miRNA是一種單股的非編碼微小RNA片段，長度18-23個核甘苷酸，近幾年的研究已證實miRNA的表達與癌症疾病有很強的關聯性，且可被視為診斷和預後的生物標誌。為了要更準確判斷miRNA表達，有幾種常見方法用來檢測miRNA，例如，即時聚合酶連鎖反應、微型核糖核酸晶片、次世代定序或是原位雜交。然而，由於miRNA其序列長度較短，家族成員之間的序列相似性，造成檢測上的困難。近年來，科學家已提出許多新方法，例如鎖核酸（LNA）和肽核酸（PNA）以獲得更好的靈敏度和特異性信號。在本研究中，我們使用本實驗室開發研究的核酸類似物，其磷酸骨幹上的氧可以經由甲基化的改質，使帶負電的磷酸根轉變成甲基磷酸三酯鍵methyl phosphotriester (MPTE)而成為電中性，使用MPTE修飾的DNA序列可以降低靜電排斥效應，同時增強雙股雜交時的親和力，因此可以提高其microRNA檢測的靈敏性和專一性，並將其應用於ISH和qPCR的方法。
在上述幾種檢測miRNA的方法中，原位雜交技術(ISH)是唯一可以對單個細胞中miRNA表達和定位的方法。我們使用MPTE修飾的探針檢測HCT116細胞系（人結腸癌細胞系）中轉染的外源miR-524-5p和內源miR-21和miR-29a的表達。實驗結果顯示，在MPTE修飾的探針都有相較於未修飾的探針較強的染色訊號，且分別使用一個錯配和三個錯配的探針在不同的雜交溫度下皆可顯示出高的信噪比。
及時定量聚合酶鏈反應（qPCR）是一種已被廣泛用於分析miRNA表達的方法。然而，因同一家族中的miRNA常具有相似的序列或單一核苷酸多態性（SNP）。先前的研究已證實許多疾病的產生與單一核苷酸多態性有關，因不易被一般 DNA 引子或探針所辨識，而導致錯誤的治療與診斷。在本研究中，我們使用miR-29a和miR-29c作為目標序列。使用含有MPTE修飾的DNA作為引物，透過調整不同MPTE修飾數的引物和退火溫度，來增加其單核苷酸多態性的辨識能力。從實驗結果得知，經由MPTE修飾的引子在單一鹼基錯誤配對辨識能力優於一般DNA引子，且我們能透過調控退火溫度來得到MPTE修飾的引子更好的專一性。
基於成功的將MPTE修飾的序列應用在ISH和qPCR方法中並改善引子和探針的專一性及靈敏度，未來可進一步將其應用在GC rich的序列中，預期MPTE修飾的寡核苷酸具有在不同的生物分子檢測平台和疾病治療用藥的潛在能力。

MicroRNA (miRNA) is a small non-coding RNA molecule, playing an essential role in the expression and regulation of genes. The expression patterns of miRNAs are important to the verification of their predicted function. miRNA levels have been demonstrated to have a strong correlation with disease progression in cancer, which can be considered as diagnostic and prognostic biomarkers. Currently, there are many ways for analyzing miRNA, such as qRT-PCR assays, microarray assays, in situ hybridization(ISH) and the next-generation sequencing(NGS). However, the challenge of detecting specific miRNA and remains
because of its small size, sequence similarity among the miRNA family members. In recent years, many novel nucleotide derivatives have been proposed to gain better signals of sensitivity and specificity such as Locked Nucleic Acid (LNA) and Peptide nucleic acid (PNA).
In this study, we developed DNA containing neutral methyl phosphotriester internucleotide (MPTE) linkages apply in ISH and qPCR.
The use of MPTE modified DNA sequence could reduce electrostatic repulsion effect, and as a consequance, could enhance the duplex formation which can specific and sensitive detection of microRNAs.
In situ hybridization is the only method which can provides insight into both the level and localization in single cell. We used MPTE modified probe for the detection of mimic exogenous miR-524-5p that transfected into HCT116 cell lines (human colon cancer cell lines) and the expression of endogenous miR-21 and miR-29a. We successfully demonstrated improved hybridization efficiency and show the high mismatch discrimination with high signal noise ratio in different temperature.
