Biological Macromolecules Detection And Delivery Of SiRNA

Biological macromolecules including nucleic acids, proteins and polysaccharides are important components of organisms and play a very important role in the life activity of the organism. Genetic information is carried and transmitted by nucleic acids, protein was involved in every process of life activities. Generally, the concentration, composition and structure of biological macromolecules in vivo are stable, if any of which was changed, which may result in disease occurrence or potential impact on the body. Therefore, the development of convenient, sensitive and accurate methods for biological macromolecules detection is extremely important in the field of life science research and molecular diagnostic.In addition, RNA interference can efficiently silence target gene expression in the post-transcriptional level, it has important applications in the treatment of cancer and genetic diseases, functional genomics, drug development and other fields. Cationic liposomes and cationic polymers were mostly used for siRNA delivery, but their applications were limited by strong cytotoxicity.In this thesis, detection of biological macromolecules and delivery of siRNA were studied, the following research findings were completed.(1) A visual DNA detection strategy based on the hydrophilicity of DNA and the salt spot signal is developed. The self-assembled DNA nanostructure with excellent hydrophilicity could capture salt solution onto the glass slide, which could form salt spot after heating-dried and visual to naked eyes. Detection limit of 100 fM was achieved by extending the salt spot signal to LCR-RCA protocol. Because of the biocompatible salt spot signal and the simplicity of operation, this method could substitute the complicated labeling technique and sophisticated and expensive detection equipment and it would have promising applications in DNA detection. (2) The expression and purification of Copk and its inhibition in click reactions.Copk which has a high affinity for Cu â… was expressed and purified. A fluorogenic click reaction system possessing well biological compatibility was constructed, and greatly increased yield was achieved after the optimal conditional experiments. Finally Copk was introduced into this reaction. Click reaction catalyzed by two-equivalent copper ion was inhibited by Copk and peptides and amino acids generated after Copk digestion, while the inhibition of latter was stronger. While copper ion is three times the amount of Copk, click reaction could be inhibited by peptides and amino acids generated after Copk digestion, could not be inhibited by Copk. The research laid a foundation for the development of protease detection system based on Copk and fluorescence click reaction. (3) The effect of Bcl-2 siRNA-modified gold nanoparticles on Hela cells. TheBcl-2 siRNA was modified on the surface of gold nanoparticles and the gold nanoparticles were used as delivery carrier of Bcl-2 siRNA. Bcl-2 mRNA level in cell was detected by Real-time PCR, Bcl-2 mRNA level of siRNA-AuNPs group was decreased by 27% compared with the control group, large cell death which was caused by siRNA transfection was not found in siRNA-AuNPs group, siRNA-AuNPs is a non-cytotoxic siRNA delivery system with good delivery efficiency.