Neotype Biosensors And Therapy Methods Based On DNA Hybridization Networks And Functional Nucleic Acid

Nucleic acid is a unique biopolymer.Nucleic acid is not only an important material for information storage and transmission,but also has evolved into functional nucleic acid with a variety of special characteristics.Compared with other materials,nucleic acid materials have significant advantages in biosensing and therapy.Firstly,oligonucleotide is efficiently synthesized by solid phase synthesis technology.Secondly,nucleic acid is highly programmable materials because of its legible molecular mechanisms.The construction of static nanostructures and the operation of responsive dynamic DNA hybridization networks are realized.Thirdly,the development of functional nucleic acid has greatly expanded the application space of nucleic acid materials.Fourthly,Precise modification of oligonucleotides can provide signal conversion or improve the stability of nucleic acid materials.At the same time,the modified nucleic acid may disturb molecular structure,achieving the stimuli-responsive engineered nucleic acid structure.Fifthly,nucleic acid material has good biological compatibility.Therefore,in the past decade,the application of nucleic acid material in treatment and diagnosis has made rapid development.Researchers have developed oligonucleotide probes,oligonucleotide therapeutics,gene-editing tools,and even DNA and m RNA vaccines.At present,nucleic acid materials are widely used in nanotechnology,materials science,molecular computing,data storage,biosensors,therapy,synthetic biology and other fields.In this paper,we have developed several neotype biosensor and therapy methods that based on responsive DNA hybridization networks and functional nucleic acid.The details are as follows:In Chapter 2,we have developed a self-tracking multifunctional nanotherapeutic system（g-C₃N₄@H1/H2）for sensitive micro RNA imaging guided Photodynamic therapy（PDT）.g-C₃N₄nanosheets were not only used as a fluorescence nanocarrier for nucleic acid delivery but also as a photosensitizer for imaging guided PDT.After the g-C₃N₄@H1/H2 nanoassembly was transfected into living cells,HCR was triggered by micro RNA-21（mi RNA-21）,enabling ultrasensitive detection of a low abundance of biomolecules in live cells.Moreover,the fluorescence of g-C₃N₄nanosheets allowed assessment of the delivery efficiency to mitigate the false negative responses.In addition,the g-C₃N₄nanosheets could generate ROS in live cells upon light irradiation,affording a useful PDT agent for tumor therapy.Benefiting from these advantages,the proposed g-C₃N₄@H1/H2 nanotheranostics may provide a potential platform for sensitive detection of biomarkers and imaging guided therapy.In Chapter 3,we have developed a programmable and activatable DNA cascade circuit for accurate cancer cell recognition and gene silencing via AND logic operations.The DNA cascade circuits were activated by structure switching of the sgc4f aptamer,and then the reconstructed DNA nanodevices were labelled on the surface via sgc8c aptamer–receptor binding for accurate cancer cell imaging and were subsequently delivered into cells for precise gene silencing.Compared to the single-receptor based imaging and therapy methods,the DNA cascade circuits exhibited high specificity and decreased off-target toxicity via AND logic operations.Moreover,our approach was general and could serve as a precise platform for other functional nucleic acid delivery.We also showed that the DNA cascade circuits enabled accurate labelling and gene silencing of the narrow population of target cancer cells.In Chapter 4,A"repaired and activated"DNAzyme in which the functional domain was modified with a single O⁶Me G lesion,was developed for monitoring of MGMT-mediated DNA repair pathways in live cells.Firstly,we systematically investigated the effect of a single O⁶Me G lesion on the catalytic activity of 8-17DNAzyme through position screening.We found that the 8-17 DNAzyme with a single O⁶Me G lesion at specific position could suppress the catalytic activity,and restored into active DNAzyme via enzyme-mediated DNA repair.We also utilized molecular dynamic simulations to gain a better understanding of the impact of different nucleotide positions on the catalytic activity of 8-17 DNAzyme.Based on the above studies,we developed a fluorogenic O⁶Me G14-RADz sensor that enable direct and real-time monitoring of MGMT-mediated DNA repair and inhibitors screening.The proposed sensor also provided the ability to distinguish MGMT mediated DNA repair activity and evaluate the drug response in glioma cells.In Chapter 5,A"repaired and activated"DNAzyme in which the functional domain was modified with a single 3Me C lesion,was developed for monitoring of ALKBH2-mediated DNA repair pathways in live cells.We systematically investigated the effect of a single 3Me C or 1Me A lesion on the catalytic activity of 8-17 DNAzyme through position screening.We found that the 8-17 DNAzyme with a single 3Me C or1Me A lesion at specific position could suppress the catalytic activity,and restored into active DNAzyme via ALKBH2-mediated DNA repair.Although both ALKBH2and ALKBH3 repair enzymes could repair 3Me C or 1Me A lesion,However,in the process of repairing the 3Me C or 1Me A lesion on 8-17 DNAzyme,experimental results showed that ALKBH2 has higher repair ability than ALKBH3.Possible reason for the above phenomenon is that the three dimensional structure of 8-17 DNAzyme is a twisted dense pseudoknot.Based on the above system discussion,we further engineered and demonstrated a fluorogenic 3Me C₁₃-RADz sensor for the selective imaging of ALKBH2 mediated DNA repair in live cells.Our"repaired and activated"DNAzyme sensing strategy was facile and modulable,providing a promising tool for cancer diagnosis,drug discovery and therapeutic evaluation.