Construction Of Resonance Energy Transfer-Based DNA Walker And Its Application In The Measurement Of Nucleic Acid Disease Markers

Detection of disease markers is an effective method for early diagnosis of diseases.It can provide real-time and accurate biological information for early diagnosis.As important carriers of genetic information stored,transmitted and expressed by organisms,abnormally expressed nucleic acids（DNA and RNA）are an important class of disease markers.Therefore,a large number of nucleic acid biosensors have been developed by researchers in order to monitor changes in the level of nucleic acid content during disease development.Thanks to the strict complementary base pairing DNA,strong ability to recognize and highly programmability,DNA walkers that built based on resonance energy transfer（RET）have become a hot research topic in the field of biosensors recently.The DNA walker is capable of walking precisely along a predetermined programmable trajectory,outputting a"one-to-many"measurement signal.Compared with traditional biosensors,DNA walkers can offer more flexible operation and multifunctional design.However,most of the existing preparation methods require time-consuming and complex operational processes and higher costs.At the same time,they have high background signals and are susceptible to false positive signals.Therefore,a cost-effective,simple,rapid,sensitive and highly specific DNA walker developed for the analysis of nucleic acid disease markers is highly in demand.To address these problems,two type of DNA walkers based on RET are constructed and applied to the detection of disease-related nucleic acids in this paper.The main research contents are as follows:1.DNA walker on graphene oxide surface driven by Exonuclease III for hepatitis B virus DNA measurement.In this study,a two-dimensional（2D）DNA walker propelled by Exonuclease III（Exo III）was constructed on graphene oxide（GO）surface for the objective analysis of hepatitis B virus DNA（HBV-DNA）.Firstly,FAM-DNA was hybridized with BHQ1-DNA to form double-stranded DNA（dsDNA）.poly-A extending from the end of BHQ1-DNA was fixed on the surface of GO by hydrogen bonding andπ–πstacking.The fluorescence of FAM was quenched due to the dual energy transfer between FAM,BHQ1 and GO.When HBV-DNA existed,it could bind to FAM-DNA through a chain displacement reaction mediated by the naked toehold of FAM-DNA,and gradually formed a new dsDNA.Exo III could splice FAM-DNA bound to HBV-DNA into single nucleotides from the 3’end.The released HBV-DNA gradually bond to the next FAM-DNA,and the fluorescence recovery of FAM（F/F₀）was taken as the signal,realizing sensitive detection of HBV-DNA.In the range of 0.5–125 nmol/L,the response of the DNA walker to HBV-DNA showed a good linear relationship with the concentration of HBV-DNA,and the detection limit was 50.4pmol/L.The innovation of this study is reflected in that we assembled a 2D DNA walker with simple operation and high cost performance through simple adsorption.The walking speed of the DNA walker can be adjusted by adjusting the density of dsDNA loaded on GO.Due to the use of dual energy transfer,the background signal is greatly reduced and the sensitivity is greatly improved.2.ATP-driven DNA walker on the surface of DNA nanospheres for miR-21measurement.In this study an endogenous adenosine triphosphate（ATP）-driven three-dimensional（3D）DNA walker was built on the surface of DNA nanosphere（DS）for the detection of miR-21 in live cells.First,a DS synthesized by annealing of single-stranded DNA with four palindromic sequences in the middle and extended sequences at both ends was used as a carrier.The locked walking strand（W/B）and DNA double strand containing ATP aptamer were grafted to the surface of DS by hybridization reaction with extended sequences.Thus,a DS based DNA walker（DS walker）was successfully constructed.miR-21 bond to the blocking strand B and released the walking strand W,triggering the ATP-driven walking process.ATP continuously binding to its aptamer provided fuel for DS walker walking,generated cumulative Cy3 fluorescence signals.In the range of 0.5–25 nmol/L,the response of DS walker to miR-21（F-F₀）showed a good linear relationship with miR-21 concentration,and the detection limit was 17.0 pmol/L.Also,the strategy proposed in this study allows real-time in situ imaging of miR-21 at the cellular level,which has great potential in the early diagnosis of cancer.The innovation of this study is reflected in that the DS walker was simplely prepared with high assembly efficiency,avoiding the tedious process of using hard nanoparticles as the carrier,and the goods can be efficiently transported into living cells.At the same time,endogenous ATP was the driving force without additional auxiliary factors,which avoided the problem of low walking efficiency caused by asynchronous transmission of components.To sum up,in this paper,using RET as the measurement signal,two types of DNA walkers are constructed based on FRET method for nucleic acid biomarkers detection and imaging analysis.DNA walker constructed based on GO had the advantages of low cost,simple operation,low background signal,fast,adjustable walking speed,which provided a new idea for designing simple and efficient DNA walker.The DS walker assembled by simple DNA hybridization realized the sensitive detection and highly specific imaging of miR-21,and high intracellular ATP was used as the driving force,to ensure efficient intracellular walking.