以磷酸根甲基化之反義去氧核醣核酸探針藉由多孔性二氧化矽奈米粒子作為載體進行基因靜默調控之研究

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姓名

吳佳嶸(Chia-Jung Wu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

以磷酸根甲基化之反義去氧核醣核酸探針藉由多孔性二氧化矽奈米粒子作為載體進行基因靜默調控之研究
(Studies of Methyl Phosphotriester Oligonucleotide as Anti-sense Oligo by Mesoporous Silica Nanoparticles for Gene Silencing)

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至系統瀏覽論文 (2024-8-1以後開放)

摘要(中)

在許多疾病包括癌症中，細胞內的蛋白質調控產生失衡，影響著蛋白質的合成與降解、訊號傳遞和代謝途徑等，甚至影響腫瘤的惡化及癌症發展。在這其 messenger RNA (mRNA) 在蛋白質轉譯形成及調節基因表達都發揮重要的作用。藥物的發展進程中，許多研究會使用反義寡核苷酸 (Antisense oligonucleotide, ASO) 做為探針，以達成基因調控的目標。ASO 與目標基因 (DNA,mRNAandmicroRNA) 進行鹼基配對，通過不同的生物機制對目標基因進行表達調控，因此從生物辨識工程的角度上反義寡核苷酸的專一性及親和力將成為一項關鍵因素。
為了實現此目標，本研究使用本實驗室所研發之磷酸根甲基化 DNA (Phosphate-methylated DNA) 作為反義寡核酸探針。nDNA 因磷酸骨幹經過特定位置的甲基化修飾，使帶電的磷酸根轉變成為三酯鍵而形成電中性 (Neutralized DNA, nDNA)，其可減少核酸雙股同時帶負電的靜電排斥效應，期待不會影響專一性外並能提升雜交穩定性。且經甲基化修飾的核酸探針，擁有抵抗核酸脢降解的能力，因此更具有成為反義核酸藥物的潛力。
為了使反義寡核酸探針進入細胞內，本研究使用多孔性二氧化矽奈米粒子(Mesoporous Silica Nanoparticle, MSN) 作為核酸類似物載體。並經 PEI 修飾，使表面帶正電，可透過靜電吸附力與核酸探針結合，再經由包吞作用進入細胞，進行基因抑制。
首先，為確保經 PEI 修飾之 MSN 不會造成細胞毒性，以 Cell Counting
Kit-8(CCK-8) 試劑進行細胞毒性檢測。結果表明，當奈米粒子濃度低於50 μg/mL 時不會引起細胞毒性，並且在 24、48 及 72 小時的共培養時間下， MSN-PEG/PEI 濃度低於 75 μg/mL 條件下，其測得生物活性皆高於 90%。並且由電泳實驗，探討 MSN-PEG/PEI 對 nDNA 吸附能力。結果顯示，使用 nDNA 與 MSN 重量百分比 1:128 為最佳吸附條件，且 MSN-PEG/PEI 在此條件下對 nDNA 與 DNA 吸附能力相當。
於 Human plasma-like medium 環境中，以 nDNA 作為反義寡核苷酸探針對 mRNA 進行抑制逆轉錄，其結果顯示，與 DNA 相比，nDNA 能更有效的抑制逆轉錄反應，其中又以修飾於序列片段中間部分的 N3-mid nDNA 效果最佳。
在基因靜默調控實驗中，本研究使用人類結腸癌細胞-綠螢光蛋白 (HCT116-GFP) 細胞，並以綠色螢光蛋白 (Greenfluorescentprotein,GFP) 作為報告基因 (Reporter gene) 。使用 nDNA 探針或 DNA 探針與 HCT116-GFP 細胞中 GFP mRNA 進行雜交，以抑制 mRNA 轉譯形成 GFP，並使用螢光顯微鏡及微量分光光度計對綠色螢光蛋白進行定量及定性分析。
結果所示，部分甲基化的反義 nDNA 探針，因擁有較佳的專一性及雜交能力，因此比 DNA 探針更有效地抑制綠色螢光蛋白之表達。且修飾於核酸
探針中間時，因提升中間雜交穩定性，因此不易與 mRNA 解旋，擁有更佳的抑制能力。同時比較市售轉染試劑 (TransIT-X2) 與 MSN-PEG/PEI 之傳遞核酸探針之能力，結果顯示在 30 nM 時，兩著對於核酸傳遞能力相當，但當濃度提高時，MSN-PEG/PEI 有較佳的傳遞能力。
本研究已經成功利用 MSN-PEG/PEI 作為 nDNA 載體，在細胞層次研究中利用 50 nM 之 N3-mid nDNA@MSN 與 HCT116-GFP 細胞經 24 小時共培養，可靜默 50% 綠螢光蛋白表達，並且可持續對綠螢光蛋白進行表達抑制達 48 小時，並期望在未來能夠以 nDNA 作為探針對人體內基因進行調控，以作為核酸藥物。

