Bioactive Molecule Detection Research Based On CRISPR/Cas12a Activity Regulation

Bioactive molecule(DNA,RNA and DNA repair enzymes)associated with malignant diseases integrate genetic and epigenetic regulatory mechanisms to regulate cancer gene expression and tumorigenesis through epigenetic,transcriptional and post-transcriptional levels.They are expressed in specific physiological states and biological processes,and their intracellular expression levels are closely related to the occurrence,development and metastasis of malignant tumors,and can be used as early diagnosis indicators and new therapeutic targets for cancer.Therefore,by carrying out research on sensitive monitoring methods of bioactive molecules related to malignant diseases,it will provide more effective theoretical basis and technical support for exploring the mechanism of cancer disease,research on early diagnosis and treatment of tumors,and prognosis evaluation.However,clinical samples(urine,serum or cell lysates)often contain very low levels of these bioactive molecule.It is difficult to directly measure them only by traditional molecular beacons.Therefore,enrichment and amplification of the signal is an important means to improve its sensitivity.Based on the signal amplification ability of CRISPR/Cas,many excellent bioactive molecules detection methods have been developed,and successfully implemented the detection of clinical samples.In particular,CRISPR/Cas12 a has unique advantages: its guide RNA(crRNA)sequence is short and easy to design,and both double-stranded DNA(ds DNA)and single-stranded DNA(ss DNA)can act as the activation strand.Its enzyme cleavage substrate can be any sequence of DNA.If coupled with nucleic acid signal amplification technology,it is expected to achieve multiple regulation of CRISPR/Cas12 a activity,thereby improving the sensitivity of the CRISPR/Cas12 a sensing platform for biologically active molecules in clinical samples.Therefore,this paper uses nucleic acid signal amplification technology(transcription amplification and rolling circle amplification),which is regulated from the crRNA and activated DNA strands of CRISPR/Cas12 a respectively,to reconstruct the CRISPR/Cas12 a system,and convert the signals of proteases and nucleic acids,and then combined with nucleic acid amplification technology and the trans-cleavage of Cas12 a to improve the detection sensitivity of trace bioactive molecules.Based on the programmability of crRNA and activated DNA strands,it is expected to become a universal new technology for bioactive molecules detection,providing new ideas for clinical diagnosis and early treatment.It mainly includes the following two parts:(1)Self-contained crRNA-regulated CRISPR/Cas12 a activity platform for sensitive detection of T4 polynucleotide kinase(T4 PNK)activity.First,a double-stranded DNA substrate probe(ds DNA)was constructed,which could be used as both a transcription template for crRNA and a T4 PNK recognition substrate.When double stranded DNA is recognized by T4 PNK,the 5’-OH end is changed to 5’-P end.Based on the specific recognition of the 5’-P end by λ exo,the ds DNA is decomposed,resulting in the degradation of the nucleic acid transcription template,and the pathway of the transcription product crRNA is blocked.After adding the CRISPR/Cas12 a protein,it is activated due to the lack of crRNA.As a result,CRISPR/Cas12 a activity was inhibited,and finally the double labeled molecular beacon could not be cut,and the fluorescence signal was turned off.In the absence of T4 PNK,the transcription template ds DNA structure remains intact,so it can be transcribed normally and produce a large number of crRNAs that activate CRISPR/Cas12 a activity,so that the rapid cleavage of molecular beacons can be achieved,and the fluorescent signal of the system is turned on.Based on this “on” to “off”fluorescence signal output mode,sensitive detection of the target T4 PNK can be achieved by detecting the degree of fluorescence signal reduction in the system In addition,the constructed system was used for the detection of T4 PNK in cell lysates,and a satisfactory recovery rate was obtained,indicating that the constructed target-regulated self-supplied Cas12a-crRNA biosensing platform is expected to be applied in the detection of actual samples,and provide a new idea for clinical medicine.(2)Multiplex DNA activation strand regulates CRISPR/Cas12 a activity platform for label-free detection of DNA.The selected target biomolecular model is hepatitis B virus DNA(HBV DNA).First of all,the long strand DNA of nucleic acid obtained through target recognition triggered RCA reaction is the activation strand of CRISPR/Cas12 a.And the crRNA recognizes the long strand DNA and forms the three-element complex of Cas12a-crRNA-ss DNA to shear the biotin-modified DNA strand.Then,two different ss DNA were added for self-assembly reaction to form macromolecules with a large number of ds DNA structures.Magnetic spheres were added to separate macromolecular structures formed by self-assembly of unbroken biotin DNA under the action of magnetic field.The macromolecular structure in solution was incubated with dye to increase the fluorescence signal.In this paper,self-assembled macromolecules were used as the“substrate” for signal output,and a large number of ds DNA structures were aggregated together to enhance the fluorescence signal.The RCA,CRISPR/Cas12 a systems’ trans-cutting and self-assembly co-amplification,improving the sensitivity of HBV DNA detection in serum.Breaking the commonly used fluorescently labeled signal reporter probes adopt label-free to avoid the tedious modification of DNA probe.This strategy provides a new strategy for label-free detection mediated by the CRISPR/Cas12 a system,which is expected to be applied to detection in actual samples and provide new ideas for clinical medicine.