Functional Nucleic Acids-enabled Cell-selective Regulation And Biosensing

Deoxyribonucleic acid(DNA),a molecular carrier of inheritable genetic information in all living organisms,has accurate Watson-Crick base pairing and diversity of base sequence alignment.With a series of chemical syntheses and modifications,DNA has become nano-functional materials with various biochemical functions.Functional nucleic acid materials have the advantages of high designable,predictable and programmable,achieving specific nucleic acid-sensing and analysis functions and engaging fine molecular devices through accurate strands displacement reactions,cascade chemical reaction networks,and intelligent logic operations.Aptamers are chemically synthesized single-stranded oligonucleotides that fold and bind to selected molecular targets as “chemical antibodies”.They have excellent chemical properties,such as high thermostability,minimal immunogenicity,and can be chemically modified,and have been used as specific cell-surface protein recognition tools.Integrating aptamer-based recognition module,DNA-based dynamic control module and logic biocomputing function,functional nucleic acid devices can perform complex molecular tasks,indicating great potential in the application of biological recognition,sensing analysis and artificial intelligence molecular design.Ligand-receptor interactions and their dynamics and kinetics are very important for the precise regulation of extracellular and intracellular signaling events and the essential cellular events,such as cell growth,differentiation,migration,and apoptosis.Deregulation of ligand-receptor interaction causes abnormalities of cellular function,leading to different disease states.Therefore,modulation of ligand-receptor interactions has therapeutic potential against various diseases.Many proteins or peptide-ligands,such as growth factors and cytokines,as ligands physiologically function as chemical communicators between cells or tissues.Since the target receptor is not exclusively expressed in the target cell population,the clinical use of a signaling ligand is limited with therapeutic benefits.The ligands may not only trigger the receptor-mediated signaling in the target cells,but also affect undesired cell populations,causing pleiotropic effects and undesirable off-target toxicity.Hence,cellspecific activation of a signaling protein-ligand is a strategy that has the great potential to significantly narrow the target cell population and improve the precision of celltargeted therapy.According to the above challenges in cell regulation and the excellent properties of the DNA-based toolkits,we developed a cell-selective regulation strategy based on functional nucleic acids for precise cell regulation and sensitive detection of cytokine.The main research contents of this dissertation are as follows:(1)An aptamer-based molecular latch was devised for controllable ligand-receptor interactions.Human hepatocyte growth factor(HGF)was selected as the target ligand for regulation of the interaction between HGF and its natural receptor c-Met(CellularMesenchymal Transition Factor).The structural insights of the aptamer latch(AptHGF)/HGF and HGF/c-Met were studied by molecular dynamics simulation,and it was found that the aptamer bound HGF at the c-Met interacting interface,implying that Apt-HGF can conformationally modulate the HGF/Met interaction.Subsequently,the binary complex of HGF and the aptamer can be dissociated by the complementary strand via Watson–Crick base pairing,which leads to the structural switching and opening of the aptamer latch.The controllability of HGF latch switching is achieved,which provides a practical toolkit for signaling ligands' artificial control.(2)Based on the developed HGF latch tool,a cell-selective key–lock system to modulate ligand–receptor interaction for cell-selective regulation of c-Met signaling transduction in specific cell communities was developed.With the help of recognition of aptamers to the cell-specific membrane proteins,selective cell signal transduction and precise cell regulation were achieved by evaluating cell-surface protein profiles.This selective regulatory strategy can be universal by simply switching aptamers that recognize other cell-specific surface proteins as markers.Moreover,selective inhibition of ligand-receptor interaction can also be achieved by replacing the unlocking chain with the aptamer latching chain.This strategy provides a powerful paradigm for precise regulation of cellular behaviors and a promising approach to address pleiotropy and off-target toxicity of ligands as therapeutic agents.(3)In order to achieve higher-order selective cell regulation,a “Scan and Unlock”DNA automaton was rationally designed to equip a native protein-ligand with cellidentity recognition and receptor-mediated signaling in a cell-specific manner.The DNA automaton was carried out by performing intelligent Boolean logic operations through nucleic acid chemical reaction network,which can accurately analyze multiple protein components on the cell surface,and achieved high-order selective cell regulation by performing the AND and NIMPLY logic operations.The DNA automaton was further successfully applied in a cell co-culture system.Furthermore,tumor necrotic factor-a(TNFa)was chosen as another target ligand,and programmed cell death of the target cell subpopulation through cell-specific modulation of TNFR1 signaling was detected,suggesting the universality of the DNA automaton.This strategy provides an intelligent high-order selective cell regulation scheme,which provides a general research approach for solving the problem of the off-target side effects of signaling ligands and achieving precise drug delivery in biomedical research.(4)Cytokines are secreted protein/peptides that participate in various cell regulation and play important roles in cell signaling transduction.The detection of cytokines has great clinical significance in disease diagnosis and treatment.However,cytokine detection in situ or on a single cell level is limited these days.Here,an aptamer-based TNF? detection method was designed in this chapter.Three TNF?detection approaches,including conformational changes of molecular aptamer beacon,the dissociation detection assay,and the two-step molecular beacon assay,were compared.It was found that the two-step strategy has the best detection performance and can achieve the detection at the nanomolar level.The optimized assay was successfully applied for the TNF? detection at the single-cell level by cell imaging.