by the human cell.

“This paves the way for the designing of potent and selective antiviral drugs targeting viral RNA polymerases,” said Belogurov.