Interaction Between DNA Andβ-Glucan Along With The Function Based On The Denaturation And Renaturation Of The Triple Helical β-Glucan

Triple helical β-(1â†'3)-D-glucan, a naturally occurring β-glucan is not only a good candidate for the biological response modifier and immunomodulator, but also shows high anti-cancer, anti-tumor and anti-inflammatory activities. Therefore, β-(1â†'3)-D-glucan family attracts much attention in recent years due to the wide application in medical field. The immunostimulatory effect is believed to be related to the solubility, molecular weight, degree of branching and chemical structure of polysaccharides. Furthermore, the immunological activity strongly depends on the triple helical chain conformation. It has been indicated that the occurrence of the denaturation for triple helical β-glucan would result in the loss of the biological activities. The intra and intermolecular hydrogen bonds are the major driving forces to maintain the triple helical conformation, and the triplex could be broken into single chains in strong alkali and ploar solution, or at the high temperature. Conversely, by changing the pH, polarity of the solvent or temperature, will the single chains recombine each other into triple hlical structure via hydrogen bonds? How is the morphology of the renatured species controlled? Can the triple helical β-glucan, similar to protein and DNA with special secondary or tertiary structure, interact with proteins and DNA? Therefore, this paper aims to study in detail the denaturation and renaturation of the triple helical β-glucan, in particular the renaturaion conditions and dynamic process. Meanwhile, based on the significant ability of β-glucan to reform the triple helix, the interaction between triple helical β-glucan and DNA along with their bio-functions and interaction mechanism will be fully investigated. This work involves interdisciplinary fields of polymer physics, analytical chemistry, biochemistry and biomedical application, and also one of the international research frontiers of polymer science.The innovative points of this work are as follows.(1) The renaturation process for triple helical β-(1â†'3)-D-glucan was, for the first time, thoroughly investigated by using DSC combined rheometer, where an simple, swift and effective method was proposed to identify the triple helical conformation. Meanwhile, it was revealed how the triple helical structure was controlled in the renaturation process.(2) A novel complex of poly(dA)/s-LNT was fabricated by utilizing the interaction between s-LNT and poly(dA) via hydrogen bonds, and the factors affecting the formation of complex were also extensively investigated, including the concentration of s-LNT, time, pH, temperature and chemicals, et al.(3) A novel method to detect the interaction between s-LNT and poly(dA) swiftly, sensitively and precisely was developed by taking the advantage of special molecular recognition for each other.(4) An intelligent, efficient and safe gene delivery system was fabricated based on the interaction between poly(dA) and s-LNT for the first time, which can be further used in immunotherapy.(5) The key factor on the gene transfection efficiency was comprehensively evaluated, and it was firstly revealed that the internalization pathway of such carrier to RAW264.7cells was possibly through fluid phase endocytosis, but not the usual β-glucan receptors.The main contents and conclusions in this project are divided into the following parts. The conformation of the triple helical β-glucan from lentinus edodes (t-LNT) and the renaturation process of the denatured t-LNT (s-LNT) was extensively investigated by using DSC combined with circular dichroism (CD), rheometer and viscometer. The results from the DSC and rheometer curves indicated that all the triple helical β-(1â†'3)-D-glucan with side chains of β-(1â†'6)-linked glucose showed characteristic temperature (T1) in the temperature region lower than room temperature, respectively, which was proved to be an effective, simple and rapid method to identify the triple helical structure of β-glucan in contrast to the traditional method of polymer solution. The experimental results also revealed that the variable structures including perfect triple helix, defective triple helix containing duplex segment, and single chains occurred in the renaturation process of s-LNT, strongly depending on the renaturation time, solvent composition, molecular weight and ranaturation mode. When s-LNT/DMSO was added into water with the final content of DMSO equal to5%(v/v), the classic low-temperature conformation transition from triplex â…  to â…¡ at～10℃(T1) appeared within4h, indicative of a rapid renaturation process for triple helical structure. Besides, one newly endothermic peak at～43℃(T2) simultaneously occurred, which was ascribed to the melting of duplex segment in the imperfect triplex. Furthermore, the second endothermic peak disappeared when DMSO content reached50%, in which single chains co-existed with triplex. Moreover, it was shown that the rate of exchange the DMSO with water was the key factor affecting the perfect triple helical structure. The slower was the exchange rate of DMSO with water, the more perfect triple helix was obtained.The interaction between poly(dA) and s-LNT was extensively investigated by circular dichroism spectra, UV-Vis spectra, nano-DSC and dynamic light scattering. The effects on the stability of complex, including the concentration of s-LNT, storing time, pH, temperature and chemicals, were also explored. All