Scholars at the Antimicrobial Resistance (AMR) interdisciplinary study group of the Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research organization in Singapore, have created a robust instrument capable of examining thousands of biological specimens to identify transfer ribonucleic acid (tRNA) alterations — minute chemical modifications to RNA molecules that influence how cells grow, adapt to stressors, and react to illnesses such as cancer and antibiotic-resistant infections. This instrument unveils new opportunities for science, healthcare, and industry — from expediting disease research and fostering more accurate diagnostics to assisting in the development of more effective medical therapies for conditions like cancer and antibiotic-resistant infections.
For this investigation, the SMART AMR group collaborated with scholars from MIT, Nanyang Technological University in Singapore, the University of Florida, the University at Albany in New York, and Lodz University of Technology in Poland.
Tackling existing challenges in RNA modification profiling
Cancer and infectious conditions are intricate health issues where cells are compelled to operate abnormally due to mutations in their genetic makeup or directives from invading microbes. The SMART-led research team is among the global frontrunners in understanding how the epitranscriptome — the over 170 diverse chemical alterations of various forms of RNA — governs the growth of normal cells and how cells react to adverse environmental changes, such as nutrient depletion or exposure to toxic substances. The investigators are also exploring how this system is disrupted in cancer or utilized by viruses, bacteria, and parasites in infectious diseases.
Current molecular techniques employed to examine the vast epitranscriptome and all the thousands of various forms of modified RNA tend to be slow, labor-intensive, expensive, and involve hazardous chemicals, which curtails research capacity and velocity.
To address this issue, the SMART team devised a new instrument that facilitates rapid, automated profiling of tRNA modifications — molecular changes that govern how cells endure, adapt to stress, and react to diseases. This functionality allows scientists to chart cell regulatory networks, discover new enzymes, and associate molecular patterns with disease mechanisms, paving the path for enhanced drug discovery and development, as well as more precise disease diagnostics.
Deciphering the intricacies of RNA modifications
SMART’s publicly accessible research, recently published in Nucleic Acids Research and titled “tRNA modification profiling reveals epitranscriptome regulatory networks in Pseudomonas aeruginosa,” demonstrates that the tool has already facilitated the discovery of previously unidentified RNA-modifying enzymes and the mapping of intricate gene regulatory networks. These networks are essential for cellular adaptation to stress and illness, offering valuable insights into how RNA modifications govern bacterial survival mechanisms.
Utilizing robotic liquid handlers, investigators extracted tRNA from over 5,700 genetically altered strains of Pseudomonas aeruginosa, a bacterium responsible for infections such as pneumonia, urinary tract infections, bloodstream infections, and wound infections. Samples underwent enzymatic digestion and were assessed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), a method that separates molecules based on their physical characteristics and identifies them with high precision and sensitivity.
During the study, this process generated over 200,000 data points in a high-resolution approach that unveiled new tRNA-modifying enzymes and simplified gene networks governing how cells respond and adjust to stress. For instance, the data indicated that the methylthiotransferase MiaB, one of the enzymes responsible for tRNA modification ms2i6A, was sensitive to the availability of iron and sulfur and to metabolic changes when oxygen levels drop. Discoveries like this illuminate how cells react to environmental challenges, and could lead to future advancements in therapies or diagnostics.
SMART’s automated system was meticulously crafted to quickly and securely profile tRNA modifications across thousands of samples. Unlike traditional approaches, this tool merges robotics to streamline sample preparation and analysis, eliminating the necessity for hazardous chemical handling and cutting costs. This enhancement augments safety, throughput, and affordability, enabling routine large-scale application in research and clinical laboratories.
A quicker and automated method to examine RNA
As the first system capable of quantitative, system-wide profiling of tRNA modifications at this scale, the tool offers a unique and comprehensive perspective of the epitranscriptome — the total set of RNA chemical modifications within cells. This capability empowers researchers to validate hypotheses regarding RNA modifications, uncover new biological insights, and pinpoint promising molecular targets for the creation of innovative therapies.
“This groundbreaking tool signifies a transformative leap in decoding the intricate language of RNA modifications that govern cellular responses,” states Professor Peter Dedon, co-lead principal investigator at SMART AMR, professor of biological engineering at MIT, and corresponding author of the paper. “Utilizing AMR’s knowledge in mass spectrometry and RNA epitranscriptomics, our research reveals new techniques to detect complex gene networks crucial for comprehending and treating cancer, as well as antibiotic-resistant infections. By facilitating swift, large-scale analysis, the tool accelerates both fundamental scientific discovery and the creation of targeted diagnostics and therapies that will tackle pressing global health challenges.”
Expediting research, industry, and healthcare applications
This adaptable tool possesses extensive applications across scientific research, industry, and healthcare. It allows for large-scale investigations of gene regulation, RNA biology, and cellular reactions to environmental and therapeutic challenges. The pharmaceutical and biotechnology sectors can leverage it for drug discovery and biomarker screening, efficiently assessing how potential drugs influence RNA modifications and cellular behavior. This assists in the formulation of targeted therapies and personalized medical treatments.
“This is the initial tool that can expediently and quantitatively profile RNA modifications across thousands of samples,” remarks Jingjing Sun, research scientist at SMART AMR and primary author of the paper. “It has not only enabled us to identify new RNA-modifying enzymes and gene networks, but also opens the pathway to pinpointing biomarkers and therapeutic targets for diseases like cancer and antibiotic-resistant infections. For the first time, large-scale epitranscriptomic analysis is feasible and attainable.”
Looking forward: enhancing clinical and pharmaceutical applications
Going forward, SMART AMR intends to broaden the tool’s functionalities to analyze RNA modifications in human cells and tissues, transitioning beyond microbial models to enhance understanding of disease mechanisms in humans. Future initiatives will concentrate on integrating the platform into clinical investigations to hasten the discovery of biomarkers and therapeutic targets. The adaptation of the technology into a tool for epitranscriptome-wide analysis that can be utilized in pharmaceutical and healthcare environments will propel the advancement of more effective and personalized treatments.
The research conducted at SMART is supported by the National Research Foundation Singapore under its Campus for Research Excellence and Technological Enterprise program.