近年來信使核糖核酸疫苗已成為對付新冠肺炎疫情的利器，也促使此領域的蓬勃發展。要生產用於疫苗的高效率信使核糖核酸需要透過體外轉錄、5′ 端加帽、3′端多腺苷酸化等步驟。其中5′帽結構與3′端多腺苷酸結構對於轉錄後修飾、轉譯起始效率和信使核糖核酸的穩定性有很大的相關性。由於信使核糖核酸5′ 端加帽反應的量化並不容易測定，本論文嘗試使用能辨認5′端帽結構的真核起始因子4E(eIF4E)與酸性磷酸酶AphA 融合報導蛋白，以量化5′端帽結構合成的效率。最初我們在大腸桿菌中產生並過表達重組 AphA-eIF4E 融合蛋白。通過組氨酸標籤親和性鎳離子管柱，使用不同濃度的咪唑沖洗，分離出帶有組氨酸標籤的融合蛋白AphA-eIF4E。eIF4E 部分能夠特異性地結合加帽的 信使核糖核酸，而 AphA 可以作為報告酶以光學估計加帽步驟的效率。我們使用鏈親和素塗層於96 孔盤做為固定相，並加入生物素化的多聚胸腺嘧啶，與核糖核酸樣品結合，篩選出帶有3′端多腺苷酸的核糖核酸樣品，然後我們使用融合蛋白 AphA-eIF4E 來識別 5′加帽結構。 最後，我們使用 AphA 催化的螢光產物來估計 核糖核酸修飾的比例。在反應干擾物的測定上顯示三磷酸鳥苷與S-腺苷甲硫氨酸會干擾本偵測反應，並須先去除。本偵測系統可以有效地分辨出信使核糖核酸與非信使核糖核酸的測試樣品，偵測靈敏度最低偵測限制為200 奈克的信使核糖核酸，而偵測線性範圍約落在200 至1600 奈克的信使核糖核酸之間。

Messenger RNA vaccines have been demonstrated to be an effective tool against Covid-19 infections and many other infections. The synthesis of mRNA typically involves steps including in vitro transcription, 5′ capping, and 3′ A-tailing. The 5′ capping is essential for translation initiation and mRNA stability. The 3′ poly A tail is necessary for nuclear export, translation and stability of mRNA. Because mRNA capping is difficult to measure, this study planned to develop a 5′ capping detection system. Initially, a recombinant AphA (acid phosphatase)-eIF4E (eukaryotic initiation factor 4E) fusion protein was generated and overexpressed in E. coli. The eIF4E portion is capable of binding capped mRNA specifically, whereas AphA could serve as a reporter enzyme to optically estimate the efficiency of the capping step. We used a streptavidin-coated 96-well plate to immobilize biotin-labeled oligo d(T), which in turn can capture mRNA with 3′poly(A) tail. Then, AphA-eIF4E was added to quantify the 5′ cap structure that can be quantitated by adding AphA substrate 4-MUP. The results show that the detection system can effectively distinguish mRNA and non-mRNA samples. Nucleotides GTP and SAMe could interfere the assay and therefore need to be removed before conducting the assay. The limit of detection of the detection system was approximately 200 ng mRNA. The linear range of detection was approximately between 200 to 1600 ng mRNA.

1. Furuichi, Y., A. LaFiandra, and A.J. Shatkin, 5′-Terminal structure and mRNA stability. Nature, 1977. 266(5599): p. 235-239.
2. Darnell Jr, J.E., Transcription units for mRNA production in eukaryotic cells and their DNA viruses. Progress in nucleic acid research and molecular biology, 1979. 22: p. 327-353.
3. Dominissini, D. and G. Rechavi, 5-methylcytosine mediates nuclear export of mRNA. Cell research, 2017. 27(6): p. 717-719.
4. Filipowicz, W., et al., A protein binding the methylated 5'-terminal sequence, m7GpppN, of eukaryotic messenger RNA. Proceedings of the National Academy of Sciences, 1976. 73(5): p. 1559-1563.