Quantitative polymerase chain reaction (qPCR) is a powerful method, which has been widely used to quantify the miRNA expression. However, miRNAs in the same family often have overlapping targets or single nucleotide polymorphism(SNP) that are not easy to be discriminated by unmodified DNA primers. we use miR-29a and miR-29c as targets which are both miRNA-29 (miR-29) family share the same mature sequence in vitro, while differ in one nucleotide. Using DNA containing MPTE linkages as primers to enhance the specificity of the SNP discrimination. By adjusting modification number of MPTE modified primer and annealing temperature, we achieve the optimum operating conditions for precise detection of miRNAs.
Depend on the success of applying DNA containing MPTE linkages in ISH and qPCR methods, it could be expected the potential ability of MPTE modified oligonucleotides developing into different biomolecular detection platform and possibly theoretic agent in the future.
 

[1] R. J. D. b. Dahm, "Friedrich Miescher and the discovery of DNA," vol. 278, no. 2, pp. 274-288, 2005.
[2] J. D. Watson and F. H. J. N. Crick, "Molecular structure of nucleic acids," vol. 171, no. 4356, pp. 737-738, 1953.
[3] S. Hawgood, I. G. Hook-Barnard, T. C. O’Brien, and K. R. J. S. t. m. Yamamoto, "Precision medicine: beyond the inflection point," vol. 7, no. 300, pp. 300ps17-300ps17, 2015.
[4] A. Leslie, S. Arnott, R. Chandrasekaran, and R. J. J. o. m. b. Ratliff, "Polymorphism of DNA double helices," vol. 143, no. 1, pp. 49-72, 1980.
[5] R. C. Lee, R. L. Feinbaum, and V. J. c. Ambros, "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14," vol. 75, no. 5, pp. 843-854, 1993.
[6] L.-A. MacFarlane and P. J. C. g. R Murphy, "MicroRNA: biogenesis, function and role in cancer," vol. 11, no. 7, pp. 537-561, 2010.
[7] K. B. J. C. c. i. Reddy, "MicroRNA (miRNA) in cancer," vol. 15, no. 1, p. 38, 2015.
[8] F. Magri, F. Vanoli, and S. Corti, miRNA in spinal muscular atrophy pathogenesis and therapy. 2017.
[9] D. G. McDowell, N. A. Burns, and H. C. J. N. a. r. Parkes, "Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR," vol. 26, no. 14, pp. 3340-3347, 1998.
[10] J. Kang, M. S. Lee, D. G. J. J. o. b. Gorenstein, and b. methods, "The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers," vol. 64, no. 2, pp. 147-151, 2005.
[11] T. Mamedov et al., "A fundamental study of the PCR amplification of GC-rich DNA templates," vol. 32, no. 6, pp. 452-457, 2008.
[12] A. G. Wilson, J. A. Symons, T. L. McDowell, H. O. McDevitt, and G. W. J. P. o. t. N. A. o. S. Duff, "Effects of a polymorphism in the human tumor necrosis factor α promoter on transcriptional activation," vol. 94, no. 7, pp. 3195-3199, 1997.
[13] D. L. J. N. m. Thomas, "Global control of hepatitis C: where challenge meets opportunity," vol. 19, no. 7, p. 850, 2013.
[14] S. Obika et al., "Synthesis of 2′-O, 4′-C-methyleneuridine and-cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering," vol. 38, no. 50, pp. 8735-8738, 1997.
[15] E. Várallyay, J. Burgyán, and Z. J. M. Havelda, "Detection of microRNAs by Northern blot analyses using LNA probes," vol. 43, no. 2, pp. 140-145, 2007.
[16] G. Obernosterer, J. Martinez, and M. J. N. p. Alenius, "Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections," vol. 2, no. 6, p. 1508, 2007.
[17] M. J. Søe, T. Møller, M. Dufva, K. J. J. o. H. Holmstrøm, and Cytochemistry, "A sensitive alternative for microRNA in situ hybridizations using probes of 2′-O-methyl RNA+ LNA," vol. 59, no. 7, pp. 661-672, 2011.
[18] P. Mouritzen, A. T. Nielsen, H. M. Pfundheller, Y. Choleva, L. Kongsbak, and S. J. E. r. o. m. d. Møller, "Single nucleotide polymorphism genotyping using locked nucleic acid (LNA™)," vol. 3, no. 1, pp. 27-38, 2003.
