3AIM-seq: quality assessment of mRNA therapeutics using sequencing for 3′ polyA tails of in vitro transcribed mRNA

In vitro transcribed (IVT) mRNA therapeutics are promising for preventing and treating diseases, including infectious diseases and cancer, by delivering nucleic acid sequences. Assessing the stability of mRNA sequences and polyA tail lengths is crucial to minimize adverse effects and ensure drug efficacy. However, accurately measuring long homopolymeric nucleotides remains technically challenging, and a specialized method for IVT mRNAs is lacking. We introduced 3AIM-seq, a technique optimized experimentally and analytically for sequencing 3′ polyA tails in IVT mRNAs. To generate high-quality data, we used a ligation-free adapter followed by 3′ end amplification to prepare high-throughput sequencing libraries. The 3AIM-seq algorithm employed a sliding window approach to base quality scores, providing higher resolution and accuracy than base-calling method, and distinguishing polyA length differences as small as ±5 bp. Using in silico synthetic standard spike-in experiments, the method estimated the homogeneity of polyA tail lengths at the individual DNA molecule level in IVT mRNAs with various polyA tail structures. Although polyA tail of up to 70 bases were accurately measured, stretches exceeding 100 bases exhibited high heterogeneity for length, highlighting the importance of thorough assessments. Therefore, 3AIM-seq is a reliable method for evaluating the structural integrity of IVT mRNA products before clinical use.