비효소적 방법에 의한 변형 뉴클레오타이드 도입 및 mRNA 발현 연구 = Non-enzymatic Incorporation of Modified Nucleotides and Its Effect on mRNA Expression

다국어 초록 (Multilingual Abstract)

Messenger RNA (mRNA) has emerged as a promising therapeutic modality, but its inherent instability continues to limit its clinical applicability. To enhance stability and translational performance, we developed a non-enzymatic method for site-specific incorporation of chemically modified nucleotides at the 3′terminus of the poly(A) tail. Five imidazole-activated guanosine derivatives—L-GMP, IMP, 2′-O-Me-GMP, 2′-O-MOE-GMP, and 2′-F-GMP—were synthesized and incorporated into the poly(A) tail of luciferase mRNA through a guide RNA–mediated reaction. Gel electrophoresis and sequencing analyses confirmed successful incorporation, with IMP displaying the highest incorporation efficiency. In HEK293 cells, luciferase assays demonstrated enhanced translation for all modified mRNAs except the L-GMP–containing construct. Together, these findings indicate that chemical modification of the 3′poly(A) tail via non-enzymatic incorporation can improve mRNA stability and translation, highlighting its potential as a platform for next-generation mRNA therapeutics.

목차 (Table of Contents)

• 서론 1
• 실험방법 6
• 1. 구아노신 기반의 변형 뉴클레오타이드의 화학적 합성 7
• 2. Guide RNA를 이용한 short RNA의 비효소적 연장 확인 24
• 3. Luciferase mRNA 3Poly(A) Tail의 비효소적 연장 확인 및 생물학적 평가 26
• 결과 및 고찰 32
• 결론 43
• 참고문헌 45
• 분석자료 50