A bioluminescent tag that can be attached to RNA will enable scientists to track RNA in real time as it moves through the human body. To understand the central role of RNA in cellular processes, scientists must be able to track RNA in vivo. The new platform will allow scientists to observe the dynamics of RNA in numerous biological processes, from virus propagation to memory formation, in real time. The bioluminescent RNA tag was developed by researchers at the University of California, Irvine. The researchers used luciferase, the enzyme that induces bioluminescence in fireflies, glowworms, jellyfish, and other organisms to make the tags. They engineered the RNA tags to recruit light-emitting luciferase fragments, called RNA lanterns, upon the transcription of DNA into RNA. Professor Jennifer Prescher (left) and professor Andrej Lupták collaborated on developing a new technique for attaching bioluminescent molecules to RNA, which enables RNA to be tracked in real time as it moves through the body. The technique could be beneficial in a range of health applications, such as detecting viruses or mapping memories in the brain. Courtesy of Lucas Van Wyk Joel/University of California, Irvine. The RNA lanterns bind proteins containing the RNA bait to assemble a functional, light-emitting enzyme. The researchers optimized the RNA lanterns and RNA bait to maximize signal turn-on and minimize the size of the protein-RNA complex. The optimized design significantly decreases the size of previously reported RNA-protein complexes for bioimaging. The researchers observed robust photon production in the bioluminescent tags for RNA targets both in cells and in live animals. They found that only a single copy of the RNA bait was required for sensitive detection and imaging of target transcripts in cells and whole organisms. The bioluminescent tags will enable scientists to visualize RNA dynamics in vitro and in vivo, broadening their view of RNA biology in living systems. For example, the tags could be used to observe how a virus penetrates the body’s defensive mechanisms. By tagging the viral RNA, researchers could track and image the virus as it propagated in the body, infecting cells with its RNA. The bioluminescent tags could also be used to track the RNA in brain cells to allow real-time imaging of RNA dynamics in the living brain. According to professor Jennifer Prescher, RNA appears to play a key role in the formation of memories in the brain. “There’s a lot of interesting biology that’s happening at the RNA level in neurons,” she said. “Being able to see early events and the transport of RNA from the cell body out to neural synapses where connections are being made to other neurons — that directly correlates with memory formation. If you have a way to watch that in real-time, that could tell you something fundamental about the brain and memory, which has been a holy grail in science for a long time.” While researchers have a molecular-level understanding of many facets of RNA biology in vitro, in live animals, the picture is not as complete. This is due, in part, to a lack of methods for noninvasive tracking of RNA in vivo. Two human cells making RNA are made visible by the bioluminescent lantern in pink (middle). The same cells make a fluorescent protein that is distributed evenly throughout the two cells (left). An overlay of the two images shows that the RNA is distributed unevenly and is concentrated at specific locations (right). The bar in the lower right corner of the third image represents 20 μm. Courtesy of University of California, Irvine. “It turns out it’s been really quite difficult to know in living cells, and especially in living organisms, when RNA is turned on and where it goes,” professor Andrej Lupták said. “If you wanted to study the first 30 seconds or the first minute — nobody knows. But we provide a tool. You can now visualize it.” Conventional approaches to observing RNA in vivo rely on fluorescent probes that require excitation, which can be difficult to deliver in whole organisms without invasive procedures. Also, external light can induce autofluorescence, reducing detection sensitivity. Short imaging times are necessary to avoid light-induced damage to tissue. Because bioluminescence does not require excitation light, it can provide superior signal to noise ratios in vivo for visualization of low-copy transcripts. It can support serial imaging without risking phototoxicity or tissue damage. The bioluminescent tags provide a platform for visualizing RNA — the molecule that reads the genetic information stored in DNA — from the micro to the macro scale. The RNA bait is modular and can be used with other split luciferases and for multiscale imaging. Further tuning of both the RNA lantern and tag could increase the number of transcripts that can be visualized in tandem. The bioluminescent tags could advance medical research by providing insight into how RNA, critical for cell functioning, can affect human health. “The first step in saying something’s going to happen in a cell — the cell is going to grow, adapt, change, or anything like that — underlying all of that is RNA,” Lupták said. The research was published in Nature Communications (www.doi.org/10.1038/s41467-024-54263-5).