3AIM-seq: Quality assessment of mRNA therapeutics using sequencing for 3-prime poly(A) tail of in vitro transcribed mRNA

In vitro transcribed (IVT) mRNA therapeutics offer a promising strategy for preventing and treating various diseases, including infectious diseases and cancer, by delivering specific nucleic acid sequences. Assessing the stability and equivalence of mRNA sequences and poly(A) tail lengths is crucial for minimizing adverse effects and ensuring the efficacy of the drugs. However, precise measurement of homopolymer stretches, such as poly(A) tails, is technically challenging, and a specialized method for IVT mRNA is lacking. Here, we introduce 3AIM-seq, a high-accuracy sequencing technique optimized both experimentally and analytically for measuring poly(A) tail lengths in IVT mRNA. We used a ligation-free adapter followed by 3-prime end amplification to prepare high-throughput sequencing libraries to generate high-quality data. The 3AIM-seq algorithm then employed a sliding window approach to base quality scores for precise poly(A) length measurement. Using calibration curves that correlate mean absolute error with poly(A) lengths from in silico synthetic standard spike-ins experiment, the method estimated the homogeneity of poly(A) lengths at the individual DNA molecule levels to evaluate fidelity. Notably, the method provided significantly more accurate length measurements than the base-calling method, which showed considerable inaccuracies. While poly(A) stretches of up to 70 bases were accurately estimated as IVT synthesized, stretches exceeding 100 bases exhibited a high proportion of non-designed lengths, highlighting the importance of careful and warranted assessment. Therefore, 3AIM-seq offers a reliable method for evaluating the structural integrity of IVT mRNA products, ensuring quantitative equivalence before clinical use.

体外转录（in vitro transcribed, IVT）mRNA疗法通过递送特定核酸序列，为包括传染病与癌症在内的多种疾病的预防与治疗提供了极具前景的策略。评估mRNA序列与poly(A)尾（poly(A) tail）长度的稳定性及等效性，对于最小化不良反应、保障药物疗效至关重要。然而，精准测量poly(A)尾这类均聚物序列（homopolymer stretches）在技术上颇具挑战，且目前尚缺乏针对IVT mRNA的专用检测方法。在此，我们介绍3AIM-seq——一种经实验与分析优化的高精度测序技术，用于测量IVT mRNA中的poly(A)尾长度。该方法采用无需连接的接头，随后通过3'端扩增制备高通量测序文库（high-throughput sequencing libraries）以获取高质量测序数据。随后，3AIM-seq算法运用滑动窗口法（sliding window approach）处理碱基质量值（base quality scores），实现精准的poly(A)长度测量。研究人员借助关联平均绝对误差（mean absolute error）与计算机模拟（in silico）合成标准掺入品实验所得poly(A)长度的校准曲线（calibration curves），在单DNA分子水平上估算poly(A)长度的均一性，以此评估检测的保真度。值得注意的是，相较于误差显著的碱基识别方法（base-calling method），本方法可提供准确度大幅提升的长度测量结果。尽管针对IVT合成的70个碱基以内的poly(A)序列可实现精准估算，但超过100个碱基的序列则呈现出较高比例的非设计长度，这凸显了审慎且合理评估的重要性。因此，3AIM-seq为评估IVT mRNA产物的结构完整性、确保临床使用前的定量等效性提供了可靠的检测方法。