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研究生：	李宗懌 Li, Zong-Yi
論文名稱：	發展針對腸病毒A71型3D非結構蛋白具專一性結合的DNA適體以作為治療或偵測試劑 Development of DNA aptamers specifically targeting the Enterovirus A71 3D non-structural protein as therapeutic or diagnostic reagents
指導教授：	王憲威 Wang, Shainn-Wei
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	64
中文關鍵詞：	腸病毒A71型、3D聚合酶、去氧核酸適體、TALON®鈷離子樹脂、系統性配體指數擴增法、結合性實驗、下拉實驗、解離常數、酵素連結免疫吸附分析法、酵素連結寡核苷酸分析法
外文關鍵詞：	Enterovirus A71, 3D polymerase, DNA Aptamer, TALON® Cobalt beads, SELEX, Binding assay, Pull-down assay, Dissociation constant, ELISA, ELONA
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腸病毒七十一型(EV-A71)的感染，會造成手足口症並且可能使孩童引發神經性疾病或心肺併發症狀。此腸病毒是無外套膜之正股單股核醣核酸病毒，可轉譯出一條含有四個結構蛋白(VP4-VP2-VP3-VP1)與七個非結構蛋白(2A-2B-2C-3A-3B-3C-3D)的前驅多胜肽蛋白。其中病毒的3D蛋白質的功能為RNA依賴性之RNA聚合酶，其功能與序列在腸病毒屬中是具有高度保守性的，因此是發展抗病毒藥物或是病毒診斷的潛力標靶。針對3D蛋白質發展出高親和力且專一結合的小片段單股核酸適體(aptamers)可能可以提供一個相對安全且便宜的治療方法甚或診斷的替代方案。因此本研究利用系統性配體指數擴增法(SELEX)和次世代定序技術(NGS)來篩選及發展新穎的3D專一性適體。細菌衍生之重組性3D蛋白質，其碳端帶有重複6個組胺酸標籤(6xHis tag)，被鈷珠與快速蛋白質液相色層析(FPLC)純化成均質3D-His蛋白質。專一性適體的篩選乃對鈷珠上包被的3D-His經由15輪循環性親和演化(SELEX)而生成。次序代定序來自C10至C15循環增幅篩選後的適體發現了3個含有保守序列的主要族群組別（A，B和C）。這些序列在C15中按照發生頻率排序，並且將它們在C10-15中的排名和頻率與增加的進化事件進行比較。四個適體候選者（A-1，B-1，B-2和C-1）其大小為76-80個去氧核醣核酸為最適或高度演化的種類，並且它們對3D-His或病毒3D的親和力以及抑制活性被更進一步探討。實驗結果顯示，該四名適體候選者A-1，B-1，B-2和C-1均可透過包被適體的磁珠在親和力下拉(pull-down)實驗中結合到重組3D-His蛋白質和病毒3D蛋白質。這些適體候選物也在另一ELISA親和力實驗中展示了它們與3D-His的結合親和力，且其解離常數Kd值在1.5~55.8nM範圍內，接近於常規抗體。藉此，我們建立了適體塗層於ELISA 盤面的ELONA測定方法學，並成功地用於診斷檢測EV-A71感染時產成的病毒3D蛋白。最後我們的結果也顯示適體B-1和B-2有顯著的抑制功能，可使EV-A71複製減緩且不具細胞毒性，儘管適體效能尚具有改進空間，因本身為核酸其於實驗中造成的非專一性脫靶效應沒有被有效排除。作為一個前驅研究，我們討論了實驗中各項方法學中可能可以改良的細部關鍵步驟以及這些適體是否可被改進為具有結構特徵的較短序列。此研究鑑定了四個對重組3D-His和病毒3D具有高親和力而極具潛力的適體候選者。具有適體塗層的ELONA板也能夠以3D-His為標準物來檢定蛋白之有效偵測範圍座落在54ng至22μg間，而具備其可用作腸病毒感染的診斷工具的依據。然而作為抑制藥物開發，這些適體必需要再進一步的改良。