the experimental results indicated that poly(dA) has a strong ability to interact with s-LNT to form a novel complex with stiff conformation through hydrogen bonding. The dynamic behaviors of the complex demonstrated that the interaction between s-LNT and poly(dA) occurred rapidly within1h with a rather weak negative peak at265nm, and reached stable within3-7h, which also strongly depended on the concentration of s-LNT and pH. The interaction between poly(dA) and s-LNT was enhanced with an increase in s-LNT concentration, then reached the maxium at～60μg/ml, finally decreased with further increase of s-LNT concentration. The poly(dA)/s-LNT complex could exist in solution with pH5.5-11.5, and pH7-10was proved to be the optimal condition for the complex. Moreover, the thermal stability of poly(dA) was enhanced by complexation with s-LNT, until65℃will gradually dissociate.Based on the fluorescence resonance energy transfer (FRET) and fluorescence anisotropy (FA), a highly sensitive and rapid detection technique was well developed that can be precisely utilized for investigating the interaction between poly(dA) and β-glucan on the graphene oxide (GO) platform. Due to the noncovalent assembly between FAM-labeled poly(dA) and GO via Ï€-Ï€ stacking, the fluorescence of the designed poly(dA) as a molecular aptamer beacon (MAB) is completely quenched by GO. The experimental results demonstrated that the addition of s-LNT would result in the significant restoration of fluorescence due to the formation of poly(dA)/s-LNT complexes with stiff rod-like structure which had weak affinity to GO and kept the dyes away from GO. While relatively weak fluorescence restoration was observed by adding single-stranded curdlan (s-CUR) for the positive control, indicative of the complex of poly(dA)/s-CUR formation with higher binding ability to the GO. When t-LNT was added into the fluorescence quenched solution, greater fluorescence restoration was observed, indicating that t-LNT showed high affinity and could expel the poly(dA) away from GO surface. Manwhile, the result from FA demenstrated that the interaction between s-LNT and poly(dA) could finish within200s, showing higher sensitivity than CD measurement in which the interaction was observed within at least1h with a rather weak negative peak at265nm and3-7h with significant variation. Additionally, the results from FRET and FA revealed that poly(dA) can only interact with single chains of triple helical β-(1â†'3)-D-glucan rather than xanthan, amylose and pullulan; at the same time, poly(dA) instead of poly(dC) can interact with the denatured triple helical β-(1â†'3)-D-glucan, indicating the specially selective recognition between s-LNT and poly(dA).A novel gene carrier was prepared on the basis of special interaction between s-LNT and poly(dA)50. By incorporation of disulfide bonds between poly(dA)5o and the target DNA, the new formed squence of DNA-SS-poly(dA)5o can then be combined by s-LNT, with the following delivery into the cells by endocytosis. The experimental results from agarose gel retardation assays and CD spectra demonstrated the disulfide bond in the complex of DNA-SS-poly(dA)5o/s-LNT could really be broken up under the treatment of reducing agents such as GSH or DTT. The confocal microscopy and flow cytometry indicated that DNA-SS-poly(dA)50/s-LNT with molecular weight of7.0×104showed the highest transfection efficiency, which strongly depended on the concentration of s-LNT. The gene transfections could be performed not only in normal but also tumor cells without any chemical modification of s-LNT. The successful delivery of CpG DNA with immunity into the cells with an enhanced IL-12p40secretion further confirmed the feasibility and potential application as an safe gene carrrier.The influence of the sequence length of poly(dA) on the stability and gene transfection efficiency of the poly(dA)/s-LNT by agarose gel retardation assay, circular dichroism, flow cytometry and ELISA kits was evaluated.. All the results revealed that the longer base length of poly(dA) owing the stronger interaction with s-LNT was favourable for the stability and gene transfection efficiency. Furthermore, the expression of β-glucan receptors including dectin-1, TLR-2,CD11b and CD18b on the surface of RAW264.7cells was investigated from the confocal images by staining the cells with PE-conjugated anti-dectin-1antibody, FITC-conjugated anti-TLR-2antibody, PE-conjugated anti-CDllb and FITC-conjugated anti-CD18b antibody. Finally, by blocking the TLR-2, CD11b and CD18b receptors on the cell surface with the specific antibody, the possible internalization pathway of such delivery system via fluid phase endocytosis, but not the usual β-glucan receptors (dectin-1, TLR-2and CR3) was revealed.Summarily, the results of this foundation research mentioned above revealed the occurrence of variable chain conformation for s-LNT upon the renaturation process, indicating the strong ability to reform the triple helical structure for β-glucan. During the process the β-glucan can specifically recognize the unique DNA sequence, such as poly(dA) to form a novel complex. Meanwhile, based on the interaction between poly(dA) and s-LNT, an efficient and safe gene carrier with the feasiblity in immunotherapy was constructed. This work will reveal the significance of triple helical conformation in the biomedical application, and also provided scientific evidence on the relationship between the conformation and the biological activity. Therefore, it has important academic value and potential applications.