Studies On Fluoresence Recogntion Of Nucleic Acid’s Specified Structures By Small Biomolecules And Application

Nucleic acid’s specific structures, which are closely related to various diseases, frequently exist in the human body. Moreover, selective recognition of these specific structures is important in developing nucleic acid-based drugs and novel sensors. Therefore, it is necessary to develop fluorescent probes that exhibit a high selectivity to the specific structures of nucleic acids and give a switch-on fluorescence response from a low background. In this thesis, we succeed in screening out such fluorescent probes specific for the nucleic acid’s structures including G-quadruplex (G4), abasic site-containing DNA (AP DNA), and triplex DNA. Our label-free methods also have the merits of simplicity, rapidity, sensitivity and effectiveness. The main contents are as follows:1. Specific G-quadruplex structure recognition of human telomeric RNA over DNA by a fluorescently activated hyperporphyrinHuman telomeric repeat-containing RNA (TERRA), which has recently been found to play as important a role in living cells as its DNA counterpart, solely adopts a parallel G-quadruplex (G4) topology. However, developing a highly selective fluorescent probe specific for the TERRA G4 is a great challenge, since difficulty arises in differentiating it from the DNA G4s that possess polymorphic structures including parallel, (3+1) hybrid, basket, and chair topologies. In this work, 5,10,15,20-tetrakis(3,5-dihydroxyphenyl)porphyrin (TOHdPP) was selected out of various porphyrins as the most efficient fluorescent probe in targeting TERRA. We found that only the TERRA binding is effective in activating the hyperporphyrin spectrum of TOHdPP, favoring red-shifted spectral bands and an enhanced fluorescence emission. We anticipate that TOHdPP most likely interacts with the 5’ tetrads of two TERRA G4s via a 1:2 sandwich association. The ribose 2’-OH favors the loop adenine residue-extended tetrad G4 plane that is specific for TERRA, thus besides π-stacking with the G4 tetrads, TOHdPP should also interact with this substructure to trigger an efficient electron communication between the tetraphenyl substituents and the porphyrin macrocycle, as required by the hyperporphyrin effect. The hydrogen bonding interactions of the eight hydroxyl substituents in TOHdPP with the backbone phosphate oxygen atoms of TERRA most likely further contribute to the binding selectivity. Our work demonstrates the potential of TOHdPP as a selective TERRA G4 fluorescent probe and a promising TERRA-based sensor reporter.2. Recognition of DNA abasic site nanocavity by fluorophore-switched probe: Suitable for all sequence environmentsRemoval of a damaged base in DNA produces an abasic site (AP site) nanocavity. If left un-repaired in vivo by the specific enzyme, this nanocavity will result in nucleotide mutation in the following DNA replication. Therefore, selective recognition of AP site nanocavity by small molecules is important for identification of such DNA damage and development of genetic drugs. In this work, we investigate the fluorescence behavior of isoquinoline alkaloids upon binding with the AP nanocavity. PAL is screened out as the most efficient fluorophore-switched probe to recognize the AP nanocavity over the fully matched DNA. Its fluorescence enhancement occurs for all of the AP nanocavity sequence environments, which has not been achieved by the previously used probes. The bridged π conjugation effect should partially contribute to the AP nanocavity-specific fluorescence, as opposed to the solvent effect. Due to the strong binding with the AP nanocavity, PAL will find wide applications in the DNA damage recognition and sensor development.3. Triggered excited-state intramolecular proton transfer fluorescence probe of fisetin for selective triplex DNA recognitionThe triplex DNA has received much interest due to its various applications in gene regulation, molecular switch, and sensor development. However, realizing a highly selective recognition using a fluorescence probe specific only for the triplex topology is still a great challenge. Herein, we found that relative to the structural analogues and several synthetic flavonoids, fisetin (FIS) is the brightest emitter when targeting the triplex DNA in contrast to binding with ss-DNA, ds-DNA (with or without an abasic site), i-motif, and DNA/RNA G4s. Only the triplex association triggers the FIS green fluorescence that is relaxed from the tautomer favorable for excited-state intramolecular proton transfer (ESIPT), FIS can stabilize the triplex structure and primarily interact with the two terminals of the triplex via a 2:1 binding mode. This work demonstrates the potential of FIS as a DNA structure-selective switch-on ESIPT probe when evolving the triplex-forming oligonucleotides and developing the novel triplex-based sensors and switches.