5. Frye, M. and S. Blanco, Post-transcriptional modifications in development and stem cells. Development, 2016. 143(21): p. 3871-3881.
6. Furuichi, Y., Discovery of m7G-cap in eukaryotic mRNAs. Proceedings of the Japan Academy, Series B, 2015. 91(8): p. 394-409.
7. Leung, D.W. and G.K. Amarasinghe, When your cap matters: structural insights into self vs non-self recognition of 5′ RNA by immunomodulatory host proteins. Current opinion in structural biology, 2016. 36: p. 133-141.
8. Zhao, J., L. Hyman, and C. Moore, Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiology and molecular biology reviews, 1999. 63(2): p. 405-445.
9. Fechter, P. and G.G. Brownlee, Recognition of mRNA cap structures by viral and cellular proteins. Journal of General Virology, 2005. 86(5): p. 1239-1249.
10. Shatkin, A., Capping of eucaryotic mRNAs. Cell, 1976. 9(4): p. 645-653.
11. Wei, C.-M., A. Gershowitz, and B. Moss, N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature, 1975. 257(5523): p. 251-253.
12. Bienroth, S., W. Keller, and E. Wahle, Assembly of a processive messenger RNA polyadenylation complex. The EMBO journal, 1993. 12(2): p. 585-594.
13. Balbo, P.B. and A. Bohm, Mechanism of poly (A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis. Structure, 2007. 15(9): p. 1117-1131.
14. Pardi, N., et al., mRNA vaccines—a new era in vaccinology. Nature reviews Drug discovery, 2018. 17(4): p. 261-279.
15. Rosa, S.S., et al., mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine, 2021. 39(16): p. 2190-2200.
16. Bustin, S.A., Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of molecular endocrinology, 2000. 25(2): p. 169-193.
17. Mora, J.R. and R.C. Getts, High-sensitivity detection methods for low-abundance RNA species: applications for functional genomics research. Expert review of molecular diagnostics, 2007. 7(6): p. 775-785.
18. Carrascosa, L.G., C.S. Huertas, and L.M. Lechuga, Prospects of optical biosensors for emerging label-free RNA analysis. Trac Trends in Analytical Chemistry, 2016. 80: p. 177-189.
19. Vlatkovic, I., et al., Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics, 2022. 14(2): p. 328.
20. Beverly, M., et al., Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Analytical and bioanalytical chemistry, 2016. 408(18): p. 5021-5030.
21. Russo, J., et al., Engineered viral RNA decay intermediates to assess XRN1-mediated decay. Methods, 2019. 155: p. 116-123.
22. Efimov, V., et al., Detection of the 5′-cap structure of messenger RNAs with the use of the cap-jumping approach. Nucleic acids research, 2001. 29(22): p. 4751-4759.
23. Fuchs, A.-L., A. Neu, and R. Sprangers, A general method for rapid and cost-efficient large-scale production of 5′ capped RNA. RNA, 2016. 22(9): p. 1454-1466.
24. Moya-Ramírez, I., et al., High resolution biosensor to test the capping level and integrity of mRNAs. Nucleic acids research, 2020. 48(22): p. e129-e129.
25. Grifo, J., et al., New initiation factor activity required for globin mRNA translation. Journal of Biological Chemistry, 1983. 258(9): p. 5804-5810.
26. Sonenberg, N., et al., Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP. Proceedings of the National Academy of Sciences, 1979. 76(9): p. 4345-4349.
27. Sonenberg, N., et al., A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA. Proceedings of the National Academy of Sciences, 1978. 75(10): p. 4843-4847.
28. Marcotrigiano, J., et al., Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell, 1997. 89(6): p. 951-961.
29. Rogers Jr, G.W., A.A. Komar, and W.C. Merrick, eIF4A: the godfather of the DEAD box helicases. 2002.
30. Schütz, P., et al., Crystal structure of the yeast eIF4A-eIF4G complex: An RNA-helicase controlled by protein–protein interactions. Proceedings of the National Academy of Sciences, 2008. 105(28): p. 9564-9569.
