本文選題：甲基化轉(zhuǎn)移酶 + DNAzyme　； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：在本論文中,我們對核酸的一些特殊二級結(jié)構(gòu)(脫氧核酶和適配子)及其特有的功能進(jìn)行了探討,并通過利用這些特殊二級結(jié)構(gòu)及其特有的功能,建立了兩種與癌癥疾病診斷相關(guān)的新方法,并對這些檢測方法進(jìn)行了深入的研究。本論文的研究內(nèi)容主要分為兩個大的部分,第一部分是利用DNAzyme能夠選擇性切割底物序列的特點(diǎn),設(shè)計了一種基于DNAzyme切割信號放大來對DamDNA甲基轉(zhuǎn)移酶活性進(jìn)行檢測的新方法。在第二部分工作中,我們利用核酸適配子對靶標(biāo)的選擇性,建立了一種結(jié)合適配子靶向和光激活熒光信號輸出的新方法,并成功運(yùn)用于乳腺癌的相關(guān)檢測中。第一部分的內(nèi)容包括：DNA甲基化是通過甲基轉(zhuǎn)移酶的作用,將供體SAM上的甲基轉(zhuǎn)移到受體胞嘧啶或腺嘌呤上的,它是一種非常重要表觀遺傳學(xué)修飾。由于DNA甲基化與基因激活、基因印記、染色質(zhì)穩(wěn)定性、女性中X-染色體失活和眾多疾病的發(fā)生密切相關(guān),DNA甲基化的研究在近些年中備受關(guān)注。有報道表明DNA甲基化的程度在不同的物種和不同的器官各不相同,且均有自身準(zhǔn)確的調(diào)控。由于甲基化的失調(diào)會造成各種疾病發(fā)生,DNA甲基化被視為是臨床診斷中一種潛在的重要標(biāo)志物。因為甲基轉(zhuǎn)移酶的活性直接影響著DNA甲基化程度,所以甲基轉(zhuǎn)移酶活性的檢測對基礎(chǔ)研究和臨床診斷都至關(guān)重要。我們利用DNAzyme這種特殊的核酸二級結(jié)構(gòu),并設(shè)計了一種新的發(fā)卡結(jié)構(gòu)的探針,用于Dam甲基轉(zhuǎn)移酶的檢測。一旦探針中的特定序列被Dam甲基轉(zhuǎn)移酶所識別,序列中的腺嘌呤會被甲基化,而甲基化后的序列可以被DpnI限制性內(nèi)切酶識別并切割,從而釋放出含有8-17 DNAzyme序列的單鏈,使DNAzyme的切割活性恢復(fù),對分子信標(biāo)結(jié)構(gòu)底物進(jìn)行切割,使熒光強(qiáng)度提高。同時,切割底物后的DNAzyme序列可以再與新的底物結(jié)合并將其切割,產(chǎn)生循環(huán)信號放大。最終,輸出的熒光信號即為Dam甲基轉(zhuǎn)移酶的活性。我們通過熒光動力學(xué)的方法驗證了該策略的可行性,并且通過凝膠電泳實(shí)驗證明了甲基化后的探針被成功切割,最后通過甲基轉(zhuǎn)移酶的抑制劑實(shí)驗證實(shí)了該策略得到的信號即為甲基轉(zhuǎn)移酶的活性。因此,我們這種基于8-17 DNAzyme來檢測Dam甲基轉(zhuǎn)移酶的方法具有簡單、靈敏、高效的特點(diǎn)。第二部分的主要內(nèi)容包括：癌癥被認(rèn)為世界范圍內(nèi)發(fā)病和死亡的主要原因之一。惡性腫瘤能夠無限制地增長,進(jìn)而干預(yù)到消化系統(tǒng)、神經(jīng)系統(tǒng)和循環(huán)系統(tǒng)。更糟糕的是,它們能通過釋放激素來改變身體的機(jī)能。癌癥難以檢測,因為它生長于器官本身,人體的免疫系統(tǒng)不能將其識別和清除。大部分癌癥患者在病發(fā)前,并沒有明顯的臨床癥狀。然而一旦惡性腫瘤形成,病人的條件會急劇惡化。因此,早期癌癥診斷對于癌癥的治療異常重要。傳統(tǒng)的癌癥檢測方法需要從腫瘤形態(tài)學(xué)變化上來進(jìn)行診斷,不能滿足癌癥早期檢測的要求。發(fā)展從分子水平上區(qū)分癌癥部位與正常部位的方法,對于癌癥的早期診斷和及時治療都有著非常重要的意義。我們選取了AS1411這種具有特殊二級結(jié)構(gòu)的核酸適配子序列,結(jié)合光激活熒光成像技術(shù),建立起了一種對該適配子的靶標(biāo)——核仁素高特異性和高靈敏度的檢測方法,并將其運(yùn)用到乳腺癌的檢測中。在這項工作中,我們首先合成了具有光激活響應(yīng)的熒光小分子,并將其成功標(biāo)記到AS1411核酸適配子上,制備出具有光激活響應(yīng)的適配子熒光探針。然后,我們通過熒光檢測、凝膠電泳和DNA質(zhì)譜證明了該探針具有光激活熒光響應(yīng)的性質(zhì)。最后,我們通過細(xì)胞實(shí)驗和組織切片實(shí)驗證明了該策略對乳腺癌細(xì)胞具有高靈敏度和高選擇性,能夠達(dá)到和臨床上一些金標(biāo)準(zhǔn)診斷方法相同的準(zhǔn)確程度,通過對比光激活前后的熒光圖像,可以得到更加準(zhǔn)確和持續(xù)性的信號。
[Abstract]:In this paper, we explored some special two structures of nucleic acid (deoxy ribozyme and aptamer) and their specific functions. Through the use of these special two grade structures and their specific functions, we have established two new methods related to the diagnosis of cancer diseases, and studied these methods in depth. The main part of the study is divided into two major parts. The first part is the design of a new method for detecting the activity of DamDNA methyltransferase based on DNAzyme cutting signal amplification by using DNAzyme to selectively cut the substrate sequence. In the second part, we use the selectivity of the nucleic acid aptamer to target the target. A new method combining aptamer targeting and light activated fluorescence signal output is successfully applied to the detection of breast cancer. The first part includes: DNA methylation is a very important epigenetic inheritance of methyl transferase on the receptor cytosine or adenine through the role of methyl transferase on the donor SAM. The study of DNA methylation, gene activation, gene imprinting, chromatin stability, X- chromosome inactivation in women is closely related to the occurrence of many diseases. The study of DNA methylation has attracted much attention in recent years. It has been reported that the degree of DNA methylation is different in different species and different organs, and has its own accuracy. Regulation. Due to maladjustment of methylation can cause a variety of diseases, DNA methylation is regarded as a