New Fluorescent Strategies Based On Chemical Reaction To Detect 5-Hydroxymethylcytosine And 5-Formylcytosine

In this thesis, we studied the application of small fluorescent probes that were used in the detection of DNA damage. Our study focused on the problem that how can we develop selective and sensitive small fluorescent probes which can be used for the qualitative and quantitative detection of 5-formylcytosine and 5-hydroxymethylcytosine. DNA methylation, in its various forms, is the key content of epigenetics. DNA damage has close relationship with many diseases, so study of its distribution can help us to know the relationship between DNA damage and many kinds of diseases deeply. As a result, there is high significance to carry out the detection of 5-hydroxymethylcytosine and 5-formylcytosine in DNA using small fluorescent probes, and it will help us a lot in the field of diagnosis and treatment of diseases.In this thesis, we discussed three strategies based on fluorescence that were used for the qualitative and quantitative detection of 5-formylcytosine and 5-hydroxymethycytosine:strategy one was based on direct fluorescent labeling; strategy two was based on fluorescent "Turn off’; strategy three was based on fluorescent "Turn on".In the first strategy, fluorescent probes can be labeled to the specific genomic loci through one step. This method has the advantage of convenience, direction and sensitiveness. Strategies of fluorescent "Turn off’ and "Turn on" can be used as a tracer for the detection of DNA damage in vivo and it can eliminate the interference of biomolecules that contains aldehyde group as 5-formylcytosine. However, the change of fluorescence should be carried out at the help of specific metal ions. So these three strategies can be used to supplement and complement to each other.Strategy based on fluorescent labeling:We designed and synthesized small fluorescent probes that contain specific groups, the probes can specificly and effectively react with 5-formylcytosine. After labeling, DNA sequence has strong fluorescent signals. The signals can be detected through PAGE analysis or fluorescent spectroscopy. The polarity of DNA differs a lot before and after labeling by fuorescent probe, so it can be distinguished through HPLC and the reaction yield can also be calculated. Usually, the reaction to form Schiff base is reversible, so the product is unstable and has a potensial to hydrolysis. We think the reason is that:The double bond between the carbon and the nitrogen of Schiff base can form a intromolecular hydrogen bond with the amino group of 5-formylcytosine at the position four. So the Schiff base is very stable and hydrolysis rarely happens. Oxidants, such as manganese dioxide and potassium perruthenate can both oxidize 5-hydroxymethylcytosine to 5-formylcytosine selectively. Potassium perruthenate can oxidize 5-hydroxymethylcytosine in DNA thoroughly. However, manganese dioxide can only oxidize 5-hydroxymethylcytosine in DNA sequence partly. So, we used potassium perruthenate to oxidize 5-hydroxymethylcytosine in DNA sequence in water firstly, and then we used amino group-cantaining probes to label the DNA sequence. Through this two step process, we can combine the detection of 5-hydroxymethylcytosine and 5-formylcytosine together.Fluorescent turn off strategy:Based on the fluorescent labeling strategy, we synthesized another probe that can reacte with 5-formylcytosine and form a cage-like structure. The structure is sensitive to Cu2+ and the fluorescence will turn off when it is coordinated with Cu2+. The fluorescence of the probe itself can not be quenched by Cu2+, so this strategy can distinguish 5-formylcytosine from other bases and the probe itself.Fluorescent turn on strategy:Designed and synthesized another fluorescent probe that can react with 5-formylcytosine to form another kind of cage-like structure. The structure is sensitive to Zn2+ and the fluorescence will turn on when it is coordinated with Zn2+. The probe itself can also turn on the fluorescence after it coordinate with Zn2+. However, the wavelength differs a lot from the fluorescence described above. So this strategy can also distinguish 5-formylcytosine from other bases and the probe itself.All these three strategies described above can be used to supplement each other. Theses strategies should be used in different systems according to the actual situation of different.