Studies On The Structure-activity Relationship Of Isoquinoline Alkaloids In Binding With Nucleic Acids And Applications

The physiological activity of many small biological molecules is closely related to the interaction with nucleic acids of various structures,and it is very important to explore the structure-activity relationship between small biological molecules and nucleic acids.In this thesis,three DNAs,such as the triplex DNA,molecular beacon and abasic site DNA,are utilized as the objects of study.According to the study on the interaction between isoquinoline alkaloids of various structures and nucleic acids,we developed a wide pH-and metal-ion tunable lable-free triplex nanoswitch,a lable-free tMB with tunable sensitivity,and enantioselective fluorescent sensor,respectively.The main contents are as follows:1.Fluorescently sensing DNA triplex assembly using an isoquinoline alkaloid as selector,stabilizer,inducer,and switch-on emitter.DNA triplex assembly has caused various interests in regulation of genetic expression,drug screening,molecular switches,and sensors.However,these achievements are essentially dependent on the formation and stability of the triplex assembly.Herein,the recognition of the DNA triplex assembly with variant isoquinoline alkaloids was investigated.We found that the natural chelerythrine(CHE)exhibits highest selectivity in recognizing the triplex structure.The DNA triplex stability is substantially increased upon the CHE binding,as opposed to innocence to the duplex counterpart.Concurrently,CHE also favors the assembly of the triplex-forming oligonucleotide(TFO)with its duplex counterpart.More importantly,the triplex binding also switches CHE to a strongly fluorescent emitter,suggesting CHE as a useful probe in following the triplex assembly.As a unique triplex selector,inducer,and emitter,CHE successfully reports the wide pH-and metal ion-dependent tunability of the triplex nanoswitch in a label-free manner.2.Label-free triplex molecular beacon for tunable DNA analysis.Detecting nucleic acid sequences is of central importance in modern life sciences.Molecular beacons(MBs)has been a research hotspot for simple operation,high sensitivity and good selectivity.Herein,we design a label-free triplex molecular beacon(tMB)with tunable selectivity.The natural chelerythrine(CHE)is utilized as fluorescent signal probe,which can bind with the triplex and lead to fluorescence enhancement.CHE also favors the assembly of the triplex-forming oligonucleotide(TFO)with its duplex counterpart.The tMB in the absence of the target can bind with CHE and emit fluorescence.While in the presence of the target,the loop part of tMB binds with target and forms a duplex structure.As a result,the hairpin structure opens and the triplex structure is broken into the duplex structure leading to fluoresence quenching.However,when the target containing the mutated base(G/C)is added,the loop part of tMB can't bind with it and form a stable duplex,then the triplex in the stem isn't broken and fluorescence keeps almost unchanged.Changing the solution pH allows the stability of the tMB's triplex structure to be changed,then the selectivity of the DNA detection is regulated.3.DNA Duplex Engineering for Enantioselective Fluorescent Sensor.Finding the biomacromolecule structure that has a specific recognition with chiral enantiomers especially from natural sources will be helpful in developing enantioselective sensor and in speeding up the step towards drug exploitation.Herein,abasic site(AP site)was first introduced into ds-DNA duplex to explore the enantiomer selectivity due to its existence in living cells.An AP site-specific fluorophore was employed as the enantioselective probe to develop a straightforward chiral sensor using natural tetrahydropalmatine(L-and D-THP)as the enantiomer representatives.We found that only L-THP can efficiently replace the pre-bound ATMND to cause a significant fluorescence increase due to its specific binding with the AP site(two orders magnitude higher in affinity than binding with D-THP).The AP site binding specificity of L-THP over D-THP was assessed by intrinsic fluorescence,isothermal titration calorimetry,DNA stability,and DFT calculation.The enantioselective performance can be easily tuned by the sequences nearby the AP site and the number of the involved AP site.A single AP site provides a perfect binding pocket to differentiate the chiral atom-induced structure discrepancy.We expect that our work will inspire the interest of the ds-DNA local structure in enantioselective sensor development.