MicroRNAs (miRNAs)為一種非編碼的小片段RNA，在調節基因表達中發揮著重要的作用。然而，目前使用反義寡核苷酸與目標miRNA雜交作為檢測或抑制miRNA的方法，而這些方法包含原位雜交技術(ISH)、RT-qPCR和miRNA微陣列分析技術，但上述這些方法皆缺乏雜交的專一性及親和力。因此為了改善這些問題，我們在核酸序列探針上修飾中性去氧核醣核酸 (nDNA)，nDNA為一種人工DNA類似物，其磷酸骨幹經過特定位置的甲基化改質後，使帶負電的磷酸根轉變成磷酸三酯鍵而形成電中性，其可減少核酸雙股同時帶負電造成的靜電排斥效應，因此具有較佳的雜交穩定性。 本研究表明，藉由RT-qPCR檢測結果所示，部分甲基化的反義nDNA探針(N4 nDNA，具有四個nDNA修飾)可比DNA 探針更有效地抑制在人體血漿培養基中的miR-21進行逆轉錄反應(N4 nDNA探針具有較高的Ct值)。接著，我們使用具有miR-21表達的人類結腸癌細胞(HCT116)進行原位雜交技術(ISH)的檢測，使用N4 nDNA探針檢測HCT116細胞中的miR-21，可顯示出比使用一般DNA探針檢測到更高的染色訊號強度且具有檢測專一性。同樣地，藉由酵素結合免疫吸附分析法(ELISA)表明N4 nDNA探針與miRNA雜交的效率高於DNA探針。此外，N4 nDNA探針在細胞中不會引起免疫反應(與國家衛生研究院紀雅惠博士合作)，並且N4 nDNA探針在體外無細胞蛋白質合成(CFPS)系統中可抑制GFP mRNA進行轉譯(與日本東京工業大學地球生命研究所的Kosuke Fujishima教授合作)。 由於N4 nDNA探針對於DNase I具有抵抗能力並且不會在細胞中引發強烈的免疫反應，因此未來在開發nDNA探針作為調節體內基因表達的治療性核酸藥物是具有可能性的。;MicroRNAs (miRNAs), a class of short non-coding RNA, play important roles in regulating gene expression. However, current miRNA hybridization methods for miRNA detection/inhibition using antisense oligonucleotides, including in situ hybridization (ISH), RT-qPCR, and miRNA microarray profiling technology, are lacking hybridization specificity and affinity. As such, to ameliorate these problems, the neutralized DNA, an emerging class of DNA oligonucleotides chemically synthesized with site-specific internucleoside methyl phosphotriester linkages was used as a hybridization probe instead of DNA. The reduction of inter-strand charge repulsion of nucleotide duplexes results in stronger binding between nDNA and other nucleic acids. This study suggested that partially methylated antisense nDNA (N4 nDNA, with four modifications) probes inhibited reverse transcription of miR-21 more efficiently (higher Ct value) than DNA probes in the human plasma-like medium, as demonstrated by the RT-qPCR assays. Subsequently, we performed in situ hybridization analysis using a miR-21-expressing colorectal cancer cell line (HCT116). HCT116 cells stained with N4 nDNA antisense probes revealed a greater detection intensity and specificity than cells stained with DNA probes. Consistently, enzyme-linked immunosorbent assays (ELISA) suggested that the miRNA hybridization efficiency of N4 nDNA antisense probes was greater than that of DNA probes. In addition, the cell-based immune response of the N4 nDNA antisense probe was immune-negative (cooperated with Dr. Ya-Hui Chi at National Health Research Institutes) and the N4 nDNA antisense prob was also inhibited in vitro GFP mRNA translation (cooperated with Prof. Kosuke Fujishima at Earth-Life Science Institute). As N4 nDNA probes are resistant to DNase I and do not elicit a strong immune response in cells, future development of nDNA probes as therapeutic nucleic acid drugs for in vivo gene expression modulation is possible.
