(Image description and credits: The binding arms on a DNAzyme (red) specifies where it binds to an RNA strand (yellow), which is then cleaved.
Credit: HHU/Manuel Etzkorn)
DNAzymes are precision biocatalysts with the ability to destroy unwanted RNA molecules. However, major obstacles to their use in medicine remain. The way DNAzymes work in real-time was now investigated with atomic-resolution by a multidisciplinary team including the Jülich Research Centre (FZJ) and the University of Bonn, a research team from Heinrich Heine University Düsseldorf (HHU), and Aldino Viegas, currently Assistant Researcher at the (Bio)molecular Structure and Interactions by NMR Lab at UCIBIO-NOVA. The important fundamental findings and their application were now published in the renowned journal “Nature”: Time-resolved structural analysis of an RNA-cleaving DNA catalyst.
DNAzymes – a word made up of DNA and enzyme – are catalytically active DNA sequences. They consist of a catalytic core of around 15 nucleic acids flanked by short binding arms on the right- and left-hand sides, each with around ten nucleic acids. While the sequence of the core is fixed, the binding arms can be modified to specifically match virtually any RNA target sequence.
The aim is to target unwanted RNA molecules of viruses, cancer, or damaged nerve cells, using DNAzymes to attack and destroy them. This is achieved via binding sequences that match a sequence of nucleotides on the targeted RNA molecule. The DNAzyme docks precisely to the matching position and the core cleaves the RNA molecule, whose fragments are then quickly degraded in the cell. The binding arms can be quickly and easily tuned to a wide range of RNA targets.
The benefits are obvious: unwanted RNA can be destroyed selectively, entailing a substantial therapeutic and biotechnological potential. For instance, in some viruses, like SARS-CoV2 and Ebola, the genetic material is coded on an RNA molecule. Additionally, like healthy cells, also cancer cells use the so-called messenger RNA (mRNA) to copy the blueprints for proteins from their DNA and transfer them to the molecule factories. The mRNA sequence in cancer cells is often slightly different from that of healthy cells, meaning that DNAzymes can be used to specifically attack cancer cells while sparing others.
“What sounds outstanding in theory and was already been proposed 20 years ago, unfortunately, doesn’t work like that in medical practice,” says Manuel Etzkorn, working group leader at the HHU Institute of Physical Biology and last author of the study. “In a test tube, the DNAzymes are highly effective at destroying the RNA molecules, but this rarely happens in a cell. There must be a competing process that blocks the DNAzymes. However, without a fundamental understanding of how they function, it is very difficult to develop improved DNAzyme variants that can accomplish their work in cells. Our insights have now brought movement into this deadlocked situation.”
In this study, the authors sought to understand how the system as a whole functions, what steps occur in the binding and cleaving process and what cofactors support the reaction. The researchers observed the processes at atomic resolution and in part in real time using high-resolution nuclear magnetic resonance (NMR) spectroscopy. This enabled them to depict the three-dimensional atomic arrangement assumed by the DNAzyme to bind to and cleave the RNA: The core wraps around the RNA strand in a highly effective way, cleaving it into two pieces in several intermediate steps. After cleaving, the DNAzyme releases the fragments and can bind again elsewhere.
Jan Borggräfe, doctoral researcher in Etzkorn’s working group and first author of the study, explains why the DNAzymes do not work well in cells: “We established that magnesium, as a key cofactor, plays various essential roles in the mechanism, but that it binds relatively poorly and only briefly to the DNAzyme. There are other components in the cell with a greater affinity for magnesium that “steal” the magnesium from the DNAzyme so to speak.”
Aldino Viegas, co-author of the work says that “the presented atomic-level mechanistic description provides a strong base to overcome the current limitations of in vivo DNAzyme applications and might lay the foundation for the knowledge-based development of next-generation Dz-derived therapeutics.”
The next step is to conduct structural investigations into cell cultures and organoids. The goal for therapeutic applications is to improve the magnesium affinity of the DNAzymes through targeted modifications to increase their activity in biological tissue.
(adapted from the Heinrich-Heine University Duesseldorf Press Release)
Original publication:
Jan Borggräfe, Julian Victor, Hannah Rosenbach, Aldino Viegas, Christoph G.W. Gertzen, Christine Wuebben, Helena Kovacs, Mohanraj Gopalswamy, Detlev Riesner, Gerhard Steger, Olav Schiemann, Holger Gohlke, Ingrid Span, Manuel Etzkorn. Time-resolved structural analysis of an RNA-cleaving DNA catalyst, Nature (2021).
DOI: 10.1038/s41586-021-04225-4