Decoding RNA's Hidden Shapes: A*STAR's Game-Changing Advance

Ribonucleic acid, or RNA, has long been recognized as the messenger that carries genetic instructions from DNA to produce proteins essential for life. However, recent discoveries highlight that RNA's three-dimensional structure plays a critical role in regulating gene expression, influencing everything from protein production efficiency to molecular stability. In a landmark achievement, scientists at Singapore's Agency for Science, Technology and Research (A*STAR) Genome Institute of Singapore (GIS) have unveiled sm-PORE-cupine, a pioneering method that maps these intricate RNA folds at the single-molecule level. Published in the prestigious journal Nature Methods, this breakthrough promises to transform our understanding of RNA's contributions to health and disease.

The method addresses a longstanding challenge: traditional techniques average RNA structures across millions of molecules, masking vital variations in individual RNAs. sm-PORE-cupine changes that by reading full-length RNA sequences one by one, revealing dynamic ensembles of shapes that dictate biological outcomes. For Singapore's vibrant biomedical research scene, this innovation underscores A*STAR's leadership in genomics, fostering collaborations with local universities and paving the way for next-generation therapies.

The Science of RNA Structures: From Messenger to Master Regulator

RNA exists in various forms—messenger RNA (mRNA) transcribes genes, transfer RNA (tRNA) delivers amino acids, and non-coding RNAs fine-tune cellular processes. Yet, RNA is not rigid; it folds into complex three-dimensional shapes influenced by temperature, cellular environment, and chemical modifications. These conformations determine how RNA interacts with proteins, ribosomes, and other molecules, directly impacting protein synthesis rates and RNA lifespan.

In health, precise RNA folding ensures balanced gene expression. Disruptions, however, underpin diseases: misfolded RNA in viruses like SARS-CoV-2 enables rapid replication, while structural anomalies in fungi like Candida albicans promote infections. In humans, aberrant RNA structures contribute to cancer, neurodegenerative disorders, and genetic diseases. Singapore researchers have noted that RNA structural insights could revolutionize diagnostics and treatments, aligning with the nation's push toward precision medicine.

Overcoming Limitations: Why sm-PORE-cupine Stands Out

Prior methods, such as SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or DMS probing, rely on ensemble averaging, losing resolution on heterogeneous populations. Nanopore sequencing offered promise but struggled with full-length reads and precise modification detection. sm-PORE-cupine integrates optimized SHAPE chemistry (NAI-N3 labeling of unpaired bases) with Oxford Nanopore direct RNA sequencing and novel signal alignment via dynamic time warping (DTW).

This rescues over 28% of previously unmappable reads, enabling high-fidelity clustering of structure ensembles using Bernoulli mixture models. The result? Unprecedented views of RNA dynamics in living cells, far surpassing bulk analyses.

Step-by-Step: How sm-PORE-cupine Maps RNA Worlds

1. Chemical Probing: RNA samples are treated with NAI-N3, which covalently binds to flexible, unpaired bases, creating detectable modifications.
2. Direct Sequencing: Nanopore technology threads native RNA through a protein pore, generating electrical signals altered by modifications.
3. Signal Alignment: DTW aligns raw signals to reference sequences, boosting mappability.
4. Modification Calling: One-class SVM identifies modification sites per molecule.
5. Ensemble Clustering: Bernoulli mixture models group molecules by structure profiles, quantifying homogeneity and conformations.

Validated on riboswitches, it distinguishes ligand-bound from free states with near-perfect accuracy.

Key Discoveries: RNA Ensembles in Action

Applied to SARS-CoV-2, sm-PORE-cupine exposed high heterogeneity at the 3' end across subgenomic RNAs, particularly the nucleocapsid gene, suggesting adaptive folding for viral packaging. In C. albicans, in vivo RNAs proved more heterogeneous than in vitro, with coding sequences (CDS) more variable than 3' untranslated regions (UTRs). Notably, 3' UTRs of highly translated transcripts grew homogeneous at 37°C, acting as 'molecular thermometers' for hyphal growth—a virulence factor in infections.