potential important marker in clinical diagnosis. As the activity of methyltransferase directly affects the degree of DNA methylation, the detection of methyltransferase activity is essential for both basic and clinical diagnosis. We use DNAzyme Special nucleic acid two structure and a new hairpin structure probe is designed for the detection of Dam methyltransferase. Once the specific sequence in the probe is identified by Dam methyltransferase, the adenine in the sequence can be methylated, and the sequence after methylation can be identified and cut by the DpnI restrictive endonuclease. The single strand of 8-17 DNAzyme sequences restores the cleavage activity of DNAzyme, cutting the molecular beacon structure substrate and improving the fluorescence intensity. At the same time, the DNAzyme sequence after the cutting substrate can be combined with the new substrate and cut it to produce the cyclic signal amplification. Finally, the output fluorescence signal is the activity of the Dam methyltransferase. We verified the feasibility of the strategy by fluorodynamic method and proved that the methylation probe was successfully cut through the gel electrophoresis experiment. Finally, the signal obtained by the methyltransferase inhibitor was proved to be the activity of the methyltransferase. Therefore, we detect Dam based on 8-17 DNAzyme. The methylation enzyme method has a simple, sensitive, and efficient feature. The main contents of the second part include: cancer is considered to be one of the major causes of disease and death worldwide. Malignant tumors can grow unrestricted, and then interfere with the digestive system, the nervous system and the loop system. And, worse, they can release hormones. It is difficult to detect the function of the body. Cancer is difficult to detect because it grows in the organ itself, the immune system of the human body can not identify and remove it. Most cancer patients have no obvious clinical symptoms before the onset of the disease. However, once the malignant tumor is formed, the condition of the patient will be drastically evil. Therefore, early cancer diagnosis of cancer treatment The traditional cancer detection methods need to be diagnosed from the morphological changes of cancer and can not meet the requirements of the early detection of cancer. It is very important for the early diagnosis and timely treatment of cancer to develop the methods to distinguish the cancer sites from the normal parts at the molecular level. We have selected the AS1411. With a special two stage structure of nucleic acid aptamer sequence, combined with light activated fluorescence imaging, a detection method for the high specificity and sensitivity of the target, the target of the aptamer, is established and applied to the detection of breast cancer. In this work, we first synthesized a small fluorescent molecule with light activation response. By labeling its success to the AS1411 nucleic acid aptamer, the aptamer fluorescent probe with light activation response was prepared. Then, we demonstrated the properties of the photoactivated fluorescence response by fluorescence detection, gel electrophoresis and DNA mass spectrometry. Finally, we proved the strategy to breast cancer by cell test and tissue section experiment. The cells have high sensitivity and selectivity, and can achieve the same accuracy as some of the clinical gold standard diagnostic methods. By comparing the fluorescence images before and after light activation, more accurate and persistent signals can be obtained.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：O657.3;R737.9

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