mRNA Fragmentation Pattern Detected by SHAPE

• Published in: Current Issues in Molecular Biology Vol. 46; no. 9; pp. 10249 - 10258
• Main Authors: Feng, Shanshan, Chen, Ting, Zhang, Yunlong, Lu, Changrui
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.09.2024; MDPI
• Subjects: Antigens; Backup software; degradation; freeze–thaw cycles; Innovations; long-term storage; Messenger RNA; mRNA stability; mRNA structure; Quality control; Vaccines; degradation; long-term storage; freeze–thaw cycles; mRNA stability; mRNA structure
• ISSN: 1467-3045; 1467-3037; 1467-3045
• DOI: 10.3390/cimb46090610

The success of messenger RNA (mRNA) vaccines in controlling COVID-19 has warranted further developments in new technology. Currently, their quality control process largely relies on low-resolution electrophoresis for detecting chain breaks. Here, we present an approach using multi-primer reverse transcription sequencing (MPRT-seq) to identify degradation fragments in mRNA products. Using this in-house-made mRNA containing two antigens and untranslated regions (UTRs), we analyzed the mRNA completeness and degradation pattern at a nucleotide resolution. We then analyzed the sensitive base sequence and its correlation with the secondary structure. Our MPRT-seq mapping shows that certain sequences on the 5' of bulge-stem-loop structures can result in preferential chain breaks. Our results agree with commonly used capillary electrophoresis (CE) integrity analysis but at a much higher resolution, and can improve mRNA stability by providing information to remove sensitive structures or sequences in the mRNA sequence design.