摘要(英)

Most diseases, including cancers, are diagnosed by protein dysregulation within cells, which impacts protein synthesis and degradation, signaling, metabolic pathways, and even tumor progression. In this context, messenger RNA (mRNA) plays a critical role in protein translation formation and the regulation of gene expression. Many therapeutic drug development studies use antisense oligonucleotides (ASO) to achieve gene regulation targets.
Our laboratory has developed phosphate-methylated DNA (designated as neutralized DNA or nDNA) antisense oligonucleotides by modifying the phosphate backbones with site-specific methyl groups to reduce the inter-strand repulsion force and manipulate the stability of nucleic acid duplexes without losing the duplex formation specificity.
This study exploited mesoporous silica nanoparticles (MSN) as a carrier to deliver ASO into cells by positively charging their surfaces using polyethyleneimine (PEI), enabling them to adsorb nucleic acid probes electrostatically and to penetrate cells via endocytosis and endosomal escape to suppress gene expression.
Initial trials ensured that the PEI-modified MSNs concentrations below 50 μg/mL has no cytotoxicity. While the electrophoresis measurements showed that a weight ratio of nDNA: MSN = 1:128 was optimal for nDNA to be adsorbed by MSNs.
Moreover, despite an equivalent loading efficiency at the dosage of 30 nM, MSNs were more capable in delivering nucleic acids than commercially available transfection reagents (TransIT-X2) at the higher dosages.
In the human plasma-like medium environment, using nDNA as antisense oligonucleotide probes to inhibit reverse transcription of GFP-mRNA showed that nDNA was more effective in inhibiting the reverse transcription reaction compared to DNA. As a result of the modifications in the middle portion of the sequence fragment, N3-mid nDNA exhibited the best effect.
Experimentally, the nDNA probes were compared with DNA probes silencing gene regulation by hybridizing with GFP mRNA in HCT116-GFP cells to inhibit mRNA translation to form GFP. Empirical results demonstrated profoundly that the nDNA probes were more potent than the DNA antisense in suppressing the expression of green fluorescent protein, owing to their enhanced specificity and hybridization stability, especially the ASOs with center- methylated positions. With different designs of the nDNA probes, we also observed different potency of the antisense.
In summary, MSN-PEG/PEI was successfully employed as an nDNA carrier, and 50 nM of N3-mid nDNA@MSN was co-cultured with HCT116-GFP cells for 24 hours, resulting in a 50% reduction in green fluorescent protein expression. The silencing effect persisted for 48 hours.

關鍵字(中)

★ 核酸類似物
★ 奈米粒子
★ 基因靜默

關鍵字(英)

