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Look inside a brain cell with Huntington’s disease or ALS and you are likely to find RNA clumped together.

These solid-like clusters, thought to be irreversible, can act as sponges that soak up surrounding proteins key for brain health, contributing to neurological disorders.

How these harmful RNA clusters form in the first place has remained an open question.

Now, University at Buffalo researchers have not only uncovered that tiny droplets of protein and nucleic acids in cells contribute to the formation of RNA clusters but also demonstrated a way to prevent and disassemble the clusters.

Their findings, described in a study published recently in Nature Chemistry, uses an engineered strand of RNA known as an antisense oligonucleotide that can bind to RNA clusters and disperse them.

“It’s fascinating to watch these clusters form over time inside dense, droplet-like mixtures of protein and RNA under the microscope. Just as striking, the clusters dissolve when antisense oligonucleotides pull the RNA aggregates apart,” says the study’s corresponding author, Priya Banerjee, PhD, associate professor in the Department of Physics, within the UB College of Arts and Sciences. “What’s exciting about this discovery is that we not only figured out how these clusters form but also found a way to break them apart.”

The work was supported by the U.S. National Institutes of Health and the St. Jude Children’s Research Hospital.

How RNA clusters form

The study sheds new light on how RNA clusters form within biomolecular condensates.

Cells make these liquid-like droplets from RNA, DNA and proteins — or a combination of all three. Banerjee’s team has researched them extensively, investigating their role in both cellular function and disease, as well as their fundamental material properties that present new opportunities for synthetic biology applications.

The condensates are essentially used as hosts by repeat RNAs, disease-linked RNA molecules with abnormally long strands of repeated sequences. At an early timepoint, the repeat RNAs remain fully mixed inside these condensates, but as the condensates age, the RNA molecules start clumping together, creating an RNA-rich solid core surrounded by an RNA-depleted fluid shell.

“Repeat RNAs are inherently sticky, but interestingly, they don’t stick to each other just by themselves because they fold into stable 3D structures. They need the right environment to unfold and clump together, and the condensates provide that,” says the study’s first author, Tharun Selvam Mahendran, a PhD student in Banerjee’s lab.

“Crucially, we also found that the solid-like repeat RNA clusters persist even after the host condensate dissolves,” Mahendran adds. “This persistence is partly why the clusters are thought to be irreversible.”

Preventing — and reversing — clusters

The team was first able to demonstrate that repeat RNA clustering can be prevented by using an RNA-binding protein known as G3Bp1 that is present in cells.

“The RNA clusters come about from the RNA strands sticking together, but if you introduce another sticky element into the condensate, like G3BP1, then the interactions between the RNAs are frustrated and clusters stop forming,” Banerjee says. “It’s like introducing a chemical inhibitor into a crystal-growing solution, the ordered structure can no longer form properly. You can think of the G3BP1 as an observant molecular chaperone that binds to the sticky RNA molecules and makes sure that RNAs don’t stick to each other.”

In order to reverse the clusters, the team employed an antisense oligonucleotide (ASO). Because ASO is a short RNA with a sequence complementary to the repeat RNA, it was able to not only bind to the aggregation-prone RNAs but also disassemble the RNA clusters.

The team found that ASO’s disassembly abilities were highly tied to its specific sequence. Scramble the sequence in any way, and the ASO would fail to prevent clustering, let alone disassemble the clusters.

“This suggests our ASO can be tailored to only target specific repeat RNAs, which is a good sign for its viability as a potential therapeutic application,” Banerjee says.

Banerjee is also exploring RNA’s role in the origin of life, thanks to a seed grant from the Hypothesis Fund. He is studying whether biomolecular condensates may have protected RNA’s functions as biomolecular catalysts in the harsh prebiotic world.

“It really just shows how RNAs may have evolved to take these different forms of matter, some of which are extremely useful for biological functions and perhaps even life itself — and others that can bring about disease,” Banerjee says.

Understanding how atoms behave has led to many advances, including PET scans that diagnose disease and nuclear energy that powers homes. But our picture of the nucleus, the heart of the atomic world, is still incomplete. Researchers plan to improve our understanding with an advanced new instrument: GRETA, the Gamma-Ray Energy Tracking Array.

The project team, led by the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab), has now completed construction of GRETA’s key components: multiple germanium detector modules, the electronics system, the instrument’s mechanical frame and infrastructure, and the computing systems. The team includes scientists and engineers from Berkeley Lab’s nuclear science, engineering, and computing divisions; Michigan State University (MSU); and Argonne and Oak Ridge national laboratories.

