Molecular Mechanisms Of Switchable DNA Conformations And A Fluorescence Probe For Lysosome Targeting

G rich sequences in DNA have significant influence on secondary structures and conformations, as well as the alteration of macromolecular interaction. CG repeat sequences in DNA double helix are converted to left-handed conformation from right-handed conformation in high salt solution, and rarely observed near physiological condition. Traditionally, the conformations of DNA are considered to be determined by the hydration of phosphate groups, and the electrostatic repulsion drives the conformation conversion. Some modification of the bases in DNA double helix may affect the DNA conformation. G rich sequences can be folded into G-quadruplexs, which play important roles in the regulation of oncogenes and the protection of telomere structure. Lysosomes are vital organelles in living cells, so the designing and synthesis of lysosome target probes is potentially valuable in practice. The work is divided into four parts:1. The Z-DNA conformation is a transient DNA structure that is unstable and not well defined. Z-DNA-forming sequences and Z-DNA-binding molecules have attracted much attention from the scientific community. Identifying the sequences involved and the facile methods to study Z-DNA are needed. In this study, we successfully monitored various Z-DNA sequences, including alternating pyrimidine-purine (APP) sequences ((GC)n and (AT)n) and non-APP sequences (A24T24, TATA box and genomic DNA), in a low-salt solution. Seemingly, the Z conformation can be easily adopted by most double helices, despite the restriction of GC-rich sequences. These pervasive Z-DNAs were induced by a metal complex (Ru(DIP)2dppz), which was synthesised in our laboratory. This complex efficiently promoted sequence-independent B to Z transition and stabilised the Z conformation in a low-salt aqueous solution. Our study may help elucidate the functional aspects of Z-DNAs.2. The DNA double helix can exist in both right-handed and left-handed conformations depending on the ionic hydration of the phosphate groups. Here we evaluate the origination of the DNA double helical conformations by using CD and2D NOESY. The N-type form is dominant for purine deoxyribose (dGMP), whereas the S-type form of the pyrimidine deoxyribose (dCMP) is prevalent; the N/S switching occurs during the formation of the DNA double helix. We successfully synthesized a phosphate-methylated DNA to achieve left-handed helix in a low-salt solution. These findings suggest that the left-handed conformation is the free type of DNA without phosphate charges and that the deoxyribose moieties direct the origination of the DNA conformations.3. Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.4. Lysosomes are vital organelles in physiological processes, as they receive and degrade macromolecules from the secretory and endocytic procedures. Evidences have shown that lysosomes were related to oncogenic activation and cancer progression, so lysosomes targeting and imaging probes make them convenient to be observed. In this study, a lysosome specific probe8was designed and synthesized via convenient one-pot reaction and Heck reaction. This probe was derived from Troer’s base with a dimethylaminomethyl end group. The optical properties of this compound were measured.8also showed two-photon absorption (TPA) effect by using laser excitation at the wavelength of infrared light. In vivo experiment,8showed high specificity and selectivity for lysosomes in living cells (HeLa cells, MRC-5cells and NRK cells), compared with LT Red, GT Red and MT Red (R=0.96). Two-photon fluorescence images of HeLa cells stained by8were obtained. And high resolution3D reconstruction of lysosomes in one HeLa cell was provided by using two-photon confocal microscopy. The anantioseparation of racemic8was carried out by chiral-HPLC, and the two enantiomers showed no significant difference in lysosomes imaging.