Preparation,Property And Application Of Molecularly Pure Spherical Nucleic Acids

Spherical nucleic acid（SNA）is a three-dimensional nanostructure based on functional nucleic acid.It is formed by functionalizing a core with densely-surrounding nucleic acid in a spherical orientation.SNA was first made by Professor Mirkin using a gold nanoparticle（Au NP）core.However,the core of SNA can actually be replaced by plenty of materials,including other inorganic nanoparticles（Ag-NP,Fe₃O₄,Si O₂,and QD）,organic materials（liposomes,polymers and other macromolecules）,hybrid structures（MOF and infinite coordination polymers）,and even a core-less hollow SNA composed of cross-linked oligonucleotides has already been reported.The differences in the nuclear structure of these SNAs will not affect their common and characteristic properties that substantially differ from their linear strands,such as resistance to nuclease degradation,strengthen binding affinities for complementary sequences,muted innate immune responses,and cell permeability with a highly negatively charged surface via scavenger receptor-mediated endocytosis in the case of endothelial cells.These characteristic features have made SNAs useful as probes for in vitro DNA and RNA detection,or as leading compounds for gene regulation,chemotherapy,and immune system regulation,making them able to play a major role in nanomedicine.In addition,as the building block of the crystal engineering strategy based on the concept of programmable assembly,SNA can be used as a basic but versatile material in materials science,a"programmable atom equivalent"（PAE）to facilitate the construction of materials,so that novel assemblies with nanoparticles as“atoms”and DNA chains as“bonds”can be realized.However,no matter whether it is used as a biomaterial or an engineering building block,the heterogeneity of SNA materials inevitably has a negative impact on the reproducibility of its property and preparation process,difficulty in the structure-activity relationship study,and the reliability as a building block.Therefore,the synthesis of"pure"SNA which modified with a precisely number of oligo strands has great significance in the further development and research of understanding the type of new materials.In the first part of this thesis,we report the world-first case of molecular spherical nucleic acid（SNA）nanostructures.These nano-sized single molecules were synthesized from either T8 polyoctahedral silsesquioxane or buckminsterfullerene C₆₀ scaffolds,modified with 8 or 12 pendant DNA strands,respectively.These two conjugates have different DNA densities.Both of them are lower than which are on the typical SNA and are basically at the edge of the DNA density required to exhibit SNA-related characteristics.Those molecularly pure SNA shown similar properties with typical SNA,including enhanced cellular uptake and capability of gene regulations.C₆₀ SNA,due to its higher DNA density,exhibits more similar properties than the other with the traditional one（Au-NP SNA）.Meanwhile,it revealed a better nuclease resistance,cell uptake and gene regulation capabilities.With the successful preparation of these molecularly pure SNAs,a portal is opened for studying the interactions of SNA structures with cells in a more precise manner,because the control over molecularly pure SNA’s structure and property is much greater than that over conventional polydisperse SNAs.With this portal opened,it becomes possible to unveil the molecular details of SNA interactions with complementary ligands and living systems.In the second part of this thesis,we report a new synthetic strategy for the preparation of molecularly pure,mikto-arm SNAs,based on our previous synthetic method for C₆₀ SNAs.This type of SNA can have two different types of DNA strands as arms,or alternatively,other functional groups can be introduced onto the SNA core through post-functionalization.Based on this,we propose a fullerene-based strategy for the preparation of single-molecule-level,enzyme-mimicking intracellular catalyst mediating abiotic reactions.In that case,the hybrid SNA can be used as the scaffold for the intracellular macromolecular catalyst.The SNA structure can promote the cell uptake and endosomal escape of this macromolecular catalysts.Meanwhile,it improves the catalytic efficiency of the active center of catalyst by providing a stable platform and a benign environment for intracellular catalysis.Using a hetero-arm SNA containing tris（triazole）components as the model,we demonstrated that this kind of SNA macromolecular catalyst scaffold can effectively improve the stability and activity of the internal catalytic center,so that it could achieve excellent catalytic efficiency of the alkyne-azide cycloaddition（Cu AAC）reaction at a copper concentration at only nanomolar level in cells or even in vivo.This work provides a new development platform and a structural design idea for enzyme-mimicking catalysis.