Research On The Novel Fluorescent Sensors And The Mechanism Based On Abasic Sites And DNA-enzyme Conjugates

Due to their high sensitivity,good selectivity,ease in design and quantitative capability,fluorescent sensors play a significant rolein analytical chemistry and are widely applied in the fields of environment monitoring,drug screening and disease diagnosis.With high biocompatibility and versatility,functional nucleic acids have been utilized as fluorescent probes and developed into various targets.Hence,the aim of this dissertation is to develop novel fluorescent sensors based on abasic sites and DNAenzyme conjugates.Moreover,the mechanism of the 17 E and 39 E DNAzyme is studied employing the insertion of abasic sites.Firstly,a label-freefluorescent approach for the detection of UDG(Uracil-DNAglycosylase)activity based onthe removal of single uracil in each DNA substrate wasdeveloped.UDG is responsible for the elimination of uracil from DNA and generation of an abasic site.An aromatic molecule called 2-amino-5,6,7-trimethyl-1,8-naphthyridine(ATMND)bound the abasic site opposite to a uracil site in the through hydrogen bonds and underwent fluorescence quenching by p–p stacking with flank bases.By this “turn-on” method,the sensor showed a detection limit of 0.0008 U/ml in buffer.In addition,application ofthis design in cell lysates wasachieved successfully.Meanwhile,the inhibitor effect of UGI(a protein)and gentamicin was also determined.Furthermore,the biochemistry of the enzyme is studied using multiple uracil-containing DNAsubstrates.Secondly,an abasic site was systematically introduced into in the catalytic cores of 17 E and 39 E DNAzymesthrough UDG-catalyzed excision of deoxyuridine between every two adjacent nucleotides.Such an abasic site provided flexibility to the insertion region.Upon the insertion,the activity of the DNAzymechanged.The substrate was dual-modified with the fluorophore and quencher so that the activity was in proportion to the rate of the fluorescence enhancement.The conclusions of the active site were in agreement with the previous findings of the DNAzyme.Hypothetical structures of 17 E and 39 E were investigated in this study.Thirdly,a reversible fluorescent probe system which consisted of aptamers immobilized on the magnetic beads and DNA-enzymeconjugateswasdemonstrated.The aptamer and the enzyme shared the same substrate,which,in this study,is adenosine.In the presence of adenosine,the aptamer folded and the conjugate was released into the solution and fluorescence intensity enhanced.After the adenosine converted into inosine by the enzyme catalysis,the conjugates hybridized to the aptamer again.The strategy combined the merits of homogeneous and heterogeneous use of enzymes with high efficiency and can be separated and recycled easily.