| BackgroundNucleic acid amplification has been widely used in the fields of molecular biology research and recombinant DNA technology,which are the main methods for detecting and analyzing small amounts of nucleic acid.Polymerase chain reaction（PCR）is still the most widely used in vitro amplification method for low-abundance nucleic acids in various fields,such as clinically used to detect and identify infectious diseases,genetic diseases,DNA and RNA amplification for other research purposes.However,it relies on a thermocycler with precise temperature control to amplify the required nucleic acid fragments,which limits its further application.In this regard,isothermal amplification of nucleic acids has emerged as a promising alternative technology that can rapidly and efficiently amplify trace amounts of nucleic acids at constant temperature without the thermal cycling required for PCR.Dozens of isothermal amplification techniques based on different amplification mechanisms have been reported,most of which have remarkable sensitivity for nucleic acid detection and have broad application prospects in the fields of biosensing,bioimaging,and nanomedicine.Three-dimensional（3D）DNA walkers are a class of dynamic nanomachines that autonomously walk along three-dimensional nucleic acid tracks by energy-driven nucleic acid walking chains.In essence,the 3D DNA walker is a nucleic acid isothermal linear amplification method for signal transduction through the continuous walking of the walking chain.However,3D DNA walkers are far less efficient than natural molecular machines in terms of speed and persistence.To address the speed and persistence issues,a 3D lame DNA walker was constructed in this study.It can walk continuously and efficiently,perform molecular machine functions with good performance and complete the task of isothermal linear amplification of target DNA.In order to improve the detection sensitivity of the constructed method,the exponential amplification method was studied.Exponential amplification reaction（EXPAR）is a unique isothermal amplification method,which utilizes functional templates to exponentially amplify targets under the synergistic action of DNA polymerase and nickase.This template consists of two copies complementary to the target segment separated by the recognition sequence of nickase.Compared with other amplification methods,the EXPAR method has the distinct advantages of its isothermal nature,high amplification efficiency,and rapid amplification kinetics.However,its utility is limited by the template sequence complementary to the target,and the amplification specificity is poor.Therefore,this study proposes nicking-assisted entropy-driven reaction triggered exponential amplification reaction（NAED-EXPAR）.The method effectively amplifies the target micro RNA（mi RNA）and improves the ability to distinguish base mutation of targets.In order to construct a method suitable for detecting long RNA,Tt Ago protein editing system was further introduced in this study.Tt Ago,from Thermus thermophilus bacteria,is an editable endonuclease.Compared with CRISPR,the required guide nucleic acid is short,only16-18 nt single-stranded DNA,and its cost is low.Besides,the selection of target sequence is not limited by the original spacer adjacent motif（PAM）,so any single-stranded target can be edited.In this study,a simple and ingenious Tt Ago-mediated EXPAR method（Tt Ago-EXPAR）was constructed by making full use of the advantages of Tt Ago in RNA specific editing.This method can amplify RNA specifically and effectively,meeting the new demand for ultra-sensitive detection of long RNA.ObjectivesPart of linear isothermal amplification method:construct a 3D DNA walker that can walk continuously and efficiently,perform molecular machine functions with good performance,and complete the task of isothermal linear amplification of target DNA.Part of exponential isothermal amplification method:to ensure the highly sensitive detection of the established methodology and improve its ability to distinguish target base mutations.To construct isothermal amplification methods suitable for detecting target mi RNA and long RNA.Methods1.Construction and detection of enzyme-powered 3D lame DNA walker（1）The nucleic acid sequences involved in the 3D lame DNA walker was designed by using the nucleic acid sequence analysis software.（2）Construction of 3D lame DNA walker on microspheres by the specific binding of streptavidin and biotin.Characterization of microspheres by scanning electron microscope.（3）The feasibility,dynamics and sustainability of the 3D lame DNA walker were studied by fluorescence dynamic monitoring,fluorescence endpoint detection and fluorescence microscope imaging.Study on the persistence of 3D lame DNA walker.