Next-generation sequencing has revolutionised our understanding of the genome and transcriptome by providing sequence information at unprecedented depth and scale. Yet, the functional output of DNA and RNA is defined not solely by their sequence, but by a complex landscape of chemical modifications that shape their structure, stability, and molecular interactions. Most post-transcriptional RNA modifications cannot be detected by conventional sequencing approaches.
To directly interrogate this vital layer of information, we are establishing a national capability - RNA Mass Spectrometry Platform - dedicated to the analysis of DNA and RNA and all their possible modifications. The RNA mass spectrometry platform enables comprehensive identification and quantitative analysis of modified nucleotides and provides powerful analytical tools to characterise the critical quality attributes of oligonucleotide-based therapeutics.
We demonstrate the versatility of workflows through two applications: (1) assessing the capping efficiency and polyA tail length of in vitro transcribed mRNA, and (2) identifying an unknown modification in a bacteriophage DNA.
The potency of mRNA vaccines and therapeutics depends heavily on the integrity of critical structural features, most notably the 5’ cap and the 3’ poly(A) tail. We have established a robust mass spectrometry workflow that enables accurate characterisation of both the capping efficiency and poly(A) tail length and composition, ensuring consistent quality attributes that support research and translational performance.
Bacteriophages developed diverse DNA modification strategies to evade the host immune defence system. Mass spectrometry can provide a precise and sensitive platform to detect and characterise these modifications, such as methylation, β-glucosyl-hydroxymethyl-cytosine (5ghmC), and 5-arabinosyl-hydroxy-cytosine (5ara-hC). We demonstrate the application of mass spectrometry to detect both expected DNA modifications, and for discovering unknown DNA modifications.
By establishing these RNA and DNA mass spectrometry capabilities, we aim to complement existing next-generation sequencing facilities, including long-read and short-read sequencing.