Application Of Special Secondary Structures Of Nucleic Acids In DNA Methyltransferase And Breast Cancer Detection

In this thesis, there are detailed introduction about the special secondary structures of nucleic acids (DNAzyme and aptamer) and corresponding unique functions of these structures. We have established two kinds of new disease diagnosis methods by using these special structures and functions.There are two parts of research contents in this article. In the first part, taking advantage of the substrate specific incising of DNAzyme, we have designed a new method of Dam DNA methyltransferase detection based on signal amplification of DNAzyme cleaving. In the second part, taking advantage of targets selectivity of nuleic acid aptamer, we have developed a novel approach combining aptamer targeting and photoactivatable fluorescent signal output, and applied it to breast cancer dignosis.The main content of the first part:DNA methylation is one of the most important epigenetic modifications and it is the process whereby the methyl group from donor SAM (S-adenosylmethionine) transfers to a target group on cytosine or adenine under the action of MTase. It has attract intense interest in the past decades due to its close relationship to gene activation, gene imprinting, chromatin stability, X-chromosome inactivation in females and several diseases. It has also been reported that levels of DNA methylation in different species or organs vary and are in precise regulation. Improper regulation of methylation would cause various diseases, therefore, DNA methylation is an important diagnostic biomarker and activity of MTase also becomes significant in fundamental study and clinical diagnosis due to its correlation to DNA methylation extent. Herein, using DNAzyme, the special secondary structure of nucleic acid, we designed a novel harpin shaped probe for Dam MTase detection. Once the specific sequence of the probe is recognized, the adenine in the sequence was methylated. Then the methylated sequence could be recognized by DpnI restriction endonuclease, allowing digestion at that specific site. After digestion, the 8-17 DNAzyme sequence would be released, resulting in the recovery of the cleavage activity toward its substrate. An amplified signal output could be achieved through the cycling of the released 8-17 DNAzyme. Thus, the activity of Dam MTase could be monitored by means of fluorescent signal output. The strategy was verified by fluorescent dynamic studies. Then, gel electrophoresis was performed to clarify the explicit digestion of the hairpin-shaped DNA probe in our system. Dam MTase inhibitor study showed that this strategy got was the signal of Dam MTase activity. In summary, our strategy based on 8-17 DNAzyme for detection of Dam MTase was demonstrated to be extremely simple, sensitive and effective.Cancer is considered as one of the primary causes of morbidity and mortality worldwide. Tumours can grow uncontrollably and interfere with the digestive, nervous, and circulatory systems. Even worse, they can release hormones that alter bodily functions. Cancer is difficult to detect and diagnose because it generally grows from the organ itself and is thus anti-immune, as the immune system cannot automatically recognize it and clear it. Most cancer patients have no characteristic clinical manifestation before onset; however, once malignant tumours form, the patient's condition is acute and more dangerous as a result of missing the opportunity for early treatment. Therefore, early cancer diagnosis is of crucial importance for cancer treatment. Traditional techniques have been restricted to the morphological changes of normal cells into tumours, but these methods are unable to meet the demands of early cancer diagnosis. The development of molecular-level differentiation methods has contributed to early diagnosis and prompted therapy. By combining AS 1411 aptamer, the special secondary structure of nucleic acids, and photoactivatable technique, we developed a selective and sensitive detection method to nucleolin, the target of the aptamer. At first, a sythetic photoactivatable fluorescent small molecule was successfully labeled on the AS 1411 nucleic acid aptamer, and then the photoactivatable aptamer fluorescent probe was got. Next, fluorescent analysis, polyacrylamide gel electrophoresis and DNA mass proved that this probe has the property of photoactivatable fluorescent response. And cell experiments and tissue biopsy experiments showed that this strategy is extraordinary sensitive and selective. Significantly, the application of this strategy can produce the same accurate results with gold-standard diagnostic technology. More accurate and persistent signals can be obtained by subtracting the primary untreated image signal from the light-activated signal.