Construction And Biological Application Of Valency-Controlled Molecular Spherical Nucleic Acid

Spherical Nucleic Acids(SNAs)are a type of nucleic acid nanostructures composed of a core layer and a shell layer.The core layer is composed of inorganic or organic nanoparticles,and the shell layer is composed of highly oriented and densely arranged nucleic acid chains.Compared with single-stranded nucleic acid,SNAs have the advantages of anti-nuclease digestion and high cell delivery efficiency,and have broad application prospects in the fields of gene regulation,disease diagnosis and drug delivery.However,the particle size of SNAs and the number of modified shell chains("valence bonds")are mostly uncontrollable,making SNAs exhibit polydispersity.This polydispersity greatly restricts the research on the structure-function relationship and reaction mechanism,and also hinders its application in biosensing.DNA nanotechnology has the advantages of programmability,addressability and predictability.It is helpful to construct monodisperse nanomaterials with controllable size and adjustable modification density.It can provide a new research tools for solving the above problems of SNAs.In this thesis,DNA nanotechnology was used to successfully construct monodisperse novel molecular spherical nucleic acids.By controlling the surface density of molecular spherical nucleic acids,the structure-function relationship can be studied at the molecular level.In addition,combined with the signal amplification strategy of catalytic hairpin assembly(CHA),this thesis also constructed a micro RNA(mi RNA)biosensor with controllable "valence bond".These probes have tunable biosensing performances,including sensitivity,response range,and detection limit.Finally,the probes were applied for the micro RNA imaging of in living cells.The specific research contents of this thesis are:In Chapter 2,we first proposed a new molecular spherical nucleic acid construction strategy based on framework nucleic acids(FNAs).Through reasonable base design,we have developed a series of novel molecular spherical nucleic acids with tunable valence bonds.Based on this,the structure-function relationship has been studied.We found that the density of modification of the shell of spherical nucleic acids is an important factor influencing its resistance to enzyme digestion and cell entry efficiency.In Chapter 3,based on the CHA amplification technology,we developed a series of molecular spherical nucleic acid fluorescent probes with different valences.With tunable ratio,these probes exhibited adjustable response rate,sensitivity and response range.In addition,molecular spherical nucleic acid probes enable imaging of low-abundance target molecules(mi RNA)in cells.In Chapter 4,by adjusting the distribution ratio of the two groups in the probe,we summarize the construction rule of the optimal probe and explore its universality.Besides,the probe can distinguish cell lines with different mi RNA expression levels.In summary,we have developed monodisperse spherical nucleic acids based on DNA nanotechnology,which provides new strategy for studying the structure-function relationship of spherical nucleic acids.In addition,we have also developed molecular spherical nucleic acid probes with adjustable biosensing properties,which have successfully achieved imaging and analysis of low-abundance target molecules at the cellular level.