論文目次

摘要 i
ABSTRACT iv
致謝 vi
目錄 viii
圖目錄 xii
表目錄 xv
一、緒論 1
二、文獻回顧 3
2.1 核酸分子介紹 3
2.1.1 核酸分子 3
2.1.2 去氧核醣核酸 4
2.1.3 核醣核酸 6
2.1.4 信使核糖核酸 7
2.2 核酸類似物 9
2.2.1 鎖核酸 9
2.2.2 肽核酸 11
2.2.3 中性去氧核醣核酸 13
2.3 反義寡核苷酸對基因的調控 19
2.4 奈米粒子 22
2.4.1 奈米粒子之內吞作用 23
2.4.2 多孔性二氧化矽奈米粒子 24
2.5 基因分子檢測 27
2.5.1 聚合酶連鎖反應 27
2.5.2 即時定量聚合酶連鎖反應 29
2.5.3反轉錄聚合酶連鎖反應 31
三、實驗方法與儀器設備 33
3.1 實驗藥品 33
3.1.1 細胞培養 33
3.1.2 細胞轉染 33
3.1.3 二氧化矽奈米粒子 34
3.1.4 細胞毒性分析 35
3.1.5 洋菜凝膠電泳 (Agarose gel) 35
3.1.6 模擬生物體內mRNA雜交 35
3.1.7 即時聚合酶鏈式反應 36
3.1.8 綠螢光蛋白表達抑制實驗 37
3.2 儀器設備 38
3.3實驗方法 39
3.3.1 細胞解凍 39
3.3.2 細胞冷凍保存40
3.3.3 細胞培養 41
3.3.4 MSN吸附 42
3.3.6 細胞轉染 44
3.3.7 細胞毒性分析47
3.3.8 洋菜凝膠電泳 (Agarose gel) 48
3.3.9 逆轉錄即時聚合酶鏈式反應 (RT-qPCR) 49
3.3.10 SYBR Green 螢光熔點量測 52
3.3.11 綠螢光蛋白表達抑制實驗 53
3.3.12 MSN的細胞攝取 55
四、實驗結果與討論 56
4.1以多孔性二氧化矽奈米粒子做為反義寡苷酸探針載體系統建立 56
4.2多孔性二氧化矽奈米粒子吸附能力之探討 60
4.2.1 不同MSN種類對於nDNA探針吸附能力之比較 60
4.2.2 MSN對於nDNA與DNA探針吸附能力之比較 64
4.3 細胞攝取 66
4.4 細胞增殖 (細胞毒性分析) 69
4.5 反義寡核苷酸探針在HUMAN PLASMA-LIKE MEDIUM中抑制能力 72
4.5.1不同nDNA探針位置設計對GFP-mRNA逆轉錄抑制能力 75
4.5.2不同nDNA探針數量設計對GFP-mRNA逆轉錄抑制能力 78
4.6 使用反義寡核苷酸探針對抑制GFP轉譯能力 81
4.6.1 nDNA 與 DNA 探針對抑制轉譯能力之比較 83
4.6.2不同nDNA探針設計對抑制轉譯結果之研究 88
4.6.3不同時間長度對抑制結果之研究 93
五、結論 97
六、未來展望 99
七、參考文獻 100