Enterovirus A71(EV-A71) infection causes hand, foot, and mouth disease (HFMD) and may causes fatal neurological and cardiopulmonary complications particularly in young children. EV-A71 is a non-enveloped, positive-sense, single stranded RNA virus, which consist of four structural proteins (VP4, VP2, VP3, and VP1), and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The 3D protein, which functions as RNA-dependent RNA polymerase, is highly conserved among the Enterovirus genus and thus a potential target for antiviral drugs and/or diagnostic detecting ligands. Small DNA aptamers with strong and specific binding affinity to 3D protein may offer a relatively safe and economic therapeutic and diagnostic alternative. In this study, a Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology in together with Next-Generation Sequencing (NGS) were used to screen novel 3D-specific aptamers. Recombinant 3D with C-terminus histidine tag (3D-His) were purified into homogenous species by Cobalt-beads and subsequent FPLC. Selection of 3D-His-specific aptamers was achieved by 15 rounds of cycling evolvement of aptamers on 3D-His-coated cobalt-beads. NGS of cycling-enriched aptamers from cycles C10 to C15 revealed 3 major groups (A, B, and C) of conserved sequences. These sequences were ranked in C15 by frequency of occurrence and their ranking and frequency in C10-15 were compared for increased evolving events. Four aptamer candidates (A-1, B-1, B-2, and C-1) in sizes of 76-80 mers were top or highly evolved species and their binding affinities to 3D-His or viral 3D as well as inhibitory activities toward EV-A71 replication were investigated. Results indicated that all four aptamer candidates A-1, B-1, B-2 and C-1 can target to both recombinant and viral 3D protein by aptamer-beads in pull-down assays. These aptamer candidates also demonstrated their binding affinities to 3D-His with Kd in the range of 1.5~55.8 nM in an ELISA format, which are closer to regular antibodies. Accordingly, Enzyme- Linked Oligonucleotide Assays (ELONA) in a format of aptamer-coated plates was successfully established for diagnostic detection of viral 3D during EV-A71 infection. Finally, aptamer B-1 and B-2 demonstrated significant inhibitory function to decelerate EV-A71 replication without cell toxicity, albeit with improvable potentials to limit their off-target effect as nucleic acids per se. As a pilot study, improvement of assays and/or refinement of these aptamers into shorter structure-featured elements were discussed. This study identified four promising aptamer candidates with high affinity to recombinant 3D-His and viral 3D. ELONA plates with coated aptamers were capable of detecting 3D-His as a standard in the range of 54 ng to 22 g, which may be used a diagnostic tool for Enterovirus infection. Further improvement of these aptamers is required for their development as inhibitory drugs.