（4）Based on the principle of single variable,the performance and optimum reaction conditions of 3D lame DNA walker were studied,including the length of walking chain T domain,the length of spacer sequence,the rigid structure and endonuclease concentration,orbital chain concentration,microsphere concentration and microsphere size.（5）Under the optimum growth conditions of bacteria,the objective group B streptococcus（GBS）and eight other interfering experimental bacteria were cultured.The GBS DNA fragments were extracted by enzyme digestion and asymmetric PCR,and the target DNA was verified by gel electrophoresis and fluorescence.（6）3D lame DNA walker combined with strand displacement reaction was used to detect the target GBS DNA and explore the sensitivity and specificity of this method.2.Isothermal amplification strategy for micro RNA specific and ultrasensitive detection based on nicking-assisted entropy-driven reaction triggered exponential amplification reaction（NAED-EXPAR）（1）The nucleic acid sequences related to NAED-EXPAR method was designed by using nucleic acid sequence analysis software.（2）The feasibility of NAED,EXPAR and whole NAED-EXPAR were studied by fluorescence and gel electrophoresis method,respectively.The nucleic acid templates related to EXPAR and NAED-EXPAR were designed and modified,and the performance of two methods was compared and analyzed by fluorescence kinetics.（3）Analysis of the NAED-EXPAR reaction mechanism by mathematical function and calculus.（4）Based on a single variable principle,optimization of NAED-EXPAR conditions:the ratio of core double-stranded S/F,template concentration,endonuclease concentration,polymerase concentration,d NTP concentration and reaction temperature.（5）Four kinds of cancer cells were cultured under the optimum conditions of cell growth.Total RNA of four kinds of cells were extracted by RNA extraction kit.（6）The sensitivity and specificity of target mi R-21 were analyzed by NAED-EXPAR.Design the corresponding sequences of target recognition,and study the universality of NAED-EXPAR.（7）NAED-EXPAR and RT-q PCR were used to detect the total RNA samples,and the correlation between two methods was statistically analyzed.3.Thermus thermophilus argonaute-mediated isothermal exponential amplification reaction（Tt Ago-EXPAR）for ultrasensitive RNA detection（1）The nucleic acid sequences involved in Tt Ago-EXPAR research was designed by using nucleic acid sequence analysis software.（2）Tt Ago recombinant plasmid was constructed in Escherichia coli to express Tt Ago protein.The Tt Ago protein was extracted and purified.Identification of Tt Ago protein by liquid chromatography-mass spectrometry.（3）Gel electrophoresis and fluorescence kinetics analysis of Tt Ago’s function on RNA specific cleavage.（4）High-temperature resistant endonuclease and polymerase were selected.The feasibility of EXPAR and Tt Ago-EXPAR was analyzed by fluorescence kinetics and gel electrophoresis.（5）On the principle of single variable,the conditions of Tt Ago-EXPAR were optimized:reaction temperature,the ratio of guiding nucleic acid to Tt Ago,the concentration of polymerase and the concentration of endonuclease.（6）Sensitivity and specificity analysis of Tt Ago-EXPAR in detecting lnc RNA HOTAIR.（7）Tt Ago-EXPAR and RT-q PCR were used to detect the actual samples of RNA extracted from four cancer cells and their methodological correlation analysis.（8）To verify the universality of Tt Ago-EXPAR,and to detect SARS-Co V-2 N gene RNA.Single base specificity analysis of Tt Ago-EXPAR and its detection for mutant KRAS-G12D m RNA.（9）The multiplex detection of RNA mediated by Tt Ago-EXPAR combined with SERS.Results:1.In this study,a novel enzyme-driven 3D lame DNA walker was designed.The whole system was composed of only one type of particle-bound substrate,lame catalyst and enzyme.Unlike the construction of most double-legged walkers,the lame walker had a long leg mainly responsible for persistent movement and a short leg cutting substrates rapidly.Kinetic study showed that the lame DNA walker enables reaction equilibrium at 30 min and can persistently move on 3D tracks with an average rate of 6.467×10-11 M s-1.Besides,study on the persistence of the lame walker confirmed that the lame catalyst does not dissociate—it was only transferred from a split substrate to an adjacent intact substrate on the same microspheres,and was bound to the substrate by hybridization of at least 14 nucleotides.The optimum experimental conditions of lame walker were as follows:89 n M orbital chain,5 U endonuclease,0.5 mg/m L microsphere,and 0.86μm microsphere diameter.Under the optimal conditions,the detection limit of lame walker was 10 p M and the linear range was 10 p M–5n M（R2=0.995）.The specificity study showed that the fluorescence intensity of 50 