[19] P. E. Nielsen, M. Egholm, R. H. Berg, and O. J. S. Buchardt, "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide," vol. 254, no. 5037, pp. 1497-1500, 1991.
[20] A. Ray and B. J. T. F. J. Norden, "Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future," vol. 14, no. 9, pp. 1041-1060, 2000.
[21] H. Stender et al., "Direct detection and identification of Mycobacterium tuberculosis in smear-positive sputum samples by fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes," vol. 3, no. 9, pp. 830-837, 1999.
[22] K. Oliveira, G. W. Procop, D. Wilson, J. Coull, and H. J. J. o. c. m. Stender, "Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes," vol. 40, no. 1, pp. 247-251, 2002.
[23] T.-L. Li et al., "Designed phosphate-methylated oligonucleotides as PCR primers for SNP discrimination," pp. 1-10, 2019.
[24] Y.-n. Lin, "Phosphate-Methylated DNA as Neutralized DNA (nDNA)：Synthesis, Properties and Potential Applications," Department of Chemical and Materials Engineering,National Central University, 2015.
[25] L. H. Koole et al., "Synthesis of phosphate-methylated DNA fragments using 9-fluorenylmethoxycarbonyl as transient base protecting group," vol. 54, no. 7, pp. 1657-1664, 1989.
[26] Y.-J. W. C. Chen, "Studies of thermodynamic and mechanism for neutralized DNA (nDNA)/DNA and DNA/DNA duplex formation. ," Master degree, Department of Chemical and Materials Engineering, National Central University, 2016.
[27] M. P. R. Paul S. Miller, * Akira Murakami, Kathleen R. Blake, Shwu-Bin Lin, and Cheryl H. Agris, "Solid-Phase Syntheses of Oligodeoxyribonucleoside Methylphosphonates1," Biochemistry, 1986.
[28] H. M. Moody, M. H. van Genderen, L. H. Koole, H. J. Kocken, E. M. Meijer, and H. M. Buck, "Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides," Nucleic Acids Research, vol. 17, no. 12, pp. 4769-4782, 1989.
[29] B. R. Meade et al., "Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications," Nat Biotechnol, vol. 32, no. 12, pp. 1256-61, Dec 2014.
[30] Y.-H. Chang, "Specificity enhancement of PCR and qPCR by using neutralized DNA (nDNA) as primer or targeting probe," master degree, Department of Chemical and Materials Engineering., National Central University 2017.
[31] L.-C. Li et al., "A single nucleotide polymorphism in the E-cadherin gene promoter alters transcriptional activities," vol. 60, no. 4, pp. 873-876, 2000.
[32] D. Fraga, T. Meulia, and S. J. C. p. e. l. t. Fenster, "Real‐time PCR," no. 1, pp. 10.3. 1-10.3. 34, 2008.
[33] J. G. J. M. Gall, "The origin of in situ hybridization–a personal history," vol. 98, pp. 4-9, 2016.
[34] M. Tanner et al., "Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples," vol. 157, no. 5, pp. 1467-1472, 2000.
[35] J. M. Levsky and R. H. J. J. o. c. s. Singer, "Fluorescence in situ hybridization: past, present and future," vol. 116, no. 14, pp. 2833-2838, 2003.
[36] J. B. Bramsen et al., "Improved silencing properties using small internally segmented interfering RNAs," vol. 35, no. 17, pp. 5886-5897, 2007.
[37] J. T. Pena et al., "miRNA in situ hybridization in formaldehyde and EDC–fixed tissues," vol. 6, no. 2, p. 139, 2009.
[38] M. O Urbanek, A. U Nawrocka, and W. J Krzyzosiak, Small RNA Detection by in Situ Hybridization Methods. 2015, pp. 13259-13286.
[39] S. Fontenete et al., "Application of locked nucleic acid-based probes in fluorescence in situ hybridization," Appl Microbiol Biotechnol, vol. 100, no. 13, pp. 5897-906, Jul 2016.
[40] M. Bogdanovska-Todorovska, G. Petrushevska, V. Janevska, L. Spasevska, and S. J. B. j. o. b. m. s. Kostadinova-Kunovska, "Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience," vol. 18, no. 2, p. 132, 2018.
[41] A. N. Silahtaroglu et al., "Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification," vol. 2, no. 10, p. 2520, 2007.
