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Updated: Sep 17, 2025
3' End Sequencing Library Preparation with A-seq2
Published on: October 10, 2017
3AIM-seq: Quality assessment of mRNA therapeutics using sequencing for 3' poly(A) tails of in vitro-transcribed mRNA
Jina Seo1, Hyo-Jung Park2, Ayoung Oh2
1Department of Molecular Science and Technology, Ajou University, Suwon 16499, South Korea; Ajou Energy Science Research Center, Ajou University, Suwon 16499, South Korea.
Abstract:
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 poly(A) tail length 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 introduce 3AIM-seq, a technique optimized experimentally and analytically for sequencing 3' poly(A) 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 the base-calling method and distinguishing poly(A) length differences as small as ±5 bp. Using in silico synthetic standard spike-in experiments, the method estimated the homogeneity of poly(A) tail lengths at the individual DNA molecule level in IVT mRNAs with various poly(A) tail structures. Although poly(A) tails 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.
Insights
A new sequencing method, 3AIM-seq, accurately measures the poly(A) tail length of in vitro-transcribed messenger RNA (IVT mRNA) therapeutics. This ensures the stability and efficacy of mRNA drugs for clinical use.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomic Medicine
Background:
- In vitro-transcribed (IVT) mRNA therapeutics offer significant potential for treating diseases like cancer and infectious diseases.
- Assessing mRNA stability and poly(A) tail length is critical for ensuring therapeutic efficacy and minimizing adverse effects.
- Current methods struggle to accurately measure long homopolymeric nucleotide sequences, particularly in IVT mRNAs.
Purpose of the Study:
- To develop and validate a specialized sequencing technique for accurately measuring 3' poly(A) tails in IVT mRNAs.
- To provide a reliable method for assessing the structural integrity of mRNA therapeutics before clinical application.
Main Methods:
- Developed 3AIM-seq, a ligation-free adapter and 3' end amplification method for high-throughput sequencing library preparation.
- Utilized a sliding window approach in the 3AIM-seq algorithm for enhanced base quality scoring and resolution.
- Employed in silico synthetic standard spike-in experiments to assess poly(A) tail homogeneity at the single-molecule level.
Main Results:
- 3AIM-seq accurately measures poly(A) tail lengths up to 70 bases with a resolution of ±5 bp.
- The method revealed high heterogeneity in poly(A) tail lengths exceeding 100 bases for IVT mRNAs.
- Demonstrated the capability to estimate poly(A) tail homogeneity in various IVT mRNA structures.
Conclusions:
- 3AIM-seq is a highly accurate and reliable method for sequencing 3' poly(A) tails in IVT mRNAs.
- The technique is essential for evaluating the structural integrity and quality control of mRNA therapeutics.
- Findings underscore the importance of thorough poly(A) tail assessment for mRNA drug development.
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