The company aims to make RNA molecules visible and druggable – opening the door to study RNA-based diseases.
Lucerna President and Co-founder Dr. Karen Wu/Courtesy of Lucerna Inc.
Apparently, the key to visually seeing RNA, the elusive cousin of DNA, is Spinach. Or Broccoli, or Corn. Maybe even pepper.
These “vegetables” make up the fluorescent technology developed by the biotechnology company Lucerna, Inc. Led by Dr. Karen Wu, Ph.D., the company aims to make RNA molecules visible and druggable – opening the door to study RNA-based diseases, to develop therapies and to simply understand how RNA works inside the cell.
Born from the laboratory of Dr. Samie Jaffrey at Cornell University, Lucerna’s Spinach technology is an RNA aptamer or an oligonucleotide sequence that can bind to and activate a specific molecule. The aptamer mimics the activity of green fluorescent protein (GFP). In GFP, a beta-barrel (protein sheets intertwined in a cylindrical structure) contains a tightly bound fluorophore that can only release energy through fluorescence.
“If you look at our fluorophores, they look almost identical to the fluorophore inside GFP,” Wu explained. “We then evolved an RNA sequence that binds to it tightly, similarly to GFP, so that the compound can only release energy through fluorescence.”
By screening billions of RNA molecules, scientists discovered the most optimal RNA aptamer sequence that would induce the highest level of fluorescence when selectively bound to the fluorophore. After attaching this aptamer tag to an RNA sequence of interest, the resulting plasmid can be genetically encoded inside a mammalian cell. The tagged RNA transcript then binds the fluorophore inside of the cell and generates bright fluorescent signals that can be easily detected through microscopy. This technology allows scientists to view in real-time where exactly the RNA of interest is located and how it travels inside the cell, via the aptamer’s fluorescence.
One advantage of this approach, as described by Wu, is the specificity of the aptamer tag. Since the selectively overexpressed RNA of interest is the only one attached to a fluorescent tag, any fluorescence seen inside the cell can be attributed to that specific RNA.
Being able to tag and visualize the precise activity of RNA molecules would be extremely informative for a wide variety of diseases. “I would like to say that almost every single disease is affected by RNA,” Wu mused. “Even if the disease is protein-based, the RNA comes first before the protein translation. Now, scientists are thinking one step forward – if they can’t drug the protein, they might be able to drug the RNA. If there’s no RNA, then there’s no protein.”
One example of such an RNA-based illness is Huntington’s disease, a neurodegenerative disorder characterized by “repeat expansion,” where the huntingtin gene uncontrollably expresses transcripts that contain repeats of three RNA nucleotides (in this case, the sequence CAG). In addition to translating into mutant proteins, these repeat-containing transcripts also form sticky RNA clumps, called foci, that trap important cellular proteins to cause massive cell death or dysfunction.
In the case of these repeat expansion diseases, drug screening often focuses on finding candidates that can destroy the mutant protein. Wu explained that another approach is looking for candidates that will destroy the repeat RNA itself. “That’s why we decided to tag these [repeat] transcripts,” she said. “You can see really bright dots in the cell where the toxic RNA foci are formed. When we add drugs, you can actually see the dots go away – so, you know that the drug is dissolving the foci and doing its job.”
Using this technique, the company aims to develop high-throughput drug screening assays to identify strong candidates for RNA-based diseases. By expressing a Spinach tagged repeat RNA sequence inside a cell, scientists can now visually see how effective a drug is in eliminating the RNA foci in real-time. And, because the Spinach tag does not negatively affect cell survival, scientists can follow these cells over several hours or even days.
Conveniently buoyed by growing public interest in RNA garnered through the COVID-19 pandemic, Lucerna has also focused its efforts on developing drug screening assays to detect anti-viral drugs. Here, the approach is beautifully simple. First, a Broccoli (the brighter version of Spinach) tag is added to a viral promoter that is responsible for generating new viruses through RNA transcription. The more the virus replicates, the more Broccoli is made and the stronger the fluorescent signal becomes. Conversely, if the virus stops replicating, the fluorescent signal disappears as Broccoli is eventually degraded by the cell.
“When we drug it, you can see a decrease in the Broccoli signal,” Wu said. “And that’s how we know if the drug works or not. We’re basically looking at RNA transcription activity through our technology.”
Lucerna has also adapted its aptamer technology to create RNA sensors, where a probe sequence attached to the aptamer tag is delivered inside a cell to bind to, or hybridize, specific endogenous sequences inside the cell. “The sensors can only fluoresce when they detect whatever the target of interest is,” Wu said. “The sensor will bind to the target sequence very specifically, and only upon that binding will the Spinach tag be activated and fluoresce.”
Ultimately, Wu hopes to leverage Lucerna’s technology to allow scientists to see and drug all RNA. After all, seeing can be a prerequisite to understanding. As Wu sees it, science has rapidly traversed the genome and proteome – now, it seems to be the transcriptome’s turn. “I’m really excited that this will be the new RNA world, I think, in the near future,” she said with a smile.