[42] J. Ge, L.-L. Zhang, S.-J. Liu, R.-Q. Yu, and X. J. A. c. Chu, "A highly sensitive target-primed rolling circle amplification (TPRCA) method for fluorescent in situ hybridization detection of microRNA in tumor cells," vol. 86, no. 3, pp. 1808-1815, 2014.
[43] F. Wang et al., "RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues," vol. 14, no. 1, pp. 22-29, 2012.
[44] I. Balcells, S. Cirera, and P. K. J. B. b. Busk, "Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers," vol. 11, no. 1, p. 70, 2011.
[45] B. S. Nielsen et al., "High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients," vol. 28, no. 1, pp. 27-38, 2011.
[46] G. Obernosterer, P. J. Leuschner, M. Alenius, and J. J. R. Martinez, "Post-transcriptional regulation of microRNA expression," vol. 12, no. 7, pp. 1161-1167, 2006.
[47] J.-J. Zhao et al., "Identification of miRNAs associated with tumorigenesis of retinoblastoma by miRNA microarray analysis," vol. 25, no. 1, pp. 13-20, 2009.
[48] M.-W. Wu, "The thermodynamic aspects of the Na+ and the mismatch discrimination on the formation of double stranded DNA containing site-specific methyl phosphotriester linkages," 2018.
[49] A. Válóczi, C. Hornyik, N. Varga, J. Burgyan, S. Kauppinen, and Z. J. N. a. r. Havelda, "Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes," vol. 32, no. 22, pp. e175-e175, 2004.
[50] T.-L. Li, "MicroRNA In Situ Hybridization with Phosphate-methylated oligonucleotides (nDNA) Probe," Master degree, Department of Chemical and Materials Engineering, National Central University, 2018.
[51] K. Ziomek, E. Kierzek, E. Biała, and R. J. B. c. Kierzek, "The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides," vol. 97, no. 2-3, pp. 233-241, 2002.
[52] U. Singh et al., "General principles and methods for routine automated microRNA in situ hybridization and double labeling with immunohistochemistry," vol. 89, no. 4, pp. 259-266, 2014.
[53] W. Tang et al., "MicroRNA-29a promotes colorectal cancer metastasis by regulating matrix metalloproteinase 2 and E-cadherin via KLF4," vol. 110, no. 2, p. 450, 2014.
[54] J. Zhao, Y. Zhang, and G. J. C. B. Zhao, "Emerging role of microRNA-21 in colorectal cancer," vol. 15, no. 3, pp. 219-226, 2015.
[55] S. Bommarito, N. Peyret, and J. S. J. N. a. r. Jr, "Thermodynamic parameters for DNA sequences with dangling ends," vol. 28, no. 9, pp. 1929-1934, 2000.
[56] P. S. Miller, L. T. Braiterman, and P. O. J. B. Ts’o, "Effects of a trinucleotide ethyl phosphotriester, Gmp (Et) Gmp (Et) U, on mammalian cells in culture," vol. 16, no. 9, pp. 1988-1996, 1977.
[57] M. Y. Shah, A. Ferrajoli, A. K. Sood, G. Lopez-Berestein, and G. A. Calin, "microRNA Therapeutics in Cancer — An Emerging Concept," EBioMedicine, vol. 12, pp. 34-42, 2016.
[58] C. K. Raymond, B. S. Roberts, P. Garrett-Engele, L. P. Lim, and J. M. J. R. Johnson, "Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs," vol. 11, no. 11, pp. 1737-1744, 2005.
[59] A. J. Kriegel, Y. Liu, Y. Fang, X. Ding, and M. J. P. g. Liang, "The miR-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury," vol. 44, no. 4, pp. 237-244, 2012.
[60] J. Zhang et al., "MiR-29c mediates epithelial-to-mesenchymal transition in human colorectal carcinoma metastasis via PTP4A and GNA13 regulation of β-catenin signaling," vol. 25, no. 11, pp. 2196-2204, 2014.
[61] S. Kwok et al., "Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies," vol. 18, no. 4, pp. 999-1005, 1990.
[62] S. J. Johnson and L. S. J. C. Beese, "Structures of mismatch replication errors observed in a DNA polymerase," vol. 116, no. 6, pp. 803-816, 2004.
[63] M.-M. Huang, N. Arnheim, and M. F. J. N. a. r. Goodman, "Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR," vol. 20, no. 17, pp. 4567-4573, 1992.