Bioanalytical Methods Based On DNA Circuit And CRISPR-Cas12a

The CRISPR-Cas modules are adaptive immune systems of bacteria and provide sequence-specific protection against foreign DNA and viral genes.Some class II type V,such as Cas12a,can activate the non-specific Collateral activity,enabling cleavage of indiscriminate single-stranded DNA in solution.The above characteristics make the CRISPR-Cas12a system both molecular recognition and signal amplification.The Cas12a system has become a powerful biosensing tool widely used in virus infection,gene detection,etc.However,the Cas12a system cannot directly target RNA or other targets.Therefore,it can only be converted into DNA targets reverse transcription or complex designs,which significantly limits the applications of Cas12a in detecting non-DNA analytes.DNA circuits serve as programmable intermediates between inputs and outputs,which can perform various tasks,such as logical operation,feedback,and signal-transducing amplification isothermally without the participation of protein enzymes.Inspired by this phenomenon,we use DNA circuits as signal converters to convert RNA signals to dsDNA signal output.And the dsDNA coupled with Cas12a to construct a universal miRNA sensing and detection method(CRISPR-CHA).At the same time,the combination of CRISPR-CHA with the molecular recognition of aptamers to construct bioanalytical sensors that meet different targets or needs.The main points are summarized as follows:(1)Exploring the expression and properties of Cas12a.In this section,Cas12a was expressed and purified in a prokaryotic expression system,and the activity was verified.We verified the specific cleavage and collaterally cleavage activity of the Cas12a protein and the preference for the target.We also explored the effects of DTT and heparin sodium on the collateral cleavage activity of Cas12a;the results showed that DTT could enhance the collateral cleavage activity within a specific concentration range,while heparin sodium could reduce it.And we introduced unpaired bases in the seed region of dsDNA and found that the collateral cleavage activity of Cas12a increased with the number of unpaired bases(0-6 nt).In addition,we also found that Cas12a can stabilize the enzyme cleavage activity of Cas12a after forming a binary complex with sgRNA.(2)Construction of a highly sensitive miRNA analysis method based on Cas12a and CHA nucleic acid circuit(CRISPR-CHA).In this section,we rationally designed the CHA circuit and sgRNA to convert the signal input of miRNA through the CHA circuits into dsDNA output.The output dsDNA was recognized by Cas12a as a target and activated the Cas12a collateral cleavage activity.We optimized the length toehold of H1,CHA reaction time,and reaction temperature and investigated the CRISPR reaction time of the CRISPR-CHA reaction.In addition,we also introduced mutated bases into the H1 hairpin so that the output H1/H2 duplex formed a predissociation region in the seed region.We found that introducing a base predissociation in the seed region can effectively improve the fluorescence of the system signal response.Based on optimized reaction conditions,the detection limit of miRNA21 of the CRISPR-CHA method was as low as 0.07 f M,and the linear detection range was 100 p M-0.1 f M(R~2=0.99).Compared with the CHA method,the detection sensitivity of CRISPR-CHA is improved by about 6 orders of magnitude,proving that the CRISPR-CHA method is a bioanalytical method with high sensitivity and a wide dynamic detection range.(3)Construction of modular analysis method for CRISPR-CHA.In this section,the CRISPR-CHA method was divided into the CHA molecular recognition module,the CRISPR signal amplification,and the output module.Only changing the variable region and highly sensitive detection of different target miRNAs can be achieved.Without any optimization,we changed a few sequences of the variable regions to achieve highly sensitive detection of miRNA155 and miRNA141,with detection limits of 0.1 and 0.15 f M and a linear range of 100 p M-0.1 f M,respectively.In addition,we used the CRISPR-CHA method to analyze the expression levels of miRNA21 in different cells.The results were consistent with the RT-qPCR method,proving that the CRISPR-CHA method has good reliability.We also used the CRISPR-CHA method to detect miRNA21 in clinical serum samples.The results showed that the CRISPR-CHA method could distinguish healthy serum from lung cancer serum,providing a high-quality,sensitive,and versatile method for analyzing miRNA disease markers in clinical samples.(4)Construction of a universal sensor based on CRISPR-CHA and aptamer molecular recognition.In this section,we combine the molecular recognition switch module of aptamer with the CRISPR-CHA method to construct a new strategy for general bioanalysis(A-CRISPR-CHA).The A-CRISPR-CHA method through a conformational change by recognizing the aptamer and the target,exposing the trigger chain of the CHA,thereby activating the collateral cleavage activity of Cas12a,and realizing detection of the target.We used dopamine as a model target to verify the feasibility of the A-CRISPR-CHA method and optimized parameters such as the length of the inhibitory strand,the hybridization ratio,the hairpin probe ratio,and the concentration of heparin sodium in the buffer.Under optimal conditions,the detection limit of A-CRISPR-CHA is 0.4 n M with a linear range of 5μM-1.6 n M.In addition,the specificity of the method and its application in actual complex samples were also verified,proving that the A-CRISPR-CHA method can be used for the analysis and detection of targets in complex samples.At the same time,by changing the recognition module of the aptamer,the detection methods of targets such as 5-HT and ATP were established,respectively.The detection limits of the method were 0.027 n M and 1.4n M,and its specificity and application ability in actual samples were verified,proving the generality of the A-CRISPR-CHA method.