Studies Of Fluorescent Biosensing Methods For The Detection Of DNA Repair Enzymes

DNA repair enzymes can participate in the repair process to DNA lesions, which play crucial roles in protecting the genome from DNA lesions and maintaining the integrity of the genetic information. Abnormal activities of the DNA repair enzymes would interrupt the repair process to DNA lesions and directly relate to various diseases, including premature aging, developmental disorders, tumors, neurodegenerative diseases and et al. Therefore, detection of DNA repair enzyme activity is beneficial for properly evaluating the repair process to DNA lesions in function studies and clinical diagnoses.In recent years, due to the advantages of simplicity and good biocompatibility, DNA-based fluorescent biosensing methods have been widely applied in the quantitive detection of biomolecules. In the methods, DNA was used as the probe to recognize the target, and then the recognition event could be transduced into the fluorescent signal which can be detected. Currently, the DNA-based fluorescent biosensing methods have been used for detecting the activities of DNA repair enzymes. However, there are still some limits:1) the requirement of labeled or modified DNA probe, as well as the low sensitivity,2) many methods are based on simultaneous removal of multiple damaged bases for signal transductions, which limite the further improvement of detection sensitivity,3) each method can only detect one of the DNA repair enzymes, which is not enough to evaluate the repair process to DNA lesions.Here, we have developed the novel fluorescent biosensing methods for detecting the activities of DNA repair enzymes. And we have chosen uracil-DNA glycosylase (UDG), endonuclease IV (Endo IV) and human apurinic/apyrimidinic endonuclease 1 (APE1) as the models of DNA repair enzymes. The main contents are as follows:Chapter one is an introduction section, and it summarizs the concept, category, function, research significance and the existing detection methods of the DNA repair enzymes. In addition, the existing problems for the DNA repair enzymes activities assay at the present stage are also summarized.In chapter two, a fluorescence amplification method based on a label-free DNA machine has been developed for sensitive detection of UDG activity. In this method, we have designed a double-strand DNA probe with uracil bases and trigger sequence. It can be used as the trigger part of DNA machine, which could recognize UDG and transduce signal. And we also designed two hairpin probes which are partially complementary. They can be used as the operation part of DNA machine, which could amplify signal. Under the action of UDG, uracil bases are removed from the double-strand DNA probe, generating apurinic/apyrimidinicabasic (AP) sites. This results in the dissociation of the double-strand DNA probe. Accordingly, the trigger sequence is released. Subsequently, the trigger sequence can activate the DNA machine, generating numerous G-quadruplex (G4) structures. Finally, the G4 structures can bind with N-methyl-mesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This method can detect UDG activity as low as 0.00044 U/mL. The method is also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this method is successfully applied for assaying the inhibition of UDG activity by inhibitor.In chapter three, a fluorescence amplification method based on toehold-mediated stand displacement reaction (TSDR) is proposed for sensitive detection of UDG activity. This method can make response to the removal of a few uracil bases in the probe. A single strand DNA probe with two uracil bases and a trigger sequence is designed. A hairpin probe with toehold domain and a report probe are also designed. Under the action of UDG, two uracil bases are removed from the single strand DNA probe, generating AP sites. Then, the single stand DNA probe with AP sites can not initiate TSDR, leaving the trigger sequence in this probe still free. Subsequently, the trigger sequence is annealed with the report probe, initiating the polymerization and nicking isothermal amplification reaction. As result, the method can make sensitive response to the removal of a few uracil bases and improve the detection sensitivity. However, in the absence of UDG, the single strand DNA probe can hybridize with the toehold domain of the hairpin probe to initiate the TSDR, resulting in the blocking of the trigger sequence. Accordingly, the subsequent isothermal amplification process will not occur, which reduces the negative signal. The proposed method has been successfully implemented for the detection of UDG activity with a detection limit of 0.000027 U/mL.In chapter four, a dual recognition hairpin probe mediated fluorescence amplification method is developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe is excised by the target enzyme to generate an AP site, achieving the UDG recognition. Then, the AP site is cleaved by a tool enzyme Endo IV, releasing a trigger sequence to initiate the rolling circle amplification (RCA) reaction. Finally, the RCA reaction produces numerous repeated G4 sequences, which could be used for signal output. Alternatively, for detecting Endo IV activity, the uracil base in the probe is first converted into an AP site by a tool enzyme UDG. Next, the AP site is cleaved by the target enzyme, achieving the Endo IV recognition. Finally, the signal is then generated and amplified by RCA, achieving the sensitive detection of Endo IV. The detection limits are 0.00017 U/mL for UDG and 0.11 U/mL for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. Additionally, the proposed method can also detect the activity of APE 1.In chapter five, a fluorescence amplification method is proposed for sensitive detection of UDG activity by using the ligase reaction. Under the action of UDG, two uracil bases are removed from the recognition probe to generate AP sites, forming mismatches on the 3’-side of the nick. The nick with mismatches on the 3’-side can not be ligated by DNA ligase, and the hairpin strcture of the recognition probe is still maintained. Then, the hairpin probe can initiate the subsequent amplification process to generate the fluorescent signal. As result, the method can make sensitive response to the removal of a few uracil bases and be beneficial for improving the detection sensitivity.Chapter six is a conclusion section, and it mainly summarizs the innovation of the paper.