Feng Shanshan, Chen Ting, Zhang Yunlong, Lu Changrui
College of Biological Science and Medical Engineering, Donghua University, Shanghai 201620, China.
Curr Issues Mol Biol. 2024 Sep 16;46(9):10249-10258. 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.
信使核糖核酸（mRNA）疫苗在控制新冠疫情方面的成功促使新技术得到进一步发展。目前，它们的质量控制过程很大程度上依赖于低分辨率电泳来检测链断裂。在此，我们提出一种使用多引物逆转录测序（MPRT-seq）的方法来识别mRNA产物中的降解片段。使用这种包含两种抗原和非翻译区（UTR）的自制mRNA，我们在核苷酸分辨率下分析了mRNA的完整性和降解模式。然后，我们分析了敏感碱基序列及其与二级结构的相关性。我们的MPRT-seq图谱显示，凸起-茎环结构5'端的某些序列可导致优先链断裂。我们的结果与常用的毛细管电泳（CE）完整性分析结果一致，但分辨率要高得多，并且可以通过提供信息以去除mRNA序列设计中的敏感结构或序列来提高mRNA的稳定性。
