Applications Of Functional Protein-based Nucleic Acid Signal Conversion Elements In Biosensing

Biosensors are widely used in disease monitoring,food analysis and public health safety due to their advantages of simple,low cost,high sensitivity and specificity.Molecular recognition element and signal conversion element are two important parts,which determine the specificity and sensitivity of biosensor respectively.In recent years,with the rapid development of biotechnology,a new interdisciplinary,synthetic biology,has emerged.The aim of synthetic biology is to design biological components,networks and systems with new functions based on engineering concept and biological technology.Therefore,synthetic biology provides a large number of molecular recognition and signal conversion elements,which greatly promotes the rapid development of biosensors.In this paper,a series of biosensors were developed by using CRISPR-Cas12 a and responsive RNA polymerase elements.And these biosensors were applied to the detection of viral nucleic acid,oncogene mutation,protease and other biomarkers in clinical samples with high sensitivity and specificity.The main research contents are as follows:(1)To explore the effect of nucleic acids in CRISPR-Cas12 a on its “collateral”cleavage activity.In CRISPR-Cas12 a,guide RNA(g RNA)guides Cas12 a protein to specifically recognize and cleave target double stranded DNA(ds DNA),and then activates the “collateral” cleavage activity to cleave any single stranded DNA(ss DNA).In this section,we selected Lb Cas12 a protein as an example to explore the influence of the composition and structure of ds DNA,g RNA and other nucleic acid components in CRISPR-Cas12 a on its “collateral” cleavage activity.We investigated the effects of the length and composition of the protospacer adjacent motif(PAM)sequence,the upstream sequence of PAM and the target sequence(TS)on the“collateral” cleavage activity of Lb Cas12 a.In addition,we designed a series of inhibitor DNA(i DNA),which affect the secondary structure and activity of g RNA through nucleic acid hybridization,thereby indirectly inhibiting the cleavage activity of Cas12 a.Moreover,we explored the “collateral” cleavage efficiency of Cas12 a to ss DNA bulge in double strand,including the influence of ss DNA bulge size and base composition.Finally,we succeed in regulating the “collateral” cleavage of Cas12 a through the rational design of i DNA,which laid the foundation for the subsequent design of biosensors.(2)Construction of positive feedback nucleic acid circuits driven by CRISPR-Cas12 a autocatalysis.Artificial nucleic acid loop has the advantages of precise control,dynamic and functional,which has great application potential in the field of biosensor.However,in molecular diagnosis,artificial nucleic acid circuit is faced with the challenge of not dealing with genomic DNA directly and lack of detection sensitivity.To solve this problem,we developed CRISPR-Cas-only amplification network(CONAN)based on the study of Cas12 a “collateral” cleavage activity regulation,and realized isothermal amplification detection of genomic DNA.In CONAN,we integrated the precise target recognition ability,helicase activity and efficient “collateral” cleavage activity of Cas12 a to design the artificial positive feedback chemical reaction network by autocatalysis-driven,and realized exponential signal amplification.As a result,CONAN could specifically detect synthetic target DNA as low as 5.0 a M.Due to the cumulative effect of signal difference in the positive feedback process,CONAN significantly improved the sensitivity of CRISPR-Cas12 a to single nucleotide mutation,especially to PAM distal mismatch.In conclusion,CONAN could not only detect target DNA with ultrasensitivity and high specificity,but also provide a strategy for the application of artificial nucleic acid circuit in molecular diagnosis.(3)Application of CONAN in clinical molecular diagnosis.In this section,we explored the application potential of CONAN to the detection of genomic DNA in clinical biological samples.The results showed that CONAN could not only detect the genomic DNA of HBV infected cells as low as 5.0 a M,but also detect the clinical serum samples of HBV infected and uninfected patients.And the detection results were consistent with q RT-PCR.In addition,CONAN could accurately detect PIK3 CA gene mutation(1633 G > A)in bladder cancer cell samples,and the detection limit is as low as 10 a M.More importantly,CONAN could distinguish single nucleotide mutations with different mutation frequencies in clinical samples of bladder cancer,which were consistent with next-generation sequencing.Therefore,CONAN showed a good prospect for clinical molecular diagnosis.(4)Electrochemical protease sensor based on responsive RNA polymerase element.In this section,we combined a responsive RNA polymerase(PR)element with an electrochemical to construct a highly sensitive electrochemical biosensor for the detection of proteases.We used the PR element to convert a single hydrolysis event of the target protease into multiple programmable RNA outputs,which were designed as G-quadruplex sequences that specifically bind to hemin,thus enabling a label-free electrochemical detection of protease activity.Taking the tumor marker matrix metalloproteinase-2(MMP-2)as an example,the electrochemical sensor showed a good linear response to MMP-2 in the concentration range of 10 f M-1.0 n M,with the detection limit as low as 7.1 f M.The sensor could also be applied to the assessment of MMP-2 activity in different cell cultures and human osteosarcoma tissue samples,demonstrating its potential in the analysis of protease biomarkers in complex clinical samples.