Index

摘要 ……………………………………………………………………….…………………...…….i
Abstract……………………………………………………………….………………….………….iii
Acknowledgments ……………………………………………….…...………………………..…….v
Index………………………………………………………………………..……………………….vii
Figure Index………………………………………………….……..………….……………….…….x
Table Index………………..………………………………………...…….…………………...…….xi
Introduction …………………………………………………………………….…...……………….1
Enterovirus A71-Historical outbreaks and pathogenesis ………………….……………………1
Vaccine trials to fight against Enterovirus ...………………………………..…………….…….1
EV-A71 genome and translational products…………………………………………………….2
Inhibitory drug development for EV-A71…………………..…………………………….…….2
EV-A71 3D polymerase …………………………………….………………………………….3
Nucleic acid aptamers as drugs………………………………………………………...……….4
G-quadruplex structure for high affinity binding……………………………………………….4
NGS (Next Generation Sequencing) by Illumina®…………………………………………….5
Goal and specific aims…………………………….……………….….……………………….…….6
Materials and Methods…………………………….………………………...……………………….7
Plasmids and 3D-His protein expression construct……………………………………………..7
Recombinant protein expression and purification………………………………………………7
SDS-PAGE gel staining and Western blot analysis………………….………………………….8
SELEX (systematic evolution of ligands by exponential enrichment)…………………...…….9
		Random single-strand DNA Aptamer………………….……….…..………..…………….9
		Cycling enrichment of aptamers by SELEX……………...……………………………….9
Next-generation sequencing (NGS) …………………….………..……….…….…………….10
Cells and viruses………………………………..…………………………..………………….11
Plaque assay……………………………………………………………….………..………….11
TCID50……………………………………………………………………….……..………….12
Aptamer transfection for functional assays…………………….……………………..……….13
Cell viability assay………………..………………………………………………..………….13
Pull-down assays………………………………………….………………………..………….14
Binding affinity measurements of aptamer candidates by ELISA…….….……………..…….14
ELONA (Enzyme- Linked Oligonucleotide Assays) development for viral detection………..15
Functional inhibition of viral replication and extracellular viral titer determination.................15
Reagents and antibodies………………………………………..…………………………..….16
		Antibodies: ………………………….………………………………………..………….16
		Reagents:…………………………………….………………………………..………….17
Instruments………………………………………..……………………………………..…….18
Primer list………………………………………………………………..…………………….18
Results…………………………………………………….…………..…………………………….19
Construction of pET28a-EV-A71-3D plasmid and optimization of recombinant 
protein expression with 0.5 mM IPTG in E. coli Rosetta strain…………….…………..…….19
Enterovirus A71-3D-His recombinant protein is homogeneously purified after 
FPLC……………………………..……………………………………………………………20
3D-His specific aptamers were cycling enriched by Systematic Evolution of Ligands 
by EXponential enrichment (SELEX) ……………………..………………………………….21
NGS analysis of cycling enriched aptamers and selection of promising aptamer 
candidates……………………………………………………………………..……………….22
Dissociation constants (Kd) of 4 aptamer candidates to 3D-His protein………………...…….23
Binding specifities of aptamer candidates to 3D-His and EV-A71 3D protein but not 
to EV-A71 structural protein and Dengue virus NS5 polymerase ……………………...….….24
Development of Enzyme Linked Oligonucleotide Assay (ELONA) as a detecting tool 
for Enterovirus infection………………………………….……………………………...…….25
B-1 and B-2 aptamer candidates had promising inhibitory effect to reduce EV-A71 
progression in cells…………………………………......…………………………..………….25
Discussion………………………………………….....……………………………...……..……….27
References……………………………………….....…………………………..………..………….32
Figures…………………………………………….....…………….....……...……………..……….35
Tables……………………………………………..………………………….…….….....………….48
Supporting information……………….....……………………........……..……………..………….52
Appendix……………………………………………………………........................…...………….64













Figure index

Figure 1. The plasmid map of pET28a-EV-A71-3D and its expression in E. coli. …………..….....35
Figure 2. Purification of the 3D-His recombinant protein through cobalt resin and 
subsequent filtration fast protein liquid chromatography (FPLC)……………………….37
Figure 3. The flowchart of Systematic Evolution of Ligands by EXponential enrichment 
(SELEX) and cycling enrichment of 3D-His specific DNA aptamers…………….….….39
Figure 4. Determination of dissociation constants of 4 candidate aptamers to recombinant 3D-His protein on ELISA plates…………………………………………………...……..………41
Figure 5. Specificity of 4 aptamer candidates to reversely bind to recombinant 3D-His 
protein and viral 3D protein. …………………………………………………..…..….…43
Figure 6.  Development of Enzyme Linked Oligonucleotide Assay for EV-A71 3D 
detection Streptavidin-coated ELONA…………………….………………..……………44
Figure 7. Effects of aptamer candidates to antagonize EV-A71 replication in RD cells………....…46



 
Table index

Table 1. Total reads and varieties of cycling enriched aptamers (C10-C15) after NGS……………48
Table 2. Ranking and grouping of highly occurrent aptamer sequences between SELEX…...…….49
Table 3. Percent occurrence and Weblogo sequences of three major groups of cycling 
enriched aptamers………………………..…………………...…………….….……...….50
Table 4. Secondary structures of 4 aptamer candidates……………………………..…..…….…….51

                                    