參考文獻

1. Dahm, R., Friedrich Miescher and the discovery of DNA. Developmental Biology, 2005. 278(2): p. 274-288.
2. Watson, J.D. and F.H. Crick, Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature, 1953. 171(4356): p. 737-8.
3. Watson, J.D. and F.H.C. Crick, Molecular Structure of Nucleic Acids - a Structure for Deoxyribose Nucleic Acid. Nature, 1953. 171(4356): p. 737-738.
4. Herbert, A., et al., Special Issue: A, B and Z: The Structure, Function and Genetics of Z-DNA and Z-RNA. International Journal of Molecular Sciences, 2021. 22(14).
5. Ashikawa, I., K. Kinosita, and A. Ikegami, Dynamics of Z-Form DNA. Biochimica Et Biophysica Acta, 1984. 782(1): p. 87-93.
6. Sharp, P.A., The Centrality of RNA. Cell, 2009. 136(4): p. 577-580.
7. Vandivier, L.E., et al., The Conservation and Function of RNA Secondary Structure in Plants. Annual Review of Plant Biology, Vol 67, 2016. 67: p. 463-488.
8. Butcher, S.E. and A.M. Pyle, The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks. Accounts of Chemical Research, 2011. 44(12): p. 1302-1311.
9. Xu, S.Q., et al., mRNA Vaccine Era-Mechanisms, Drug Platform and Clinical Prospection. International Journal of Molecular Sciences, 2020. 21(18).
10. Hollams, E.M., et al., mRNA stability and the control of gene expression: Implications for human disease. Neurochemical Research, 2002. 27(10): p. 957-980.
11. Kaur, H., B.R. Babu, and S. Maiti, Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem Rev, 2007. 107(11): p. 4672-97.
12. Fakhfakh, K., et al., Molecular thermodynamics of LNA: LNA base pairs and the hyperstabilizing effect of 5′‐proximal LNA: DNA base pairs. AIChE Journal, 2015. 61(9): p. 2711-2731.
13. Singh, S.K., et al., LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chemical Communications, 1998(4): p. 455-456.
14. Alfeghaly, C., et al., Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection. Methods Mol Biol, 2021. 2300: p. 31-37.
15. Kurreck, J., et al., Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Research, 2002. 30(9): p. 1911-1918.
16. Burel, S.A., et al., Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Research, 2016. 44(5): p. 2093-2109.
17. Wahlestedt, C., et al., Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(10): p. 5633-5638.
18. Gupta, A., A. Mishra, and N. Puri, Peptide nucleic acids: Advanced tools for biomedical applications. Journal of Biotechnology, 2017. 259: p. 148-159.
19. Nielsen, P.E. and M. Egholm, An introduction to peptide nucleic acid. Current issues in molecular biology, 1999. 1(2): p. 89-104.
20. Gambari, R., Applications of peptide nucleic acids (PNA) in molecular medicine and biotechnology - Preface. Minerva Biotecnologica, 1999. 11(3): p. 161-162.
21. Marchelli, R., et al., Gene modulation by peptide nucleic acids (PNAs) targeting microRNAs (miRs), in Targets in Gene Therapy. 2011, IntechOpen.
22. Nulf, C.J. and D. Corey, Intracellular inhibition of hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) (vol 32, pg 3792, 2004). Nucleic Acids Research, 2004. 32(16): p. 4954-4954.
23. Moody, H.M., et al., Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides. Nucleic acids research, 1989. 17(12): p. 4769-4782.
24. van Genderen, M.H., L.H. Koole, and H.M. Buck, Hybridization of phosphate‐methylated DNA and natural oligonucleotides. Implications for protein‐induced DNA duplex destabilization. Recueil des Travaux Chimiques des Pays‐Bas, 1989. 108(1): p. 28-35.
25. Kuo, T.-C., et al., Reduction of interstrand charge repulsion of DNA duplexes by salts and by neutral phosphotriesters–Contrary effects for harnessing duplex formation. Journal of the Taiwan Institute of Chemical Engineers, 2020. 110: p. 1-7.
26. Chou, S.-C., et al., Increasing the λ-Red mediated gene deletion efficiency in Escherichia coli using methyl phosphotriester-modified DNA. Journal of the Taiwan Institute of Chemical Engineers, 2022. 137: p. 104297.
27. Miller, P.S., et al., Solid-phase synthesis of oligodeoxyribonucleoside methylphosphonates. Biochemistry, 1986. 25(18): p. 5092-5097.
28. Wang, P.-H., et al., Sensitive and Specific MicroRNA In Situ Hybridization Using Partially Methylated Phosphotriester Antisense DNA Probes. GEN Biotechnology, 2022. 1(5): p. 447-455.
29. Buck, H.M., Phosphate-methylated oligonucleotides past, present and future. Journal of Biophysical Chemistry, 2020. 11(03): p. 27-42.
30. 陳奕儒, 探討中性DNA與一般DNA雜交反應熱力學與結合機制之研究, in 化學工程與材料工程學系. 2016, 國立中央大學: 桃園縣. p. 115.
31. Caruthers, M.H., Gene synthesis machines: DNA chemistry and its uses. Science, 1985. 230(4723): p. 281-285.
