利用質譜儀進行DNA修飾的酵素學研究

Study of enzymatic DNA modifications by MALDI-TOF MS analysis

曹禮醫(LI-I TASO)

指導教授 ： 方偉宏

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2020年)

https://doi.org/10.6342/NTU202003051

摘要

MALDI-TOF質譜儀被廣泛運用於許多領域，包括:胺基酸、蛋白質、核酸、微生物等分析，又其高解析力特點可偵測DNA上不同鹼基中微小質量的差異，已被應用於單核苷酸引子延伸法中，亦即使用無標記的雙去氧核醣核苷酸三磷酸(ddNTPs)進行引子延伸反應，再以MALDI-TOF分析，即可得知基因特定位置的多形性。先前實驗室學長姊將單核苷酸引子延伸的概念延伸應用於探討第一型聚合酶對於internal mismatch的校正能力。因此我想探討MALDI-TOF質譜儀在檢測DNA修飾上的可行性，包括:DNA甲基化、DNA磷酸化與DNA去磷酸化。首先，先以EcoRI methyltransferase測試，發現給予8 U EcoRI methyltransferase反應16小時可以使單股含一特異性甲基化位點的寡核苷酸DNA序列全部加上甲基，於圖譜上呈現比原訊號多14分子量的訊號。同樣給予8 U EcoRI methyltransferase反應10、20、30分鐘，可分別得到32.43%、51.26%、58.86%的甲基化反應效率。確定MALDI-TOF質譜儀可成功檢測DNA甲基化後，我接著測試CpG methyltransferase (M. SssI)，給予2 U M. SssI反應30分鐘可以發現有30.1%的引子股被甲基化，給予4 U反應30、60分鐘則分別有67.2%和76.5%的引子股被甲基化，並發現2 U與4 U反應30分鐘時存在劑量依賴性的現象。
我也對DNA磷酸化修飾以MALTI-TOF MS進行偵測，給予0.1、0.5的T4 polynucleotide kinase分別反應10分鐘，都發現於圖譜上顯示比原訊號向右位移78的分子量，表示於單股寡核苷酸序列的5’端加上一個磷酸根。給予0.05、0.1 U分別反應1、3分鐘，發現0.1 U反應1分鐘有56.06%的反應產物，3分鐘則全反應；0.05 U反應 1分鐘有 8.43%的反應產物，3分鐘有27.73%的反應產物。
最後測試DNA的去磷酸化，我以於5’端有磷酸化修飾的P3U-5'P單股寡核苷酸序列為受質，給予0.1 U的Calf intestinal alkaline phosphatase反應1分鐘，可於圖譜上發現比原訊號向左位移78的分子量，代表原本的寡核苷酸上的磷酸根被去除。給予0.0001 U反應1分鐘有12.43±0.62%，2分鐘有18.26±0.80%，3分鐘有22.93±1.27%的反應產物；0.0002 U反應1分鐘有21.99±3.37%，2分鐘有34.20±2.58%，3分鐘有50.30±9.18%的反應產物；0.0004 U反應1分鐘有67.15±9.61%，2分鐘有74.66±6.43%，3分鐘有83.61±2.27%的反應產物。
經初步研究顯示，MALDI-TOF質譜儀適用於測定核酸修飾蛋白的活性。

關鍵字

基質協助雷射去吸附離子化-飛行時間質譜儀 ； DNA甲基化 ； DNA磷酸酸化 ； DNA去磷酸化 ； EcoRI甲基化酶 ； CpG甲基化酶 ； T4多核苷酸激酶 ； 小牛腸鹼性磷酸酶

並列摘要

MALDI-TOF has been widely used in many fields, including analysis of amino acids, protein, nucleic acid, microorganisms and so on. Its high resolution can detect the small mass of various bases on DNA, and has been used in single-nucleotide primer extension method, that is, the use of unlabeled dideoxyribonucleotide triphosphates (ddNTPs) for primer extension reaction, followed by MALDI-TOF to analyze the polymorphism of the specific location of the gene. In our previous experiment of our lab, we extend the concept of single-nucleotide primer extension to explore the proofreading characteristic of polymerase I for internal mismatch correction.
Here, we try to explore the practicability of MALDI-TOF MS to detect different types of DNA modification, including DNA methylation, DNA phosphorylation, and DNA dephosphorylation. First, we test the activity of EcoRI methyltransferase. In the present of 8 U EcoRI methyltransferase and reacted for 16 hours, all the single strand oligonucleotide DNA substrates containing a specific enzyme recognition site are methylated. A signal with extra 14 daltons compared to the original signal is displayed on the spectrum. Reducing the reaction time to 10, 20, and 30 minutes result in the methylation reaction efficiency is 32.43%, 51.26%, and 58.86%, respectively. After ensuring MALDI-TOF can successfully detect DNA methylation, I than tested the CpG methyltransferase (M.Sssl) activity assay. By adding 2 U M.Sssl in a 30 minutes reaction, I found that about 30.1% of primer are methylated. In the presented of 4 U M.Sssl for 30 and 60 minutes, and the primer methylation reaction efficiency is 67.2% and 76.5%, respectively. I found that there was a dose-dependent phenomenon between 2 U and 4 U enzyme reaction when reacted for 30 minutes.
After testing DNA methylation, I further want to know whether DNA phosphorylation modification can be detected by MALTI-TOF MS. I applied 0.1, 0.5 and 1 U of T4 PNK and react for 10 minutes, and all of reaction products showed a molecular weight shifted to the right by 78 daltons from the original signal, indicating that a phosphate group was added to the single stranded oligonucleotide substrate. I than reduced the enzyme amount to 0.05 and 0.1 U and react for 1 and 3 minutes. The result showed that 0.05 U produce 8.43% of reaction product in 1 minutes and 27.73% reaction product in 3 minute; 0.1 U T4 PNK produced 56.06% of reaction product in 1 minute, and complete reaction in 3 minutes.
Then we tested the dephosphorylation of DNA. We used the P3U-5'P single-stranded oligonucleotide sequence with phosphorylation modification at the 5'end as the substrate and gave 0.1 U of CIP in reaction for 1 minutes, we observed the molecular weight shift to the left by 78 daltons from the original substrate signal on the spectrum, which means that the phosphate on the oligonucleotide substrate was removed. I than reduced the enzyme amount to 0.0001、0.0002 and 0.0004 U react for 1、2 and 3 minutes. The result showed that 0.0001 U CIP produced 12.43±0.62% of reaction product in 1 minute, 18.26±0.80% of reaction product in 2 minutes, 22.93±1.27% of reaction product in 3 minutes; 0.0002 U CIP produced 21.99±3.37% of reaction product in 1 minute, 34.20±2.58% of reaction product in 2 minutes, 50.30±9.18% of reaction product in 3 minutes; 0.0004 U CIP produced 67.15±9.61% of reaction product in 1 minute, 74.66±6.43% of reaction product in 2 minutes, 83.61±2.27% of reaction product in 3 minutes.
In conclusion, MALDI-TOF MS is an excellent tool for DNA modification enzyme analysis.

並列關鍵字

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry ； DNA methylation ； DNA phosphorylation ； DNA dephosphorylation ； EcoRI methyltransferase ； CpG methyltransferase ； T4 polynucleotide kinase ； Calf intestinal alkaline phosphatase

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