3AIM-seq : Examining poly(A) tail length of mRNA therapeutics via 3’ end targeted sequencing
3AIM-seq: 3' 말단 타겟 시퀀싱을 통한 mRNA 치료제의 폴리(A) 꼬리 길이 평가
- 주제(키워드) mRNA therapeutics , poly(A) , In vitro transcribed mRNA , Bioinformatics
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Daechan Park
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033787
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
COVID-19 unprecedentedly facilitated scientific research and commercial development of in vitro transcribed (IVT) mRNA therapeutics, requiring methodologies to ensure their stability and equivalence as therapeutics. Most of all, the verification of nucleic acid sequences in synthetic mRNA therapeutics is indispensable to prevent from eliciting unintended immunogenicity. Also, resolving sequence structures of mRNA drug is crucial because structural complexities are growing upon rapid expansion of mRNA therapeutics. Particularly, accurate measurement of poly(A) tail length is an important but still challenging issue by using current sequencing methods. Here, I present a novel sequencing approach, 3AIM-seq: examining poly(A) tail length of mRNA therapeutics via 3' end targeted sequencing. This is an efficient 3’ end targeted sequencing method to quantitatively compute poly(A) lengths for IVT mRNA. To amplify 3’ end region of mRNA drugs, 3' ligation-free anchored primer (3' LAP) was used for ligation-free addition of adapter sequences, resulting in improvement of amplicon quality and quantity. After next generation sequencing of the 3’ end amplicon, 3AIM-seq algorithm utilized base quality information rather than base calling to calculate poly(A) tail length with a sliding window approach. 3AIM-seq accurately measured the poly(A) lengths of standard controls, synthetic ssDNA that copied 3’ end of mRNA drug. For mRNA vaccines with four different poly(A) structures, 3AIM-seq was able to estimate the poly(A) lengths at the resolution of 5 bp. This experimentally and computationally straightforward method will be useful for precise measurement of poly(A) lengths of mRNA drugs, eventually aiding in development of high-efficacy mRNA drug with high yield.
more목차
I. Introduction 1
II. Materials and Methods 4
A. 3’ end targeted amplification by ligation-free method 4
B. 3’ end targeted amplification by direct ligation method 8
C. Preparation of spike-ins 8
D. Next-generation sequencing library construction 11
E. Process of read filtering 14
F. The algorithm strategy of poly(A) tail length measurement in 3AIM-seq 14
G. The method of poly(A) tail length measurement based on base calling 15
III. Results 16
A. Base quality scores indicated the poly(A) length 16
B. Strategies for optimal poly(A) targeted sequencing: experimental and analytic approaches 19
C. NGS library preparation from poly(A) targeted amplicons 23
D. Ligation-free method was more consistent and efficient than direct ligation method 28
E. 3AIM-seq estimates poly(A) tail length with high accuracy and resolution 38
F. IVT mRNA with long poly(A) included truncated poly(A) 49
IV. Discussion 58
V. Reference 61
