Novel Functional Nucleic Acid Fluorescent Probes For Recognition Of Metal Ions And Tumor Cells

In order to quickly obtain accurate information to guide the prevention,therapy and prognosis of a disease,the development of highly sensitive and selective biosensors with simple operation and low cost is urgently needed.Functional nucleic acids are a type of nucleic acids with special functions?e.g.molecular recognition?.They mainly include three major classes:DNAzymes,aptamers and molecular beacons.DNAzymes are a class of nucleic acids that have similar catalytic activity in the presence of cofactors as protein based enzymes do.Aptamers are a kind of nucleic acids that can fold into secondary structures and specifically recognize various target molecules,such as metal ions,small molecules,drugs,proteins and even whole cells.DNAzymes and aptamers are generated via in vitro directed evolution techniques.Molecular beacons are a kind of nucleic acids with stem-loop hairpin structure,in which a quencher group?e.g.dabcyl?and a fluorescent group are conjugated to its terminus,respectively.Molecular beacons can be designed to specifically detect any DNA/RNA sequences.Based on good selectivity,wide target range,good biocompatibility,easy synthesis and functional modification,functional nucleic acids provide a powerful class of molecular recognition tools for the life science.Natural functional nucleic acids are generally D-type nucleic acids,Their recognition ability can be disturbed by non-target proteins,causing off-target effects and false positive signals.Moreover,D-type nucleic acids can be easily degraded by proteinase in physiological fluid.These disadvantages severely hamper the applications of functional nucleic acids in complex samples.Therefore,in order to extend the applications of functional nucleic acids,emphasis has been placed on improving the selectivity and stability of natural nucleic acids.Based on the principle of enantiomer of nucleic acid,non-natural L-type nucleic acids have been used to prepare functional nucleic acids.L-type nucleic acids have similar thermal stability to D-type nucleic acids but have better biostability and are ideal materials for constructing biosensors for complex system detection.The existing fluorescent probes have many disadvantages,such as poor biological stability,high cost,high background and poor biocompatibility.To resolve these problems,novel functional nucleic acid-based fluorescent probes have been developed through the use of molecular engineering technology and nanotechnology.Their application in a series of complex biological systems have also been tested.The details are as follows:?1?In Chapter 2,a simple and unlabeled fluorescent probe was reported for the detection of lead ions.The probe consisted of Pb²⁺-DNAzyme and its hairpin substrate:Pb²⁺-DNAzyme substrate flanked with a G-rich DNA sequence and a C-rich DNA sequence.In the absence of lead ions,the hairpin substrate remained integrated and G-rich DNA sequences could not be released.In the presence of Pb²⁺,the enzymatic cleavage reaction of hairpin substrates was triggered and many G-rich DNA sequences were released,resulting in the formation of Zn PPIX/G-quadruplex fluorescent complexes.The detection limitation of the probe is 3 n M.Furthermore,the probe based functional nucleic acids could successfully detect Pb²⁺in river water samples with high sensitivity and selectivity.?2?In chapter 3,a simple and novel L-type DNAzyme-based nucleic acid probe was designed for the detection of Cu²⁺in plasma and living cells.Compared to D-type DNAzyme-based nucleic acid probe,the L-type DNAzyme-based nucleic acid probe had high sensitivity and specificity for the detection of Cu²⁺.The limit of detection for the probe is 10 n M.Moreover,the probe was resistant to the interference of biological matrices,such as nuclease and single-stranded binding protein,and could be used for the detection of Cu²⁺in serum and cells.?3?In Chapter 4,a novel activatable aptamer/antibody had been reported to improve selectivity and limit off-target effects of aptamer/antibody.To prepare the activatable aptamer/antibody,PEG5000-azobenzene,which responds to azoreductase,had been introduced as a caged motif in the aptamer or antibody conjugate to deactivate the recognition ability of the aptamer or antibody.High-performance liquid chromatography?HPLC?,electrospray ionization-mass spectrometry?ESI-MS?,flow cytometry and fluorescence microscopy all amply demonstrated that activatable aptamer/antibody can specifically recognize its target on cells only after being activated by sodium dithionite,a surrogate of azoreductase.The sequential targeting strategy can contrast the recognition ability of aptamer/antibody on specific cells and then provide a unique handle on cells,promising an efficient strategy for the development of aptamer/antibody-based diagnostic probes and therapeutic drugs.?4?In Chapter 5,an Sgc8 aptamer-functionalized Zr-NMOFs was prepared via assembly of phosphorylated Sgc8 containing a G-rich DNA sequence on Zr-NMOFs via Zr-O-P bond for cancer diagnosis and photodynamic therapy.Aptamer Sgc8 can specifically recognize target cells.The G-rich DNA sequence could fold into G-quadruplex,providing a carrier for photosensitizers.In vitro experiments and in vivo experiments demonstrated that the nanosystem had good biocompatibility,good nucleic acid-protection ability,high drug load efficiency,good recognition selectivity,improved tumor accumulation and high therapeutic efficacy.Therefore,it could be expected that our nanosystem may provide a platform for cancer diagnosis and therapy.