Scientists have perfected a way to reveal the smallest glitches in the biochemical machinery that makes proteins in our bodies – glitches that can trigger devastating diseases.

With this technique – squeezing molecules through tiny holes in a membrane – the team have identified how a mutation in a transfer RNA (tRNA) molecule affects its real time structure. Until now scientists have struggled to observe the behaviour of molecules in their natural environment.

tRNA is a molecular messenger essential for building proteins, and a mutation in these molecules can disrupt the body’s building and repair processes. Dysregulation of tRNA has been implicated in numerous human conditions, including Alzheimer's disease, Parkinson's disease, cancer, developmental disorders, and even hearing loss.

The new ability to identify problems could enable a better understanding of disease pathways and ultimately more effective treatment, said Professor Patrick Kluth from the Department of Materials Physics.

“We are examining the machinery that builds proteins in the first place. It's like checking the assembly line rather than inspecting finished products - allowing us to catch and understand problems much earlier.

“When even a single nucleotide in the tRNA's sequence changes, just one building block swapped for another, can distort its shape and disrupt protein production. This is particularly damaging in brain cells because they have exceptionally high protein synthesis demands,” Professor Kluth said.

Led by ANU researchers, the team included international partners from Ohio State University in the United States and Jagiellonian University in Poland. Their study is published in Nucleic Acids Research.

The analysis process involves having the sample in a liquid conducting solution, in two small chambers separated by a silicon chip membrane with a nanometer-scale pore in it. A voltage is applied between the chambers, leading to an electric current and pulling the molecules through the pore. While molecules momentarily plug the hole, a dip in the current is created.

With judicious choice of pore size, the size and shape of this dip in the current becomes a molecular fingerprint, because it is strongly dependent on size and shape of the molecule. The smallest change in the molecule’s components can change its shape (known as its conformation) and disrupt its protein translation function.

In this first study, the team compared samples of standard tRNA molecule (known as wildtype), with a mutated version, using a membrane with an 8 nm pore. The team analysed over three million individual tRNA molecules – unprecedented numbers that provided statistical power impossible with conventional methods, said lead author Dr Shankar Dutt from the ANU Research School of Physics.

“This high-throughput capability, combined with the ability to observe molecules one at a time, bridges the gap between detailed single-molecule insights and population-level understanding,” he said.

Unlike existing techniques that require crystallisation or freezing, this approach measures molecules in solution, capturing their natural states. The new technique allowed the team to easily distinguish between the wildtype and the mutant molecules from their current dip profiles.

Not only that, the team could easily follow the dynamics of the conformational changes far faster and cheaper than existing single-molecule methods such as optical tweezers.

To do this, the team introduced magnesium ions to both wildtype and mutant tRNA sample, taking snapshots every seven minutes. Magnesium is important for tRNA to fold and function correctly, but it was clear that the mutant tRNA folded significantly differently under its influence.

“We can directly detect and measure shapes in real time, and see how they change their shape from one form to another, at scale – something which has been impossible until now,” said Dr Dutt.

"For example, we discovered that the mutant tRNA adopts different shapes compared to the wild-type — and importantly, it gets stuck in these alternative conformations even when the cellular environment tries to correct it."

Dr Dutt said the technology is helping researchers understand disease mechanisms.  

“Being able to spot molecular problems at their earliest stage is transformative as it also helps us to understand why diseases occur in first place,” he said.

“More than that, the technology can be used for screening potential therapeutic drugs that stabilize the functional shapes of these tRNA. This could lead to massive advances in treatment of these diseases.”