Construction Of Enzyme-free Nucleic Acid Circuit And Its Application In Biomedicine Study

It is of great significance to develop highly sensitive and highly specific molecular probes for biochemical analysis and diagnosis and treatment of tumors at early stage.However,the following two problems often occur in the practical research:(1)For the low concentration of target within biological samples,the signals often can not be detected due to insufficient sensitivity,thus cause false negative signals;(2)The interference of complex biological environment such as degradation of probes by enzymes in serum and even cells or activation of probes by non-target molecules,the off-target effect caused by single-target recognition system,which can result in false positive signals.As a signal amplification form which bases on nucleic acid hybridization,the enzyme-free(Specifically for protease-free)nucleic acid circuit encodes the required information in the designed DNA sequence,initiates a series of hybridization reactions under the trigger of various targets such as nucleic acid,protein and small molecule,and finally outputs the signal.It does not need the participation of protease,constant temperature,mild reaction conditions,so it shows a unique advantage and important application value in the bioanalysis fields.Focusing on the above key problems: "false negative" and "false positive",this dissertation aims to develop novel DNA probes and analytical methods by using AuNPs-based colorimetric and FRET as output signals.These research constructs and develops a series of probes and analytical methods with elevated sensitivity?anti-interference capability and precision in the complex biological environment for highly sensitive and specific detection of important biological molecules,and even the accurate identification and killing of tumor cells.The specific research content is as follows:1.An amplified gold nanoparticle-based colorimetric detection strategy driven by hybridization chain reactionAiming at the problems of poor sensitivity caused by conventional AuNPs-based colorimetric methods,we introduce an enzyme-free and amplified colorimetric detection strategy,which is based on gold nanoparticle(AuNP)aggregation through hybridization chain reaction(HCR),and apply it to DNA detection.In the absence of target,the two kinds of hairpins coexist in solution.In the presence of target DNA,it acts as the catalyst that fuels the DNA hairpins to open and then hybridize each other through HCR.These newly formed duplexes could assemble and induce AuNPs aggregation via hybridization of the complementary domains between the linker sequence grafted on the surface of the AuNPs and the tail sequence at the end of the hairpins.With this method,the detection limit(LOD)is 200 pM and without sacrificing it specificity towards single base mutation DNA,the sensitivity of which is about 2 orders of magnitude higher than that of conventional AuNP-based colorimetric biosensing.Meanwhile,the method is simple and does not require protease and complex instruments,thus providing a new technique for analysis research.2.An amplified gold nanoparticle-based colorimetric detection strategy driven by catalyzed hairpin assemblyHere we introduce an enzyme-free and amplified colorimetric detection strategy,which is based on gold nanoparticle(AuNP)aggregation through catalyzed hairpin assembly(CHA),and apply it to adenosine detection.The approach is based on the adenosine-aptamer binding triggered liberation of an initiator strand that triggers the CHA reaction.In the presence of adenosine,it first binds to the aptamer and then the initiator sequence T is available to catalyze consecutively hybridization of DNA hairpins.The adenosine/aptamer complex is inherently unstable and dissociates from the H1/H2 complex,completing the reaction and allowing T to act as a catalyst to trigger the hybridization of additional pairs of H1 and H2 hairpins.These newly formed duplexes could assemble and induce AuNPs aggregation via hybridization of the complementary domains between the linker sequence grafted on the surface of the AuNPs and the tail sequence at the end of the hairpins.The results show that this method is much better than the traditional colorimetric detection method with LOD as low as 300 nM.The strategy also could be applied to adenosine analysis in complicated biological samples(cell lysates).3.Cascades of catalyzed hairpin assembly-hybridization chain reaction for sensitive DNA and adenosine detectionAlthough the AuNPs-based colorimetry methos described above can realize the amplified detection of targets to a certain extent,when the concentration of target is extremely low,the linear amplification by single nucleic acid circuit can no longer meet the analysis requirements.Meanwhile,the interference of complex biological environment such as degradation of probes by enzymes in serum and even cells,activation of probes by non-target molecules,the off-target effect caused by single-target detection system,which can result in false positive signals.Focusing on these problems,this work embeds the initiation sequence of HCR into the constitution unit of CHA,and successfully constructs two-layer enzyme-free CHA-HCR nucleic acid circuit.Combined with FRET signal,the target concentration is quantified by the fluorescence ratio at different wavele ngths,so as to improve the anti-interference ability of probes and obtain more accurate results.In the cascade circuits,upstream CHA circuit yields numerous duplexes while the downstream HCR circuit yields a long nicked concatamer carrying a large number of repeat units,thus producing remarkable FRET signals.This circuit could amplify 50000-fold signal,comparable to no amplification,without sacrificing its specificity within 2 h.Subsequently,with coupling of structure-switching aptamer,as low as 200 pM adenosine is detected in buffer,as well as in human serum.4.Dual-microRNA-controlled DNAzyme-hybridization chain reaction cascades for accurate discrimination of cell subtypesThe development of reliable strategies to distinguish various cancer cell subtypes at the molecular level is particularly important for disease diagnosis.In this work,we have designed dual-microRNA-controlled double-amplified DNAzyme-HCR cascaded circuits for liver cancer cell subtype identification.MiR-122 acts as the initiator of the upstream DNAzyme circuits and activates the DNAzyme.The cleaved product enters the downstream and reacts with the hairpin for HCR triggered by miR-21,resulting in a long DNA concatamers carrying a large number of repeat units,thus producing remarkable FRET signals.The basic idea is to improve sensitivity by DNAzyme and HCR circuit,improve accuracy by the “AND” logic gate based on miR-122 and miR-21,and resist the interference of the complex.The in-tube and in-cell experimental results show that the cascaded logic DNA circuits can work and serve to differentiate the liver cancer cells Huh7 from other normal cells and cancer cells.5.Dual-RNA-controlled hybridization chain reaction-DNAzyme cascades for accurate diagnosis and gene therapyThe above works aim to improve the sensitivity,anti-interference ability and accuracy in complex biological environment,thus solve the two key problems of intracellular sensing: “false negative” and “false positive”.In this study,with the goal of designing theranostics probes,we have engineered amplified and dual-RNA-controlled HCR-DNAzyme circuits for accurate diagnosis and double-checked gene therapy of target cancer cells.HCR is employed to amplify the miR-21 recognition event which is highly expressed in the breast cancer cells as well as the therapeutic DNAzyme(tDz)activation,subsequently the active tDz is utilized to recognize and catalytic cleave EGR-1 mRNA which is also highly expressed in breast cancer cells for gene therapy.The cascade circuit overcomes the limitations of traditional theranostics methods in terms of sensitivity,speci ficity and accuracy.Moreover,it provides advanced research tools for early diagnosis and treatment of disease.