Construction Of Amplified Functional Nucleic Acid Circuits For Cell Imaging And Modulation

Cancer is one of the leading causes of morbidity and mortality worldwide,especially in developing countries.During the process of cancerous cell metabolism,the dysregulation of some important micro RNAs or proteins makes them potential biomarkers for cancer diagnosis.Thus,it is necessary to construct sensing systems for sensitive and specific detection of such biomarkers.Nucleic acids have substantially expanded their functions from natural bioinformation storage platforms to potent and designable tools for biosensing and bioengineering applications.Based on the remarkable Watson–Crick base-pairing,nucleic acids can either respond quickly to specific biomolecules or self-assemble into intelligent networks.Particularly,functional nucleic acids provide valuable means to gain insight into tumor biology and cancer therapy.As one kind of functional nucleic acids,aptamers exhibit highly specific binding affinities to their corresponding targets by mimicking the functions of antibodies.The other functional nucleic acid is known as DNAzyme,which possesses highly catalytic activity like proteases.Both them could substitute for proteins in many biological applications due to their low cost and high stability.Moreover,the programmability of nucleic acids makes them excellent candidates for performing predictably intelligent self-assembly and designing various reaction networks.DNA circuits lay a solid foundation for the signal amplification of functional nucleic acids,which inspires us to construct cascade DNA circuits for disease biomarkers detection.By virtue of isothermal enzyme-free DNA circuits,functional nucleic acids could be effectively improved for high-performance biosensing and bioengineering applications.The main research contents and results are as follows:1)Enzyme-free isothermal concatenated nucleic acid amplification circuit(CHD)was designed by integrating a lead-in catalyzed hairpin assembly(CHA),intermediate hybridization chain reaction(HCR),and ultimate DNAzyme amplifier units.The target analyte initiated the self-assembly of hairpin-like reactants into ds DNA products powered by CHA,which generated numerous trigger sequences for activating the subsequent HCR-assembly of long tandem DNAzyme nanowires.Then,the as-acquired DNAzyme successively cleaved its substrates,leading to an amplified fluorescence readout.The sophisticated design of the CHD scheme was systematically investigated in vitro and showed dramatically enhanced detection performance.As a general sensing strategy,this CHD strategy enabled the sensitive analysis of micro RNA and its precise intracellular imaging,originating from their synergistic signal amplifications.This method shows great potential for analyzing trace amounts of biomarkers in various clinical researches.2)An activatable,recombined,and amplified modulator(ARAM)was constructed on cell membranes for specific cell identification and modulation.This ARAM strategy was composed of a catalytic hairpin assembly(CHA)circuit to execute the activation,catalysis and converted functions between two specific functional aptamers for realizing the dual signal combination of membrane proteins.Additionally,a hybridization chain reaction(HCR)circuit was designed to generate polymer units for amplified signal outputting.Under the interface fluidity of the cell membrane,we have successfully realized signal transfer from one aptamer to another,forming firework-like signal amplification.We could accurately and sensitively identify target cells among the subpopulation cells via the presence or absence of different biomarkers.Furthermore,HCR operating on cell membranes also achieved cell migration behavior modulation along with the specific imaging by arranging the distribution of c-Met receptors.All in all,this ARAM strategy can provide a versatile platform for autonomous and specific cell identification only by changing aptamers and present a possibility for specifically modulate their related cellular behaviors,which helps us to understand the magical biology and shows great promise in precise disease diagnosis and therapy.