Dickinson Lab Maps RNA Molecules with Novel Proximity Labeling Method
Cells are like busy rooms filled with tiny molecules called biomolecules. In the intricate world of cellular biology, understanding the spatial organization of biomolecules is like having a detailed map of the cellular world that not only helps scientists unravel the complexities of life, but also holds immense potential for improving healthcare, advancing scientific discoveries, and driving innovation in biotechnology.
Today, with techniques such as proximity-based labeling, scientists use special tools to tag or label specific biomolecules that are close to each other within a cell. This helps scientists figure out how biomolecules interact and work together inside cells.
Today’s advancements in proximity-based labeling techniques have opened new avenues for researchers to explore these complex structures. In a recent paper published in April in Nature Chemistry,the Bryan Dickinson Lab has introduced a new set of tools that specifically allows them to see which RNA molecules are found inside cells, allowing them to study RNA's spatial organization in detail.
" A living cell has a lot of biomolecules, and it is not a homogenous mixture. The precise location of these biomolecules inside a cell often dictates its function. So, you want to understand how different biomolecules are placed in different regions or compartments of the cells,” said Shubhasree Pani, a graduate student of the Dickinson Lab who, along with co-first author Dr. Tian Qiu, shed light on their innovative methods and the implications for scientific discovery.
Pani gave this analogy about the implications for their scientific discovery:
Imagine tracking a person’s daily routine through Google locations, identifying their commute to work every morning, and visit to a taco truck at noon for lunch. To an observer, this would give insight into a person’s habits and preferences. Similarly, in biology, there are numerous non-coding RNAs whose functions remain unclear despite their identification through advanced sequencing.
“Our method, which can precisely locate these RNAs within cells, allows us to infer their potential activities,” said Pani. “These discoveries help us generate hypotheses about the functions of various RNAs, contributing significantly to scientific exploration and understanding cell dynamics. It's akin to finding clues that illuminate the intricate mechanisms within cells.”
Traditionally, methods like unbiased RNA mapping faced challenges due to biases towards certain nucleotides. The group says that this bias limited the scope of earlier research, emphasizing the need for refined, unbiased methods.
“One of the major reasons it has been biased in the past was because all the chemistries used to study this were based on radical chemistry,” said Qiu. “A radical chemistry approach is designed to be more reactive towards certain bases, but the way we envisioned our probe to react with 2' hydroxyl group based on acylation reaction which is present at every base.”
The research team's innovative approach involved developing a chemical process to control the release of acylating agent using a non-radical chemistry. This novel chemistry allowed for labeling of RNA biomolecules in a proximity-dependent manner, representing a unique signature from earlier radical-based approaches.
This breakthrough not only offers a deeper understanding of cellular organization but also paves the way for future discoveries in RNA biology and beyond.
"With more methods like this, we are more likely to notice what particular types of RNA might be doing, " said Pani.
To develop an unbiased method, the group had to develop their own tools along a path that was fraught with challenges. They highlighted the hurdles faced, from developing new reactions to adapting profiling methods.
“It was tricky because the chemistry methods we needed weren't readily available and we had to figure out how to build a chemical reaction that hadn't been reported before.
As there was little research to help develop their method, Qiu tirelessly screened until the right reaction was found. When the group did locate the desired reaction, they then had to figure out how to extract and sequence the RNA.
Overcoming these challenges required rigorous experimentation and diligent fine-tuning of their methods, but the scientists remained steadfast.
“We must make it work. We have to try do it,” said Pani.
Finally, when the right reaction had been found, Pani described what it was like when she finally saw the fruit of their efforts come to light.
“I remember the first time in the dark microscope room, I saw the labeling using our acid-chloride probes, and it was so bright. We didn't have to analyze and go back further to know that it is proximity-dependent labeling. Looking directly at it, you're seeing and believing. It was kind of an event.”
Their work now represents a paradigm shift in proximity-based labeling techniques, offering scientists unprecedented tools to explore the intricate world within our cells. With ongoing projects in cell surface proteomics and potential applications in metabolomics, Pani and Qiu say they stay excited about what the future holds for their research.
Citation - Pani, S.*, Qiu, T.*, Kentala, K. et al. Bioorthogonal masked acylating agents for proximity-dependent RNA labelling. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01493-1 (* denotes equal contribution)