Construction And Application Of MicroRNAs Sensing And Regulating Technology Based On DNA Nanotechnology

Background:MicroRNAs(miRNAs)play important roles in many processes of life and are closely related to the occurrence and development of many diseases,so they can be used as one of the biomarkers for the diagnosis and prediction of diseases such as tumors.It has been confirmed that the combined detection of abnormally expressed miRNAs and other biomarkers can effectively improve the early diagnosis rate of tumors.However,the existing miRNAs detection methods are difficult to meet the requirement of diagnosis and treatment.The reason is that,first of all,the miRNAs have the characteristics of low content,small size,similar sequences,easy degradation,and poor specificity,which is not conducive to detection.Secondly,the currently used miRNAs detection methods,such as western blot,real-time PCR and in situ hybridization,cannot meet the clinical detection requirement of high sensitivity,high specificity and in situ detection.Therefore,in order to meet the needs of clinical diagnosis,it is urged for us to development an accurate,reliable,simple and fast miRNAs detection methods.In addition to DNA as carriers of genetic information,DNA can also be used as building elements for tunable nanomaterials.Their programmed identification and assembly can schieve precise control of the nanomaterial topology and function.The single DNA strand,which served as self-assembling motifs,can be switched between single and double strands driven by changes in chemical entropy caused by base pairing.Inspired by this characteristic,controllable movement or special functions of DNA nanotechnology can be achieved by properly designing the DNA sequences and using the differences in chemical entropy of single-stranded DNA hybrids of different lengths.With the development of molecular engineering,DNA nanotechnology has become an effective molecular technology,which is applied to various aspects such as biological detection,drug delivery and logical operations.Nucleic acid isothermal amplification technology has the advantages of high sensitivity,short reaction time,simple operation,and does not require complicated instruments,which is particularly suitable for high sensitivity,high specificity biological analysis and clinical diagnosis,especially for point for care testing.Hybrid strand reaction(HCR)is a cascade hybridization reaction that is caused by a single strand and two DNA hairpin structures.At present,this technology has been successfully applied to the detection of SNP,miRNAs,etc.,and the nonlinear hybrid strand reaction(NLHCR)introduced more reaction strands,so cascade hybridization can form a dendritic structure,further enhancing the effect of signal amplification.DNA nanomachine is a direction for the development of DNA molecular nanotechnology.After being exposed to external stimuli,they can change from one state to another,and develop into nano-scale mechanical parts that can be precisely controlled by specific molecules.DNA nanomachines can achieve precise detection of specific molecules according to their working principle.Among them,DNA tweezers can be opened and closed automatically after receiving the corresponding stimulus,so as to realize the detection of the target substance.At present,DNA nanomachines are mainly used for in vitro detection.The reason may be that the complexity of the environment can affect their work and even destroy their key structures,making them impossible to exist in a stable manner.Since the DNA tetrahedron structure has been proven to maintain a relatively stable structure in living cells,the combination of DNA tweezers and the tetrahedron structure is expected to build a DNA nanomachine that can stably exist and maintain cell functions.Thus,this project aims to build methods that can accurately detect miRNAs in vivo and in vitro through DNA nanotechnology.Objectives:Combining with the advantages of high sensitivity detection of electrochemical biosensors,this study aims to construct a mi RNAs electrochemical biosensor,which is based on DNA isothermal amplification technology.This detection method is established to achieve the high sensitivity and specificity detection of extracellular miRNAs.Combining the working principle of DNA tweezers and the structural stability of DNA tetrahedron,this study aims to construct DNA nanomachines that can stably exist in living cells and have the function of detection and regulation to achieve the in situ detection and regulation of miRNAs expression in living cells.This DNA nanomachine can overcome the difficulty in indentifying the origin of abnoemal miRNAs in the diagnosis and treatment of diseases,and provide more accurate test results for clinical diagnosis.Methods:1.Construction of miRNAs electrochemical biosensor detection method based on Y-shaped probe and nonlinear hybridization chain reaction.1)Design and validate sequences for Y-shaped probe and nonlinear hybridization chain reaction.2)Validate competitive binding of mi RNAs to Y-shaped probe