A New Method Of Nucleic Acid Sensing Based On Isothermal Amplification

Cancer is a kind of disease threatening human health with high mortality and morbidity.Its early diagnosis brings great hope for timely medical intervention and reduction of mortality.Cancer cells can express specific biomarkers(Such as micro RNAs,DNAs and proteins)that are expressed in very small amounts or not in normal cells.Nucleic acid amplification is an excellent method to detect biomarkers.Polymerase chain reaction(PCR)is the most widely used nucleic acid amplification technique in clinical practice.However,PCR requires precise and complex heating cycles,which makes integrating the energy-intensive and cumbersome PCR instruments into the molecular detection platform an imperfect solution.As a result,isothermal amplification technology emerged to amplify nucleic acids without additional complex thermal cycling steps and related control mechanisms,and to accumulate nucleic acid sequences rapidly and efficiently at constant temperature,providing a powerful tool for diagnostic platforms.In this paper,three fast and sensitive nucleic acid sensing methods were constructed based on nucleic acid isothermal amplification technology,which were respectively used for the detection of human papillomavirus subtype(HPV16),exosome and micro RNAs(mi RNA-21).The research contents of this paper mainly include the following three parts:(1)We designed a CRISPR-Cas12 a and hybridization chain reaction(HCR)mediated signal amplification for human papilloma virus detection.HPV16 sequence can be recognized by protospacer adjacent motif(PAM).When it hybridized with HPV16,CRISPR-Cas12 a transcleavage activity was activated,thus cutting the initiator chain I.When I was cleaved,HCR could not open hairpin probes H1 and H2,and fluorescence on H1 would not be activated.In the absence of HPV16,when the CRISPR-Cas12 a protease was not activated,the initiator chain I would open the hairpin probes of H1 and H2 continuously,and the hybridization chain reaction would occur,forming the hybridization chain,and the fluorescence signal on the hairpin probes of H1 would be released.We can quantitatively detect the HPV16 target by detecting the fluorescence signal in the solution(0.5 p M).The method does not require any additional target amplification steps and is about 100 times more sensitive than the method using HCR alone.This low-cost,simple and rapid detection method of human papilloma virus is also suitable for the detection of other small molecules or targets,and has high application value in clinical diagnosis,biomedical research and chemical analysis.(2)An adjacent hybridization mediated hyperbranched HCR isothermal amplification method was reasonably designed for the specific detection of exosomes.Firstly,two nucleic acid probes with different functions were designed.One was designed with a CD63 aptamer at the 5’ end to specifically recognize the CD63 protein on the surface of exosomes,and the other was labeled with cholesterol at the 3’ end to facilitate its insertion into the lipid bilayer of exosomes.Subsequently,due to proximity,adjacent hybridization occurs between the two functional nucleic acid probes connected to exosomes to form initiator chain I.Initiating chain I can then open hairpin probes H1,H2,H3,and H4,resulting in a hyperbranched hybridization chain reaction.By adding a fluorescent reporter gene,the interference signal was significantly reduced,so we achieved a high specificity for exosome detection while ensuring a relatively high sensitivity(0.02 mg/m L).Near hybridization mediated hyperbranched HCR isothermal amplification can also provide a new discovery for the application of biological analysis in living systems and contribute to the diagnosis of various human diseases,especially cancer.(3)A method based on the combination of copper-DNA nanoscale self-assembly structure and catalytic hairpin self-assembly was reasonably designed to detect mi RNA-21.Firstly,hairpin probe H1 and hairpin probe H2 labeled with fluorescence groups were designed to catalyzed hairpin self-assembly reaction in response to mi RNA-21 to form fluorescence doublestranded structure.With the help of copper ions,hairpin probe H1 and self-quenching hairpin probe H2 were assembled together into uniform coordination nanoparticles through a one-pot reaction without any other complex modifications.The hybrid nanostructure has good biocompatibility and hairpin probes H1 and H2 are released from the nanoparticles after entering the cell.In the presence of mi RNA-21,the catalytic hairpin self-assembly reaction takes place for in-situ imaging.Simple operation,high loading efficiency,good biocompatibility,good specificity,effective amplification ability without involving multiple proteases,and strong stability at physiological temperature make hybrid nanostructures more attractive in biological research and disease diagnosis.Importantly,the proposed self-assembly system with efficient encapsulation capability provides a widely applicable method for in-situ visualization of low-abundance nucleic acids in cells and evaluation of gene expression(0.01 n M),which may have great potential as nanocarriers for delivery strategies and combination therapy.In conclusion,this paper designs three detection methods for small molecules based on nucleic acid amplification,providing a new idea for the diagnosis and treatment of related diseases.