Design And Application Of Novel Fluorescence Probe For G Quadruplex,Design And Application Of PH-controlled DNAzyme

Nucleic acid is the genetic material of human being's;it exists in double helix form in chromatin.Except for the sites on four bases which are participating in the formation of Watson-Crick hydrogen bonds,there are many other hydrogen/nitrogen atoms on the bases that can form hydrogen bonds,this property renders DNA can form many other secondary structures,such as G quadruplex,i-motif DNA and triplex DNA structures.In addition,DNA that own specific sequence and structure can also get catalytic properties,it is called DNAzyme.DNAzyme can catalyze DNA cleavage,DNA ligation,DNA phosphorylation,thymine dimer repair and so on.These diverse structures greatly enrich the function of nucleic acids,and make DNA can not only function as genetic material,but also can be used as tools for scientific research.Our research mainly focuses on these noncanonical DNA structures,and our thesis contain two parts:(1)Novel G quadruplex fluorescence detection probeG quadruplex is a kind of DNA secondary structure that forms by Guanine-rich DNA strand.Four guanine form one G-tetrad by Hoogsteen Hydrogen bonds,and two or more G-tetrad layers form one G quadruplex structure through ?-? stacking.G quadruplex widely exists in human genome,especially in the telomere region and the promoter region of oncogene,it is able to maintain the stability of telomere DNA,and suppress gene expression of oncogene.G quadruplex detection methods can be used for biological function study of G quadruplex.Many fluorescence probes have been developed for G quadruplex detection in vitro and in vivo,but some of them can induce the formation of G quadruplex,thus we cannot tell the G quadruplex detected by these probe is innately existed,or is formed by induction.Squaraine is a kind of cyanine dye,it has red/near-infrared excitation wavelength,which cause no damage to organisms.Squaraine has strong fluorescence intensity and stable photochemical property,it has been widely used for photoelectric device preparations.In this thesis,we have developed the first squaraine probe for G quadruplex fluorescence detection,named TSQ1.TSQ1 can specifically detect G quadruplex,the exciting wavelength and emission wavelength of TSQ1 is 610 nm(red)and 650 nm(/near-infrared),respectively.In addition,TSQ1 cannot induce the formation of G quadruplex,thus it has the potential for innate G quadruplex detection.(2)pH-controlled DNAzyme DNAzyme is DNA oligonucleotide that is capable of performing a specific chemical reaction,it owns high catalytic activity and sequence specificity,and has been widely applied in DNA nanomachines.The catalytic activity of DNAzyme is influence by several factors,such as its catalytic core and substrate sequence,little efforts have been made to design pH-controlled DNAzyme.pH is a very important physiological conditions,it maintains the normal physiological function.I-motif DNA and TC-type triplex DNA are two kinds of pH-controlled DNA structure,they can form in acid condition and be dissolved in basic condition.In virtue of these properties,we have introduced i-motif DNA and triplex DNA into the catalytic core of DNAzyme,and developed two kinds of novel pH-controlled DNAzymes,termed iMNAzyme and TNAzyme.The catalytic activities of iMNAzyme and TNAzyme can be changed with pH change,each of them has an optimal pH.By changing the sequence of the inserted i-motif DNA and triplex DNA,we have designed a series of iMNAzymes and TNAzymes,their optimal pHs range from 5.6 to 7.2.In addition,their pH sensitivities are different with each other.We have applied one iMNAzyme,which owns high catalytic activity and pH sensitivity,in DNA walker to develop a novel DNA walker which walking rate can be controlled by pH.