31. Amorim, I.S., G. Lach, and C.G. Gkogkas, The role of the eukaryotic translation initiation factor 4E (eIF4E) in neuropsychiatric disorders. Frontiers in genetics, 2018. 9: p. 561.
32. Siddiqui, N. and N. Sonenberg, Signalling to eIF4E in cancer. Biochemical Society Transactions, 2015. 43(5): p. 763-772.
33. Mamane, Y., et al., eIF4E–from translation to transformation. Oncogene, 2004. 23(18): p. 3172-3179.
34. Furic, L., et al., eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression. Proceedings of the National Academy of Sciences, 2010. 107(32): p. 14134-14139.
35. Dong, Q., et al., Tumor-derived exosomal eIF4E as a biomarker for survival prediction in patients with non-small cell lung cancer. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research, 2020. 26: p. e923210-1.
36. Choi, Y.H. and C.H. Hagedorn, Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3′ poly (A) end phenotype. Proceedings of the National Academy of Sciences, 2003. 100(12): p. 7033-7038.
37. Rahman, M. and M.M. Karim, Heterologous Expression of a Human Cap-Binding Protein eIF4E in Escherichia coli. Dhaka University Journal of Pharmaceutical Sciences, 2006. 5(1): p. 59-62.
38. Thaller, M.C., et al., Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product. FEMS Microbiology Letters, 1997. 146(2): p. 191-198.
39. Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 2003. 60(5): p. 523-533.
40. Schmidt, S.R., Fusion-proteins as biopharmaceuticals–applications and challenges. Curr Opin Drug Discov Devel, 2009. 12(2): p. 284-295.
41. Kim, B.-J., et al., Transferrin fusion technology: a novel approach to prolonging biological half-life of insulinotropic peptides. Journal of Pharmacology and Experimental Therapeutics, 2010. 334(3): p. 682-692.
42. Zhao, H.L., et al., Increasing the homogeneity, stability and activity of human serum albumin and interferon-α2b fusion protein by linker engineering. Protein expression and purification, 2008. 61(1): p. 73-77.
43. Amet, N., H.-F. Lee, and W.-C. Shen, Insertion of the designed helical linker led to increased expression of tf-based fusion proteins. Pharmaceutical research, 2009. 26(3): p. 523-528.
44. Bai, Y. and W.-C. Shen, Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharmaceutical research, 2006. 23(9): p. 2116-2121.
45. Waldo, G.S., et al., Rapid protein-folding assay using green fluorescent protein. Nature biotechnology, 1999. 17(7): p. 691-695.
46. Maeda, Y., et al., Engineering of functional chimeric protein G–VargulaLuciferase. Analytical biochemistry, 1997. 249(2): p. 147-152.
47. Chen, X., et al., Design of an in vivo cleavable disulfide linker in recombinant fusion proteins. Biotechniques, 2010. 49(1): p. 513-518.
48. Wu, J., et al., Ribogenomics: the science and knowledge of RNA. Genomics, proteomics & bioinformatics, 2014. 12(2): p. 57-63.
49. Carino, E.J., K. Scheets, and W.A. Miller, The RNA of maize chlorotic mottle virus, an obligatory component of maize lethal necrosis disease, is translated via a variant panicum mosaic virus-like cap-independent translation element. Journal of virology, 2020. 94(22): p. e01005-20.
50. Hellman, L.M. and M.G. Fried, Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nature protocols, 2007. 2(8): p. 1849-1861.
51. Von der Haar, T., P.D. Ball, and J.E. McCarthy, Stabilization of eukaryotic initiation factor 4E binding to the mRNA 5′-Cap by domains of eIF4G. Journal of Biological Chemistry, 2000. 275(39): p. 30551-30555.
52. Maria, C.T., et al., Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes. Microbiology, 1995. 141(1): p. 147-154.
53. Uerkvitz, W., Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity. Journal of Biological Chemistry, 1988. 263(30): p. 15823-15830.
54. Goss, D.J. and F.E. Kleiman, Poly (A) binding proteins: are they all created equal? Wiley Interdisciplinary Reviews: RNA, 2013. 4(2): p. 167-179.