“Our goal was to make the best high-resolution, high-efficiency gamma-ray detector we possibly could, so we can answer big questions about the nature of matter and fundamental forces,” said Paul Fallon, GRETA’s project director. “With every advance in this technology, we can improve our resolving power and can see weaker and weaker structures. GRETA will be 10 to 100 times more sensitive than previous nuclear science experiments.”

GRETA was assembled at Berkeley Lab and is now ready to ship to the Facility for Rare Isotope Beams (FRIB), a DOE Office of Science user facility at MSU. There, researchers will install and commission the device, adding additional detector modules as they become available. GRETA is built for flexibility as well as sensitivity, and will move to different stations at FRIB (and later Argonne) to use particle beams of different types and energies.

To get a glimpse inside the nucleus, researchers will smash a particle beam into a target placed at the center of GRETA, briefly creating energetic, rare atoms. GRETA’s sensitive germanium detectors measure the 3D paths and energies of emitted gamma rays, particles of light made as an excited atom returns to a more stable state. That data can reveal all sorts of interesting insights.

This timelapse video shows the construction and testing of GRETA at Berkeley Lab. (Credit: GRETA collaboration)

For example, researchers can probe the structure of rare and short-lived isotopes, atoms with different numbers of neutrons compared to the more common versions of the elements. FRIB will be able to create and study more than 1,000 new isotopes. Scientists can also test the limits of how many protons and neutrons a nucleus can hold, exploring “the drip lines,” the point beyond which neutrons or protons can no longer bind within the nucleus and instead “drip” away.

Other experiments will study pear-shaped nuclei, a way to search for subtle violations of fundamental symmetries in nature and explore why our universe is made mostly of matter (instead of antimatter). Researchers will also use GRETA to shed light on the processes within stars that forge elements heavier than iron.

“Gamma-ray spectroscopy is among our most powerful tools to learn about the fundamental nature of the atomic nucleus,” said Heather Crawford, a scientist at Berkeley Lab and deputy project director for GRETA. “The excited states and gamma rays are a fingerprint for each isotope. GRETA is the world’s most powerful microscope to examine these fingerprints and answer questions about the nucleus and the forces that govern it.”

A Sphere of Germanium Crystals

GRETA is an expansion of an earlier project, GRETINA, that used 12 germanium detector modules to capture gamma rays. GRETA will bring the total to 30 modules, completing a full sphere around the target and vastly increasing the instrument’s tracking capabilities. By catching more gamma rays, researchers will get a more accurate picture of what’s happening in the nucleus – and more quickly.

Each detector module is made of four tapered hexagonal crystals of ultra-pure germanium roughly the size of a 10-ounce coffee cup. The germanium crystals are such specialized and difficult pieces to make, only about four detector modules can be produced every year. Once tightly packed together and cooled to cryogenic temperatures (around negative 300 degrees Fahrenheit), the crystals are exceptionally good at measuring the energy and position of gamma rays, enabling researchers to reconstruct their interactions in the crystal.

GRETA’s backbone is a complex, meter-wide aluminum frame that supports the germanium detectors and electronics. The sphere is built in two halves that separate, opening space for researchers to change out targets at the center of the instrument. To make sure the sphere comes back together seamlessly, the base plate and rails are aligned within one millionth of an inch. Each half can also rotate, allowing researchers to safely install the detector modules before moving them into their final orientation for operations. The team put the support assembly through its paces with dummy weights last year before beginning integration of the other components.

Fastest Gamma-Ray Detector in the West

A key part of the project was to design compact and efficient new electronics and a dedicated computing system for GRETA. The new electronic system can perform with up to 50,000 signals per second in each crystal, and a dedicated computing cluster will process up to 480,000 gamma-ray interactions every second in real time. Tests this spring showed GRETA surpassing its design goal, processing as many as 511,000 gamma-ray interactions per second.

GRETA is also a potential first use case for an accelerated data pipeline called DELERIA, a new software platform for streaming enormous amounts of data at high speeds. Researchers will be able to transfer data through DOE’s high-speed network, ESNet, to be crunched at supercomputing facilities off-site and returned almost immediately. That rapid feedback will help researchers optimize their experiments as they take place even without a dedicated computing cluster.

GRETA will incorporate GRETINA’s detector modules and replace the instrument as the flagship gamma-ray detector at FRIB. The project team will ship the instrument to FRIB this summer, with installation expected in the fall and first experiments to begin in 2026.