and initiation of nonlinear hybridization chain reaction in solution.3)Construct the miRNAs electrochemical biosensor detection method which is based on Y-shaped probe and nonlinear hybridization chain reaction.4)Optimizing the reaction conditions of miRNAs electrochemical biosensor based on Y-shaped probe and nonlinear hybridization chain reaction.5)Detect the sensitivity,specificity and stability of miRNAs electrochemical biosensor based on Y-shaped probe and nonlinear hybridization chain reaction.6)Detection of clinical samples.2.Construction of novel three-dimensional DNA tweezers for miRNAs detection.1)Design and synthesize three-dimensional DNA tweezers.2)Thermodynamics and kinetic analysis of three-dimensional DNA tweezers.3)Verification of the sensitivity and specificity of three-dimensional DNA tweezers for detecting mi RNAs.3.The study of three-dimensional DNA tweezers for in situ detection of miRNAs in living cells.1)Preparation of K562 and other cells.2)Detection of the stability and cytotoxicity of three-dimensional DNA tweezers.3)Optimize the reaction conditions of miRNAs detection in three-dimensional DNA tweezers.4)Verify that the three-dimensional DNA tweezers can be used for semi-quantitative detection and specificity of miRNAs in living cells.5)Detection of clinical samples.4.Construction of modular DNA intelligent tweezers for detection and regulation of miRNAs in living cells.1)Design and construct the modular DNA intelligent tweezers.2)Validate the ability of modular DNA intelligent tweezers to regulate miRNAs expression in living cells.Results:1.Successfully constructed the miRNAs electrochemical biosensor based on Y-shaped probe and nonlinear hybridization chain reaction.After optimized the reaction conditions,this miRNAs electrochemical biosensor has achieved a good linear response with a linear range of1fM-10pM,the regression equation is Y=0.03252lgc+0.3121,R~2=0.9977,and the minimum detection limit is 0.3334fM.This method has good specificity and can recognize single base mutation.We also tested the clinical application ability using biological simulation samples,the results proved that it can meet the detection needs of biological complex samples.2.Three-dimensional DNA tweezers have been successfully designed and synthesized.They can detect the target miRNAs in a short time according to the analysis of thermodynamic and kinetic.This method has good sensitivity and specificity:the linear range is 1nM-50nM,the regression equation is Y=1.04x+202,R~2=0.9980 and the minimum detection limit is 1.499nM.Comparison with the DNA tweezers reported,this three-dimensional DNA tweezers hold the ability to be used as DNA nanomachines for miRNAs detection.3.Three-dimensional DNA tweezers have been verified have no obvious cytotoxicity to living cells and can stably present in living cells for more than 6 hours,which can meet the needs of in situ detection.By optimizing the reaction conditions,semi-quantitative detection of miRNAs expression in living cells can be achieved,and the abnormally expressed miRNAs and cells can be detected simultaneously.The application potential have been verified by clinical blood samples,the results indicate that three-dimensional DNA tweezers can achieve the practical detection of simple clinical samples and have the potential for application in clinical practice.4.Successfully designed and synthesized the modular DNA intelligent tweezers.The detection and regulation function to target miRNAs in living cells have been verified by RT-PCR and WB.Conclusions:1.The successful construction of miRNAs electrochemical biosensor based on Y-shaped probe and nonlinear hybridization chain reaction has improved the sensitivity and specificity of miRNAs detection.This method provides a reliable detection platform for the application of DNA nanotechnology in biological detection,so that the application of DNA nanotechnology at the molecular level is becoming more and more widespread.2.The construction of three-dimensional DNA tweezers and their good performances in miRNAs detection have proved that it can be used as a DNA nanomachine in biological detection.The structure of this novel DNA nanomachine improves the stability and environmental adaptability of DNA nanomachines and helps to expand the application range.3.The successful in situ miRNAs detection in living cells of three-dimensional DNA tweezers has overcame the problem of poor specificity of miRNAs in diseases diagnosis,which provides technical support for clinical application of mi RNAs as biomarkers.Moreover,the successful application of this detection method in living cells helps to develop more in situ detection technologies.4.The integrated operation of the modular DNA intelligent tweezers for detecting and regulating mi RNAs in living cells provides a reliable solution for the precise diagnosis and treatment of mi RNAs.This design of intelligent tweezers means that DNA nanomachines can evolve from a single function to a composite function and may have more important application value in the field of biology.