Signal Amplification Based On DNAzyme Motor For The Detection Of MiRNA-10b

Purpose:The expression level of micro RNA（miRNA）is closely related to the occurrence and development of cancer.Among them,miRNA-10b shows high expression level in advanced and metastatic breast cancer,and is closely related to the metastasis of breast cancer.Therefore,establishing a simple,stable,and reliable method for miRNA detection is of great significance for studying the biological function of miRNA and molecular diagnosis and treatment of diseases.Gold nanoparticles（AuNPs）can be used in molecular diagnosis and biomedical fields,but their lack of specificity for target substances limits their application.DNA aptamers have the property of specifically recognizing target molecules,but lack stability in vivo.This study combined the good biocompatibility of AuNPs and the programmable characteristics of DNA to construct a DNAzyme nanomachine with cascading amplification effect under isothermal conditions without additional protease addition,in order to achieve the goal of highly sensitive detection of miRNA-10b,which could provide theoretical basis and technical support for the clinical detection of miRNA-10b.Methods:1.A nucleic acid signal amplification model is constructed through optimization and design of nucleic acid sequences.A self walking DNAzyme nanomachine was prepared through an Au-S bond between nucleic acid signal amplification system and AuNPs;with poly acrylamide gel electrophoresis（PAGE）and fluorescence kinetics experiments,the sequence structure was optimized and its feasibility was verified;the synthesized nano machine was characterized by UV absorption spectroscopy,Zeta potential,dynamic light scattering,and other characterization methods;the proportion of double chains in the system during the synthesis process was optimized;the concentration of salt ions,the p H of the reaction solution,the concentration of activation factors,and the reaction temperature was optimized in the detection of miRNA-10b;and the repeatability and stability of the synthesized nanomachine for detecting miRNA-10b were investigated.2.Under the optimized conditions,the detection effacacy of DNAzyme nanomachine on miRNA-10b was evaluated.The sensitivity and detection limit of the DNAzyme nanomachine was evaluated;Using different kinds of miRNAs and base mismatched miRNA-10b sequences,explore the specificity of the constructed DNAzyme nanomachine was explored;The synthesized DNAzyme nanomachine with nucleic acid signal amplification function was used to simulate the detection of miRNA-10b in human serum and the recovery rate was calculated.Results:1.Construction of nucleic acid signal amplification systemThe annealing ratio between the DNAzyme chain and the Lock chain was adjusted based on the PAGE results through the design and optimization of the nucleic acid sequence.The PAGE results can be clearly seen that at each step of the reaction,the position of the bands produced by the chain replacement reaction changed and the light dark relationship changed.When DNAzyme chain,Lock chain,target substance,and activator were present simultaneously,a large number of cleaved DNAzyme chain and miRNA binding bands were generated.However,when additional Lock chain was added to validate the second step reaction,a certain amount of cleaved DNAzyme chain and its binding bands with Lock chain were generated,and the bands were significantly deepened.This proves that a nucleic acid signal amplification system has been constructed.2.Construction of DNA nanomachinesTo construct a 3D DNA Azyme nanomachine that can walk independently,nucleic acid amplification system with AuNPs were combined.The nucleic acid cascading signal amplification system can specifically cut the substrate chain through the DNAzyme structure which was generated after the activation of the target substance,achieving signal amplification detection of miRNA.The UV characterization results showed that the maximum absorption peak of DNA modified AuNPs shifted from 522nm to 528 nm;The Zeta potential value of AuNPs before and after DNA modification changed from-46.97±2.66 m V to-14.07±1.88 m V;The dynamic light scattering results showed that the particle size of AuNPs changed from 28.41±0.42 nm to 59.19±0.87 nm before and after DNA loading;The AuNPs loaded with DNA did not undergo aggregation after adding salt ions.The above characterization results indicated that DNA was successfully loaded onto the surface of AuNPs.3.Optimization of experimental conditions detection of miRNA-10b with DNA nanomachineThe experimental conditions for constructing a DNAzyme nanomachine with cascading signal amplification effect for detecting miRNA-10b were optimized.The synthesis ratio,salt ion concentration,p H,activation factor concentration,and reaction temperature were optimized.The optimal reaction conditions for this method included:the ratio of AuNP:double strands DNA:substrate（Au:ds DNA:Substrate）in the synthesis of nano machines was 1:25:1200;In the operating solution of the fluorescence kinetic reaction,the concentration of salt ions was 200 m M Na Cl,the buffer solution used in the experiment was p H=8.0 Tris-Ace,the concentration of the activation factor Mn²⁺was 750μM,and the optimal reaction temperature was 29℃.4.Evaluating the detection effacacy of DNA nanomachines for miRNA-10b through labeling and recovery experiments in healthy human serumThis method does not require the addition of proteases,and can achieve isothermal amplification of signals with the assistance of ribozymes alone.Isothermal amplification of miRNA-10b is achieved through cascade signal amplification.When the concentration of miRNA-10b ranged from 0 nM to 3 nM,the constructed detection method exhibited a good linear relationship.The linear equation was Y=1479X+1401822（R²=0.979）.The detection limit of miRNA-10b was calculated to be 11 p M.The specificity results of activating factor showed that Mn²⁺was the best cofactor for truncated DNAzyme and the best activating factor for DNAzyme in this experiment.This experimental method can specifically recognize miRNA-10b and initiate the autonomous walking of DNAzyme nanomachines,while there was no or only a small amount of fluorescence enhancement trend for miR-222,miR-141,miR-let-7a,miR-155,and miRNA-10b containing base mismatches.Therefore,this method demonstrated good specificity.Simulated detection of miRNA-10b in human serum was performed by adding 20,200,and 2000 p M miRNA-10b,with recoveries of 92.2%,100.3%,and 95.2%,respectively.The above results indicate that this method has a wide linear range and a low detection limit.In addition,this method only requires 90 minutes to complete the detection.Conclusions:1.In this study,a signal amplification method based on a nuclease nanomachine was established to detect miRNA-10b,which requires no additional protease.The signal amplification technology can be realized only by ribozyme under isothermal conditions.A nucleic acid nanomachine with dual signal amplification function was prepared by constructing a biosensor using AuNPs.2.This method has good specificity and high sensitivity,and requires no preprocessing process.The detection can be completed within 90 minutes.It can be used for the detection of miRNA-10b in human serum,with a wide linear range and a low detection limit,providing a scientific basis for the clinical diagnosis and prognosis of miRNA.