Homogeneous structures correlated with faster decay and efficient translation, validated by luciferase reporters showing up to twofold efficiency changes. These findings link RNA plasticity to fungal pathogenesis and host responses.

Photo by Chris Briggs on Unsplash

Singapore's RNA Research Powerhouse: A*STAR and Beyond

A*STAR GIS, Singapore's genomics hub, drives this innovation amid a $130 million National Initiative for RNA Biology and Applications (NIRBA), launched in 2025. Collaborations with National University of Singapore (NUS) Yong Loo Lin School of Medicine—where some authors hold joint appointments—bridge academia and industry. This ecosystem trains PhD students and postdocs in nanopore analytics, positioning Singapore as an RNA therapeutics leader.

Recent NATi efforts include mRNA manufacturing facilities with Wellcome Leap, enhancing vaccine responses for regional threats like dengue and COVID variants. For universities, sm-PORE-cupine tools empower NUS and NTU labs to probe local disease models, from tropical infections to aging-related disorders prevalent in Singapore's population.

From Lab to Clinic: Therapeutic Horizons

By pinpointing disease-linked conformations, sm-PORE-cupine accelerates RNA-targeted drugs. For instance, stabilizing antiviral folds could curb SARS-CoV-2 evolution; fungal UTR targeting might yield novel antifungals amid rising resistance. Globally, RNA therapies like mRNA vaccines (Pfizer-BioNTech) succeeded against COVID; Singapore's method refines designs for stability and efficacy.

Locally, implications span neurodegenerative diseases (RNA misfolding in ALS) and cancers (structure-driven oncogenes). Experts foresee integration with AI models like A*STAR's RiNALMo for predictive structure-function mapping, slashing development timelines.Read the full Nature Methods paper.

Expert Insights and Validation

"This work lays the foundation for more precise approaches to diagnosis and treatment." – Dr. Yue Wan, A*STAR GIS Executive Director.

"We now have a unique capability to study the dynamics of how RNAs shape-shift." – Dr. Niranjan Nagarajan, A*STAR GIS.

Reporter assays confirmed translation shifts, while thiolutin decay assays linked homogeneity to stability. These rigorous validations affirm sm-PORE-cupine's reliability for transcriptomic studies.

Boosting Singapore's Biomedical Talent Pipeline

Singapore's universities benefit immensely. NUS researchers, co-affiliated with A*STAR, gain access to GIS infrastructure for training. Programs like NIRBA fund PhDs in RNA engineering, addressing talent gaps in genomics. NTU's biotech labs could adapt sm-PORE-cupine for synthetic biology, while SMU's AI expertise enhances clustering algorithms.

Statistics show Singapore's R&D spend at 2.2% GDP, with biomed hub Biopolis hosting 50+ institutions. This breakthrough elevates A*STAR's profile, attracting global collaborators and retaining top minds amid brain drain risks.

Future Outlook: RNA Revolution Ahead

sm-PORE-cupine heralds an era of structure-aware RNA biology. Upcoming applications include human transcriptomes for cancer biomarkers and personalized antivirals. In Singapore, integration with NATi's mRNA platforms could yield homegrown vaccines by 2030.

For aspiring researchers, opportunities abound in A*STAR fellowships and NUS PhDs. As Dr. Wan notes, deeper RNA insights promise transformative health solutions. Explore Singapore's research jobs to join this frontier.Learn more about NATi.

Photo by jerry chen on Unsplash

Stakeholder Perspectives: From Labs to Industry

• Academics: Enables cell-type specific RNA probing, vital for developmental biology.
• Clinicians: Identifies disease-specific conformations for targeted therapies.
• Industry: Pharma giants eye RNA stabilizers; Singapore's NATi accelerates translation.
• Students: Hands-on nanopore training at GIS boosts employability.

Challenges remain: scaling to human cells and integrating with cryo-EM. Yet, Singapore's ecosystem—$20B RIE2025 plan—positions it ideally.