DNA Nanostructure Based Sirna Delivery System

Small interfering ribonucleic acid(siRNA)plays a pivotal role in gene silencing.However,delivery of naked siRNAs remains a major challenge due to several shortcomings,for instance,good hydrophilicity inhibits its effective endocytosis.It is likely to be hydrolyzed by a variety of nucleases existing in blood and cells.Additionally,the size of siRNA is too small to be excluded by kidneys easily.In order to solve these problems,a range of carriers have been designed and developed including liposomes and cationic polymers to assist the delivery of siRNAs.Although they have made remarkable achievements,they are extraneous materials which may cause toxicity.It is well known that DNA is a kind of genetic material in living organisms,with inherent biocompatibility and biodegradability.More importantly,molecular recognition between DNA strands enables the construction of one-,two-,and threedimensional DNA nanostructures in a wide range of ways.Taking the advantage of DNA,a number of DNA nanosystems have been proposed and constructed to delivery siRNA,such as DNA nanocage,spherical nucleic acid,nanowires and so forth.These DNA nanostructures exhibit excellent transfection efficiency.However,siRNAs exposed outside the structure are prone to be degraded by enzymes in blood circulation and cells.The three-dimensional network structure of the hydrogel can protect embedded siRNA.Besides that,the size is adjustable,making it enter the cell successfully.Thus far,most current gel systems could introduce additional chemicals such as polymers,and whether it is safe to cells or organism is not determined.Therefore,how to develop a safe and stable nanocarrier that can protect siRNA as well as achieve high gene silencing effect has become an important issue.Based on this consideration,we proposed a new DNA nanogel by virtue of the base recognition mechanism.The biggest innovation is that nanogel was composed entirely of pure nucleic acid.Then we evaluated its gene silencing effect through a series of cell experiments.In this thesis,we first designed and synthesized DNA tetrahedron with four sticky ends(tailed-TET),and then assembled nanogel(TET-nanogel)by complementary base pairing of sticky ends using siRNA as linkers.The nanogel was completely composed of pure nucleic acid,without introducing any other chemicals.The formation and size of nanogel were verified by biological and chemical characterization methods.Then we evaluated the stability of nanogel in the physiological environment and examinined the functional siRNA release by RNase H.Subsequently,we explored whether the entire TET-nanogel could assist siRNA to achieve high cellular uptake efficiency,and assessed the gene silencing effect at the gene level,mRNA level as well as protein level.Cell experiments demonstrated that it can effectively down-regulate over-expressed genes and inhibit relevant protein synthesis.With all experimental results,we concluded that nanogel comprised by only nucleic acid achieved excellent transfection efficiency without relying on any transfection reagents and exhibited prominent gene silencing effect.On this basis,we constructed a DNA and siRNA hybridized nanotube structure.Similarly,the nanotube was also composed entirely of nucleic acid and had geometrically well-defined size and structure.Its size was determined by gel electrophoresis and dynamic light scattering scanning.Notably,the rigidity of nanotube could protect embedded siRNA to some extent.Stability experiment in serum confirmed that nanotube could prevent siRNA from nonspecific enzyme for a long time.The results of endocytosis experiments show that nanotubes can be efficiently uptaked by cells,showing the potential of siRNA transfection.However,whether the DNAsiRNA hybrid nanotube can release siRNA successfully in cells and whether it possesses the ability to inhibit target gene expression remain to be further explored.