Aptamer-based Multivalent DNA Nanostructures For Tumor Cell Analyses

The cells can provide dynamic,rich and real information for physiological and pathological processes,which have attracted widespread attention.However,cells are often in complex environment,and some of them are usually present at very low abundance and h ave high heterogeneity,such as circulating tumor cells,circulating fetal cells and specific T cells in blood.Therefore,t here are still challenges in how to find rare cells in complex environment and even acquire relevant biological information inside the cells,which puts forward high requirements for probe s,including at least two aspects:(1)the high affinity for specific recognition with target cells;(2)the high signals for discrimination of target cells from complex background s.Multivalent binding brings a new opportunity for the construction of probes with high affinity,that is,improv ing affinity of existing probes by increasing the interaction between ligands and receptors.Meanwhile,the emergence of signal amplification methods based on nanomaterials also provides new strateg ies for target identification.DNA nanostructures are easily assembled and showed good biocompatibility,which provide powerful tool s for living cell analyses.In particular,aptamers and nucleic acid signal amplification strategies are compatible with DNA nanostructures.Herein,taking living cells as a research subject,we pay attention to the choice of nanostructures,integration mode of multivalent ligands and functional units,and biocompatibility.The multivalent DNA nanostructures based on aptamers are developed for living cell s analyses.C ontents in this dissertation include following four parts:1.A DNA nanocreeper was constructed for the specific detection of tumor cells.Firstly,DNA duplex with repeated units was prepared as the trunk of nanocreeper based on hybridization chain reaction(HCR).Then,multiple aptamers and branching probes were assembled into HCR products via streptavidin-biotin interaction,similar to the feet and leaves of plant creeper s.Due to the flexib le DNA duplex,the HCR products could bend well to adapt to receptors randomly distributed on cell membrane and result in multivalent binding,similar to the feet of creepers tightly clinging to the walls and rocks.Meanwhile,multiple branches could hybridize with fluorescein labelled signal probes to form multiplexed fluorescence supersandwiches to amplify the signals,similar to creepers covered with blossom.The dissociation constant(K _d)of DNA nanocreeper was 0.75±0.14 n M,just one tenth of monovalent aptamer(K_d=7.48±1.65 n M),which indicated the affinity was significantly improved.Meanwhile,the mean fluorescence intensity and signal to background ratio were both 10-fold of monovalent aptamer(from 2 to 21,11 to 160,respectively).Combined with microfluidic chip,it could realize the specific detection of cells in blood.This method is expected to provide a powerful tool for the detection of low abundance target cells in liquid biopsy.2.A multivalent ratiometric fluorescent DNA nanostructure(RFDN)was designed for mitochondrial adenosine triphosphate(ATP)imaging in living cells.HS1-Cy3 and HS2-Cy5 contained split aptamer fragments of ATP and are labeled with fluorescent donor s(Cy3)and fluorescent acceptor s(Cy5),respectively.The RFDN was constructed by the continuous hybridization of two hairpin DNA strands(HS1-Cy3 and HS2-Cy5)under the initiation of the trigger,which could produce RFDN with repeated units.The binding of split aptamer fragments on the RFDN to ATP shortened the distance between Cy3 and Cy5,resulting in effective energy transfer and ratiometric signal output.Since local concentration of ATP probes was improved,the limit of detection was 1/10 lower than that of conventional split aptamers.The RFDN showed good biocompatibility and could be noninvasively internalized into cells in a caveolin-dependent endocytosis pathway.Then RFDN could target the mitochondria via Cy3 and Cy5,and achieve ratiometric imaging of mitochondrial ATP.Moreover,confocal imaging results showed that the intracellular ATP changes stimulated by drugs in living cells could be indicated by the RFDN.In this way,the RFDN is expected to be a simple,flexible,and general platform for chemo/biosensing in living cells.3.A multivalent Janus triangular prism was constructed for the sens itive detection of tumor cells.We further take spatial factors into consideration when increase the ligand density for multivalent binding.Taking rigid DNA triangular prism as basic construction unit,the trigger strands for signal amplification were assembled to the top of triangular prism and the aptamers and linker strands were assembled to the bottom,namely Janus triangular prism nanostr ucture.It has two faces like Janus,the door god of ancient Rome,and can look two opposite directions at the same time.Then,multiple triangular prisms were hybridized in the form of“hand-in-hand”and formed a flexible network and recognition probe s could extend horizontally and vertically to form flexible network-like multivalent Janus DNA nanostructure.One side of nanostructure could bind to cells and the other side was used for signal amplification by multiplexed fluorescence supersandwiches.The K_d value was 0.17±0.27 n M,indicating 10-fold improvement in affinity of conventional aptamers.The mean fluorescence intensity and signal to background ratio of target cells were 182 and 1069respectively,which were both 100-fold higher than that of monovalent aptamer and 9-fold and 7-fold of DNA nanocreeper.The multivalent nanostructure realized relative independence of recognition unit and signal unit based on the structural support and spatial separation of DNA triangular prism.At the same time,multiple triangular prisms are woven in a way of“hand-in-hand”,which could match the cell surface and achi eve high affinity and signal int ensity.It is expected to provide a new idea for rare cells detection and cell surface engineering.4.A multivalent Janus DNA nanostructure was constructed to achieve specific capture of tumor cells in whole blood,to expand the application ability of multivalent DNA nanostructures in real samples.Herein,the DNA triangular prism was still used as the basic construction unit s.The biotin modified capture strands were assembled to the top of triangular prism,and the aptamers and linker strands were assembled to the bottom.The network-like multivalent DNA nanostructure was woven by“hand-in-hand”.The bottom could multivalently bind with cells,while the top could multivalent bind with magnetic beads via streptavidin-biotin interactions to capture of tumor cells by magnetic separation in blood.The capture efficiency was 90.7-94.1%.The Janus-like design could separate two functional units and perform their respective duties.It connected micron sized cells and magnetic beads through double multivalent effects,which was like“double-sided tape”.Therefore,it is expected to provide a new idea for the efficient capture of rare cells in complex biological samples.Taking living cells as the research subject,the dissertation aims at the problems of structure selection,ligand assembly and biocompatibility in the design and assembly of multivalent nanostructures.The multivalent DNA nanostructures were constructed based on the principle of ensuring high affinity of recognition probes and high performance of o ther functional probes,to realize the detection,imaging,separation,and enrichment of living cells.They are expected to provide powerful tools for cell analyses and function regulation in complex biological samples and even in vivo.