利用單分子操控術進行限制性內切核酸酶EcoRI之力化學反應速率定量量測

Quantifying the Mechanochemical Reaction Rates of the Restriction Endonuclease EcoRI by Single Molecule Techniques

蔡靜欣(Ching-Shin Tsai)

指導教授 ： 楊自森

臺北醫學大學/口腔醫學院/生醫材料暨工程研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00001

摘要

限制性內切核酸酶 (restriction endonuclease) 是由細菌體內所發現，可將外源入侵的DNA片段精準地切除。因此利用限制性內切核酸酶的特性來酶切雙股DNA (double-stranded DNA) 特殊序列，是近年來重要的分子技術。由於傳統的分子生物技術無法直接觀察限制性內切酶酶切DNA的過程，本研究將利用單分子生物技術搭配螢光顯微系統，觀察限制性內切核酸酶EcoRI在空間中如何移動尋找?軵NA上的特殊位點。當DNA兩端分別與乳膠珠結合後，使用光鉗系統將乳膠珠箝制並將乳膠珠向下拉至蓋玻片表面固定。再利用微量注射幫浦將與量子點(quantum dots)結合的限制性內切核酸酶(EcoRI)送入觀測區，即可用倒立式螢光顯微鏡觀察EcoRI在空間中移動的行為。從實驗結果中得知，對?軵NA施予1 pN以及14 pN的拉力來觀察QD-EcoRI的移動軌跡，發現施予1 pN的力量時，QD-EcoRI結合上辨識位點後所產生的位移變化量較大，?軵NA仍可在兩個乳膠珠間進行小幅度的擺盪。而施予14 pN的力量時所產生的位移變化量較小。從14 pN的X-Y-T圖中可很清楚的觀察到當QD-EcoRI結合至位點後，會對於所處的位置進行X方向小幅度的移擺盪，並隨著結合時間的增加而減少擺盪；Y方向的位移變化量則是QD-EcoRI隨著?軵NA擺盪而產生的位移量。實驗中又利用EDTA溶液進入QD-EcoRI的微通道後，成功可觀察到QD-EcoRI與?軵NA由結合狀態過渡到解離狀態的反應過程。

關鍵字

雷射光鉗 ； 限制性內切核酸酶力化學反應速率 ； 量子點

並列摘要

Restriction endonuclease is found in bacteria and can precisely cut off the DNA sequence specificity invaded from outside. Nowadays, using the restriction endonuclease to cleavage the double-stranded DNA (dsDNA) at specific recognition nucleotide sequences is regarded as an important biotechnology. However, the traditional approaches cannot directly observe the process of specific cleavage of DNA by the restriction endonuclease. In this thesis, we presented single molecule biotechnology, together with the single molecule fluorescence detection system, to directly observe how the EcoRI find the recognition binding site. After the biotinylated ends of the bacteriophage???軵NA were attached to streptavidin-coated beads, these two beads were then stuck on the cover glass surface via optical tweezers, followed by delivering the QD-EcoRI into the observation region. In this study, we found that the distributions of the QD-EcoRI trajectory are significantly different under different extension forces exerted on ?軵NA. On the other hand, we found that the dissociation of QD-EcoRI occurred while delivering EDTA buffer into the observation region, this suggested that the association is an reversible process.

並列關鍵字

Laser Tweezers ； Restriction Endonuclease ； Mechanochemical Reaction Rates ； Quantum Dots

參考文獻

1.Kim, K., Modrich, P. and Kim, S-H.. 'Interactive' recognition in EcoK1 restriction enzyme-DNA complex. Nucleic Acids Research, 12, 7285-7202 (1984)

2.Bram van den Broek, Maarten C. Noom and Gijs J. L. Wuite DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway. Nucleic Acids Research, 33:2676-2684. (2005)

3.Bensimon,A., Simon,A., Chiffaudel,A., Croquette,V., Heslot,F. and Bensimon,D. Alignment and sensitive detection of DNA by a moving interface. Science, 265, 2096–2098. (1994)

4.Blainey,P.C., van Oijen,A.M., Banerjee,A., Verdine,G.L. and Xie,X.S. A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. Proc. Natl Acad. Sci. USA, 103, 5752–5757. (2006)

5.Smith SB, Cui Y, Bustamante C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science. 271(5250):795-9. (1996)