32. 周韋成, 設計不帶電中性核酸探針於矽奈米線場效電晶體來改善富含GC鹼基核醣核酸之檢測專一性, in 化學工程與材料工程學系. 2018, 國立中央大學: 桃園縣. p. 118.
33. 洪靖雅, 應用磷酸根甲基化去氧核醣核酸引子以提升檢測單一核酸變異和微核醣核酸專一性之研究, in 化學工程與材料工程學系. 2021, 國立中央大學: 桃園縣. p. 128.
34. 李采璘, 使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究, in 化學工程與材料工程學系. 2018, 國立中央大學: 桃園縣. p. 85.
35. Gitanjali Kher, S.T., Ambikanandan Misra, 7 - Antisense Oligonucleotides and RNA Interference, in Challenges in Delivery of Therapeutic Genomics and Proteomics. 2011.
36. Jafar-Nejad, P., et al., The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration. Nucleic Acids Research, 2021. 49(2).
37. Monia, B.P., et al., Evaluation of 2 ‘-modified oligonucleotides containing 2 ‘-deoxy gaps as antisense inhibitors of gene expression. Journal of Biological Chemistry, 1993. 268(19): p. 14514-14522.
38. Crooke, S.T., Antisense drug technology: principles, strategies, and applications. 2007: CRC press.
39. Bennett, C.F. and E.E. Swayze, RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annual review of pharmacology and toxicology, 2010. 50: p. 259-293.
40. Boirivant, M., et al., Inhibition of Smad7 with a specific antisense oligonucleotide facilitates TGF-β1–mediated suppression of colitis. Gastroenterology, 2006. 131(6): p. 1786-1798.
41. Shi, S.J., et al., Solid Lipid Nanoparticles Loaded with Anti-microRNA Oligonucleotides (AMOs) for Suppression of MicroRNA-21 Functions in Human Lung Cancer Cells. Pharmaceutical Research, 2012. 29(1): p. 97-109.
42. Liang, G.F., et al., PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells. Nanoscale Research Letters, 2011. 6.
43. Li, X., et al., The packaging of siRNA within the mesoporous structure of silica nanoparticles. Biomaterials, 2011. 32(35): p. 9546-9556.
44. Sun, H.X., et al., Visualizing the down-regulation of hTERT mRNA expression using gold-nanoflare probes and verifying the correlation with cancer cell apoptosis. Analyst, 2019. 144(9): p. 2994-3004.
45. Malik, S., et al., Next generation miRNA inhibition using short anti-seed PNAs encapsulated in PLGA nanoparticles. Journal of Controlled Release, 2020. 327: p. 406-419.
46. Behzadi, S., et al., Cellular uptake of nanoparticles: journey inside the cell. Chemical Society Reviews, 2017. 46(14): p. 4218-4244.
47. Verma, A. and F. Stellacci, Effect of Surface Properties on Nanoparticle-Cell Interactions. Small, 2010. 6(1): p. 12-21.
48. Nel, A.E., et al., Understanding biophysicochemical interactions at the nano-bio interface. Nature Materials, 2009. 8(7): p. 543-557.
49. Iversen, T.G., T. Skotland, and K. Sandvig, Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today, 2011. 6(2): p. 176-185.
50. Ho, Y.T., R.D. Kamm, and J.C.Y. Kah, Influence of protein corona and caveolae-mediated endocytosis on nanoparticle uptake and transcytosis. Nanoscale, 2018. 10(26): p. 12386-12397.
51. Cheng, X.J., et al., Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and Nonphagocytic Cells in a Size-Dependent Manner. Acs Applied Materials & Interfaces, 2015. 7(37): p. 20568-20575.
52. Shapero, K., et al., Time and space resolved uptake study of silica nanoparticles by human cells. Molecular Biosystems, 2011. 7(2): p. 371-378.
53. Frohlich, E., The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. International Journal of Nanomedicine, 2012. 7: p. 5577-5591.
54. Akinc, A. and G. Battaglia, Exploiting Endocytosis for Nanomedicines. Cold Spring Harbor Perspectives in Biology, 2013. 5(11).
55. Chen, L.A., et al., The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells. Nanotechnology, 2011. 22(10).
56. AbouAitah, K. and W. Lojkowski, Delivery of Natural Agents by Means of Mesoporous Silica Nanospheres as a Promising Anticancer Strategy. Pharmaceutics, 2021. 13(2).
57. Slowing, I.I., et al., Mesoporous silica nanoparticles: structural design and applications. Journal of Materials Chemistry, 2010. 20(37): p. 7924-7937.
58. Trewyn, B.G., et al., Mesoporous silica nanoparticle based controlled release, drug delivery, and biosensor systems. Chemical Communications, 2007(31): p. 3236-3245.
59. He, Q.J. and J.L. Shi, Mesoporous silica nanoparticle based nano drug delivery systems: synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility. Journal of Materials Chemistry, 2011. 21(16): p. 5845-5855.
60. Kim, J., et al., Multifunctional Uniform Nanoparticles Composed of a Magnetite Nanocrystal Core and a Mesoporous Silica Shell for Magnetic Resonance and Fluorescence Imaging and for Drug Delivery. Angewandte Chemie-International Edition, 2008. 47(44): p. 8438-8441.