p M GBS T was five times higher than that of other eight bacteria at 5 n M.When combined with asymmetric PCR,the detection limit of GBS by lame walker was 5×104 CFU/m L.2.The exponential amplification triggered by endonuclease-assisted entropy drive was a method in which one endonuclease mediated two reactions in the same system.The optimized reaction conditions of NAED-EXPAR were as follows:S/F ratio of 1:3,50 n M template,1.6U Bst DNA polymerase,1 U Nb.Bbv CI,0.2 m M d NTPs and the reaction temperature of45°C.NAED-EXPAR can detect 100 a M mi R-21 within 60 min,and its LOD was at least five orders of magnitude higher than the standard EXPAR which directly detected the target.The method also showed good specificity of identifying single nucleotide variations and can sensitively analyze the expression levels of mi R-21 in As PC-1,MCF-7,22Rv1 and He La cells.The highly consistent detection results of mi R-21 between this method and RT-q PCR proved the reliability of this method.3.The results of PAGE showed that Tt Ago enzyme possesses the ability to distinguish wild type and mutant type with high specificity at the temperature between 65℃and 69℃.Among the candidate combinations of polymerase and nickase,an EXPAR system compatible with Tt Ago enzyme was found:Bst 3.0 DNA polymerase and Nb.Bts I nickase.Through optimization,the optimal experimental conditions for the Tt Ago-EXPAR method were obtained as follows:Tt Ago protein 0.1μM,reaction temperature 66℃,ratio of Tt Ago to g DNA 1:1,polymerase 0.2 U,and nickase 5 U.Under optimal experimental conditions,this method can detect the synthetic target lnc RNA as low as 20 a M in 100 min.The detection results of lnc RNA in As PC-1,MCF-7,22Rv1 and He La cells by Tt Ago-EXPAR were in good agreement with those of RT-q PCR,indicating that this method has the potential to detect real samples under complex conditions.In addition,it can detect SARS-Co V-2 N gene RNA with a detection limit of 50 a M,further confirming the versatility of this method.Specificity studies showed that single-nucleotide mutations located between the fifth and thirteenth nucleotides showed better discriminating effects and the eleventh nucleotide position had the highest specificity.Based on its strong ability to discriminate single nucleotide differences,Tt Ago-EXPAR was further used to detect the well-studied common oncogene KRAS m RNA G12D mutation.The results showed that Tt Ago-EXPAR was able to detect KRAS-G12D m RNA as low as 0.1%in a large amount of wild-type WT KRAS m RNA.Tt Ago-EXPAR combined with SERS was used to detect two kinds of RNA.The results showed that when two targes existed at the same time,the Raman tag characteristic peak corresponding to the target could be generated near 1095 cm-1 and 1346 cm-1.Conclusions1.The new enzyme-driven three-dimensional lame DNA walker designed in this study can move rapidly and continuously on the preset spherical orbit.Different from the reported structure of the two-legged 3D DNA walker,the walking chain of the 3D lame DNA walker consists of a long leg which is mainly responsible for continuous movement at one end and a short leg which quickly cuts the substrate at the other end.The proposed three-dimensional lame DNA walker system has high persistence,reaction rate,signal-to-noise ratio and high sensitivity and specificity in sensing and detection.This work will further expand the performance of 3D DNA walker and help to improve the understanding of DNA walking system.2.A NAED-EXPAR technology is established by ingeniously combining the traditional entropy-driven reaction with the fast and efficient amplification of EXPAR through nickase,so this technology has both the high specificity recognition of the entropy-driven reaction and the high-efficiency amplification of EXPAR.The experimental results show that NAED-EXPAR can detect mi R-21 specifically and sensitively.Compared with the standard EXPAR method,the amplification efficiency of NAED-EXPAR is not affected by the target sequence,and its detection sensitivity and specificity are better than that of standard EXPAR.This method is expected to expand the application of standard EXPAR that cannot be achieved in biosensing.3.In this study,a novel one-step isothermal amplification method,Tt Ago-EXPAR,was successfully constructed based on Ago protein mediation.This method made full use of THE RNA editing function of Tt Ago protein and the superior amplification performance of EXPAR,and could be used for RNA ultra-sensitive and specific analysis within 100 min at 66°C.The detection sensitivity and single base resolution of long chain RNA amoles are achieved.This research is expected to expand the application of nucleic acid diagnosis based on p Agos. |
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