A Novel Synthesis Method Of G-quadruplexes And Its Application In Biosensing

The main function of DNA is to carry most of the genetic instructions and monitor the information based on its special structures. With the development of multiple subject combination, DNA is no longer merely playing the character of s torage of genetic information, its role has been updated. People can obtain some functional nucleic acids through selection processes. The functional nucleic acids can be classified into two sorts: one type, like antibodies, can recognize the target, and i s called aptamers; the other type is like enzyme which has the catalytic ability, and is called DNAzymes/RNAzyme. The discovery of functional nucleic acids has expanded the understanding of nucleic acids and provided novel molecular tools for researchers. Comparing to protein enzymes or antibodies, nucleic acids are stable and adaptable, and can be easily produced. A number of analytic methods have been explored in biochemical analysis, as well as the application in biological imaging and cancer therapy in recent years.Functional nucleic acids can form particular structure when they bind to target molecule or provide an environment for catalytic reaction. On account of the obvious colour change of the substrate for colorimetric assay, horseradish peroxidase mimicking DNAzyme is one of the most widely used functional nucleic acids. This kind of DNAzyme is composed of G-quadruplex and hemin. Current studies using this DNAzyme are based on the known rigid sequence, randomly arranged sequence is seldomly reporte d. Here, a novel synthesis method of randomly arranged G-rich DNA was developed, the characters and potential applications were further studied.The details are summarized as follows: 1. A novel synthesis method of randomly arranged G-quadruplex.In this chapter, randomly arrayed G-rich sequences were obtained by employing a template-free polymerase terminal deoxynucleotidyl transferase(Td T) and an appropriate ratio of d NTP. The randomly arrayed G-rich sequence can serve as efficient peroxidase-mimic DNAzyme after incubated with hemin. We firstly investigated the products of Td T polymerization under three sets of d NTP pool. The denaturing PAGE was used to characterize the length of Td T-synthesized products. Circular dichroism(CD) spectroscopy was applied to identify the conformation of the Td T-produced DNA. The maximal activity was obtained when the pool composed 60% d GTP and 40% d ATP. We also found that the randomly arrayed G-rich DNA sequence can enhance the fluorescence intensity of G-quadruplex-specific dye(Th T) as well. We verified that the random G-rich sequence has obvious peroxidase activity and the ability to bind to specific dye. Td T combining with G-rich d NTP pool provides a new method to prepare G-quadruplex and its derived DNAzyme. 2. The application of randomly arrayed G-rich DNA sequence in biosensing.Based on the studies of randomly arrayed G-rich DNA sequence, we explored a series of biosensing platforms, whose targets included DNA, protein and enzymes. Because there is no specific sequence required for Td T-generated DNAzyme, the design criteria of this new sensing system are quite distinct from that of conventional DNAzyme-based methods. Firstly, we demonstrate a label-free and sensitive assay for Td T with a low detection limit of 0.0394 U(S/N=3). Due to the quick binding between Th T and Td T-generated G-quadruplex to form stable fluorescent complex, we developed a real-time fluorescence method to monitor the activity of Td T. The analysis was also conducted in 10% human blood serum samples which clearly indicated the potentiality of this assay in real biological samples. For the target DNA detection, 3’phosphorylated end of DNA was introduced in the assay because it could completely prohibit the Td T-mediated tailing. The 3’-OH was produced when the tareget DNA existed, which triggered the NESA. The detection of protein(thrombin) was realized by the recognition of aptamer and the released DNA sequence that hybrided with the amplification probe to amplify the signal in a similar NESA way. Our strategy achieved sensitive assaies of DNA, thrombin and enzyme based on the activation of Td T-generated randomly arrayed G-rich DNA, proving the potential versatility and feasibility of this method in various bio-analysis. 3. The detection of apoptotic cell based on the randomly arrayed G-rich DNA sequence.Here, we developed a protocol employing terminal deoxynucleotidyl transferase to incorporate nucleotides into 3’-OH recessed terminal of DNA breaks producing randomly arrayed G-rich sequence. This method determined the apoptotic cell by detecting the 180-200 bp DNA fragements during the apoptosis. First, we detected the single-stranded DNA based on the randomly arrayed G-rich sequence. Then, high weight DNAs(plasmid DNA and cell genome DNA) were analyzed after DNase I digested. Based on the aforementioned experimental results, we developed a situ-detection protocol of apoptotic cell. The results based on the randomly arrayed G-rich sequence were in accordance with flow cytometry assay and gel electrophoresis analysis. 4. The study and application of the interaction between Sc GFP and G-quadruplex/hemin complex.It’s firstly observed that the fluorescence of Sc GFP was quenched by G-quadruplex/hemin complex, while the hemin alone or DNA(which cannot form G-quadruplex)/hemin complex couldn’t elicit the quenching. To further understand their interaction, we conducted a series of experiments to investigate the mechanism of the quenching process in detail. Through the comparation of the emission spectrum of Sc G FP with the absorbance spectrum of G-quadruplex/hemin complex, the fluorescence intensity of Sc GFP after incubated with increased concentration of hemin, and the life time of Sc GFP before and after G-quadruplex/hemin complex added, we came to a conclusion that the quenching was resulted from the photoinduced electron transfer of Sc GFP. Taking advantage of the quench phenomena, we can develop label-free fluorescence sensors. We developed a heparin detection platform and a Tb3+ detection platform based on the interaction between Sc GFP and G-quadruplex /hemin complex.