61. Vivero-Escoto, J.L., et al., Photoinduced Intracellular Controlled Release Drug Delivery in Human Cells by Gold-Capped Mesoporous Silica Nanosphere. Journal of the American Chemical Society, 2009. 131(10): p. 3462-+.
62. Liong, M., et al., Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. Acs Nano, 2008. 2(5): p. 889-896.
63. Radu, D.R., et al., A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent. Journal of the American Chemical Society, 2004. 126(41): p. 13216-13217.
64. Torney, F., et al., Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nature Nanotechnology, 2007. 2(5): p. 295-300.
65. Manzano, M. and M. Vallet-Regi, New developments in ordered mesoporous materials for drug delivery. Journal of Materials Chemistry, 2010. 20(27): p. 5593-5604.
66. Kim, M.H., et al., Facile Synthesis of Monodispersed Mesoporous Silica Nanoparticles with Ultralarge Pores and Their Application in Gene Delivery. Acs Nano, 2011. 5(5): p. 3568-3576.
67. Xia, T., et al., Polyethyleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of siRNA and DNA constructs. ACS nano, 2009. 3(10): p. 3273-3286.
68. Chou, C.C., et al., Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in Vitro and in Vivo. Acs Applied Materials & Interfaces, 2017. 9(27): p. 22235-22251.
69. Dilnawaz, F. and S.K. Sahoo, Augmented Anticancer Efficacy by si-RNA Complexed Drug-Loaded Mesoporous Silica Nanoparticles in Lung Cancer Therapy. Acs Applied Nano Materials, 2018. 1(2): p. 730-740.
70. White, T.J., N. Arnheim, and H.A. Erlich, The polymerase chain reaction. Trends in genetics, 1989. 5: p. 185-189.
71. Bartlett, J.M. and D. Stirling, A short history of the polymerase chain reaction. PCR protocols, 2003: p. 3-6.
72. Saiki, R.K., et al., Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA-Polymerase. Science, 1988. 239(4839): p. 487-491.
73. Wilhelm, J. and A. Pingoud, Real‐time polymerase chain reaction. Chembiochem, 2003. 4(11): p. 1120-1128.
74. Chen, C., et al., Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 2005. 33(20): p. e179.
75. Yi, G.H., et al., Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides. Plos One, 2013. 8(10).
76. Dembélé, J., et al., Overcoming Cytosolic Delivery Barriers of Proteins Using Denatured Protein-Conjugated Mesoporous Silica Nanoparticles. ACS Applied Materials & Interfaces, 2022. 15(1): p. 432-451.
77. Chamchoy, K., et al., Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays. Bmc Biochemistry, 2019. 20.
78. Held, P., An Absorbance-based Cytotoxicity Assay using High Absorptivity, Water-soluble Tetrazolium Salts
79. Mamedov, T., et al., A fundamental study of the PCR amplification of GC-rich DNA templates. Computational biology and chemistry, 2008. 32(6): p. 452-457.
80. Chakrabarti, R. and C.E. Schutt, The enhancement of PCR amplification by low molecular weight amides. Nucleic acids research, 2001. 29(11): p. 2377-2381.
81. Sarkar, G., S. Kapelner, and S.S. Sommer, Formamide can dramatically improve the specificity of PCR. Nucleic acids research, 1990. 18(24): p. 7465.
82. Sun, Y., G. Hegamyer, and N.H. Colburn, PCR-direct sequencing of a GC-rich region by inclusion of 10% DMSO: application to mouse c-jun. Biotechniques, 1993. 15(3): p. 372-4.
83. Weissensteiner, T. and J.S. Lanchbury, Strategy for controlling preferential amplification and avoiding false negatives in PCR typing. Biotechniques, 1996. 21(6): p. 1102-8.
84. Leeman, M., et al., Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4). Analytical and bioanalytical chemistry, 2018. 410: p. 4867-4873.
85. Ralser, M., et al., An efficient and economic enhancer mix for PCR. Biochemical and Biophysical Research Communications, 2006. 347(3): p. 747-751.
86. Milo, R., What is the total number of protein molecules per cell volume? A call to rethink some published values. Bioessays, 2013. 35(12): p. 1050-1055.
87. Palazzo, A.F. and E.S. Lee, Non-coding RNA: what is functional and what is junk? Frontiers in genetics, 2015. 6: p. 2.
88. Juusola, J. and J. Ballantyne, Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Science International, 2003. 135(2): p. 85-96.
89. 張晴雯, 部分磷酸根甲基化之反義去氧核醣核酸探針與微小核糖核酸雜交靈敏度與專一性之研究, in 化學工程與材料工程學系. 2022, 國立中央大學: 桃園縣. p. 123.
90. Choi, E. and S. Kim, Surface pH buffering to promote degradation of mesoporous silica nanoparticles under a physiological condition. Journal of Colloid and Interface Science, 2019. 533: p. 463-470.
91. McHugh, M.M., et al., The antitumor enediyne C-1027 alters cell cycle progression and induces chromosomal aberrations and telomere dysfunction. Cancer Research, 2005. 65(12): p. 5344-5351.

指導教授

陳文逸

審核日期

2023-7-20

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