From the same sheet of paper, origami’s intricate folds can create diverse forms: a hopping frog, a gliding plane, or a rolling ball. Likewise, molecules of ribonucleic acid (RNA) can be folded into a range of shapes with different properties. These structural variations are thought to influence how RNA regulates a myriad of cellular processes by controlling the molecule’s stability, localisation and activity.
However, unlike origami, the secrets of RNA folding and their links to RNA function can be more difficult to unravel. “Current approaches to RNA structure studies need tens of millions of cells as starting materials,” said Yue Wan, a Principal Investigator at A*STAR’s Genome Institute of Singapore (GIS).
These limitations have hindered the study of RNA in rare cell types and heterogeneous cell populations from tumours, or biological samples with limited material.
In collaboration with GIS Junior Principal Investigator Jiaxu Wang and Senior Scientist Roland Huber from the Bioinformatics Institute (BII), Wan’s group devised new approaches to study RNA structure at the single-cell level. They aimed to gain insights into the determinants of cell identity and how RNA structure contributes to cellular functions and diversity.
This work led to the development of single-cell structure probing of RNA transcripts (sc-SPORT), a new method of RNA structural analysis designed to simultaneously map RNA secondary structure and gene expression at a single-cell resolution.
Unlike conventional approaches, sc-SPORT determines RNA structures in individual cells by chemically modifying fragments of unfolded RNA; isolating and sequencing those fragments; and analysing DNA copies to understand how the related RNA folds influence cell functions. This approach proved remarkably sensitive, enabling researchers to use single cells as starting material for sc-SPORT.
Using sc-SPORT to examine human embryonic stem cells, Wan and colleagues discovered that their RNA had mostly uniform structures, suggesting they had consistent roles in the early stages of cell development. However, those structures became more diverse as the same stem cells differentiated into neuronal precursor cells—a crucial step towards becoming fully developed neurons. This variation in RNA folding patterns highlights a complex layer of gene regulation that becomes more pronounced as stem cells become specialised.
“Beyond RNA expression, its structure provides an added layer of information on cellular identity,” said Wan, adding that sc-SPORT could aid studies on rare cell types—cancer stem cells, organoids, embryo cells and short transit cells among them—of which very limited samples tend to be available.
Moving forward, Wan’s team plan to optimise sc-SPORT to scale up the number of cells it can analyse per run. They also aim to combine sc-SPORT with spatial omics to develop a spatial RNA structure sequencing approach, allowing scientists to track RNA structures in the context of intact tissue.
The A*STAR-affiliated researchers contributing to this research are from the Genome Institute of Singapore (GIS) and the Bioinformatics Institute (BII).