1. Schmidt, N.J., E.H. Lennette, and H.H. Ho, An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis, 1974. 129(3): p. 304-9.
2. Lin, T.Y., et al., The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. (1537-6591 (Electronic)).
3. Enterovirus surveillance guidelines. 2015.
4. Chong, P., et al., Review of enterovirus 71 vaccines. Clin Infect Dis, 2015. 60(5): p. 797-803.
5. Ai-Hsiang Chou, C.-C.L., Jui-Yuan Chang, et al., Formalin-Inactivated EV71 Vaccine Candidate Induced Cross-Neutralizing Antibody against Subgenotypes B1, B4, B5 and C4A in Adult Volunteers. PLOS ONE, 2013. 8(11).
6. Shen, M., et al., Picornavirus genome replication: Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C(2)-3Dpol complex. The Journal of biological chemistry, 2008. 283(2): p. 875-888.
7. Bedard, K.M. and B.L. Semler, Regulation of picornavirus gene expression. Microbes and Infection, 2004. 6(7): p. 702-713.
8. Hung, H.C., et al., Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid. J Antimicrob Chemother, 2010. 65(4): p. 676-83.
9. Chen, T.C., et al., Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71. Antimicrob Agents Chemother, 2009. 53(7): p. 2740-7.
10. De Clercq, E., New developments in anti-HIV chemotherapy. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2002. 1587(0006-3002 (Print)): p. 258-275.
11. Yap, Y., A. Zhang X Fau - Andonov, and R. He, Structural analysis of inhibition mechanisms of aurintricarboxylic acid on SARS-CoV polymerase and other proteins. Computational Biology and Chemistry, 2005. 29(1476-9271 (Print)): p. 212-219.
12. Hung, H.C., et al., Aurintricarboxylic acid inhibits influenza virus neuraminidase. Antiviral Research, 2009. 81(1872-9096 (Electronic)): p. 123-131.
13. Wang, Y., et al., In Vitro Assessment of Combinations of Enterovirus Inhibitors against Enterovirus 71. Antimicrob Agents Chemother, 2016. 60(9): p. 5357-67.
14. Gohara, D.W., et al., Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B. J Biol Chem, 2000. 275(33): p. 25523-32.
15. Wu, Y., et al., Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China. Protein Cell, 2010. 1(5): p. 491-500.
16. Paul, A.V., et al., Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus. Journal of Virology, 2003. 77(0022-538X (Print)).
17. Chen, C., et al., Crystal structure of enterovirus 71 RNA-dependent RNA polymerase complexed with its protein primer VPg: implication for a trans mechanism of VPg uridylylation. Journal of Virology, 2013. 87(1098-5514 (Electronic)).
18. Parashar, A., Aptamers in Therapeutics. Journal of Clinical and Diagnostic Research : JCDR, 2016. 10(6): p. BE01-BE06.
19. Burge, S., et al., Quadruplex DNA: sequence, topology and structure. Nucleic Acids Research, 2006. 34(19): p. 5402-5415.
20. Tucker, W.O., J.A. Shum Kt Fau - Tanner, and J.A. Tanner, G-quadruplex DNA aptamers and their ligands: structure, function and application. Current Pharmaceutical Design, 2012. 18(1873-4286 (Electronic)): p. 2014-2026.
21. Wyatt, J.R., et al., Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion. Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(4): p. 1356-1360.
22. Reuter, J.A., D. Spacek, and M.P. Snyder, High-Throughput Sequencing Technologies. Molecular cell, 2015. 58(4): p. 586-597.
23. Bornhorst, J.A. and J.J. Falke, Purification of proteins using polyhistidine affinity tags. 2000(0076-6879 (Print)).
24. Yang, H., et al., Base editing generates substantial off-target single nucleotide variants. bioRxiv, 2018: p. 480145.
25. Landry, J.P., et al., Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform. Journal of Immunological Methods, 2015. 417: p. 86-96.
26. Kang, J., M.S. Lee, and D.G. Gorenstein, The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods, 2005. 64(2): p. 147-51.
27. Lakhin, A.V., V.Z. Tarantul, and L.V. Gening, Aptamers: Problems, Solutions and Prospects. Acta Naturae, 2013. 5(4): p. 34-43.
28. Liu, Y., et al., Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis. 2009(1431-6730 (Print)).
29. Arnau, J., et al., Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. 2006(1046-5928 (Print)).
30. Wang, Q., et al., G-quadruplex formation at the 3' end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase. 2011
(1362-4962 (Electronic)).

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