DNA Tweezers Regulated Regenerated Nanoreactors For Sensitive Detection Of RNA

MicroRNAs(miRNAs)are a class of noncoding small RNA molecules.They participate in the regulation of ontogeny,cell proliferation,differentiation and apoptosis through incomplete hybridization with target genes and inhibition of target gene translation.Mi RNAs are also closely related to pathological processes such as tumor occurrence,metastasis,and drug resistance.The development of reliable mi RNA detection methods is of great significance for the early diagnosis of disease.RNA is the genetic material of some viruses.Common RNA viruses include HIV(retrovirus),all influenza viruses,Ebola virus(Ebola Virus),dengue virus(DENV)and Zika virus(ZIKV),etc.Many diseases that seriously endanger human life,such as the new coronavirus pneumonia(COVID-19)that has swept the world in the past two years,are caused by RNA viruses.Therefore,rapid diagnosis of virus types and infections can effectively reduce the spread and infection of viral infectious diseases in a large area,and provide important scientific basis for their prevention and control.In the past two decades,various new techniques for detecting RNA have emerged,such as colorimetric analysis,electrochemiluminescence,surface-enhanced Raman,and fluorescence.Among them,fluorescence detection technology has many advantages and characteristics such as safety,fast response,high sensitivity,and simple operation.Currently,researchers have developed a series of fluorescence methods for sensitive detection of RNA.The more widely used methods can be roughly divided into nucleic acid isothermal amplification technology and fluorescence detection of nanomaterials.The current fluorescence detection method can achieve sensitive and specific detection of RNA,but it lacks"renewability",that is,once the reaction is activated,it will continue until the reactant is exhausted,making the entire detection process irreversible and uncontrollable,and it is impossible to achieve Continuous detection of targets.Therefore,it is very necessary to construct a reproducible RNA fluorescence detection system and improve the continuous detection ability of the method.DNA tweezers have a simple structure and an intuitive mechanism for rapid response.They have unique advantages in sensing applications.In addition,DNA tweezers have both open and closed state conformations,and their dynamic conformational changes can be designed to quickly respond to different types of targets.Because DNA tweezers have high continuous controllability,mechanical stiffness and dynamic adjustability,they become an ideal biomolecular tissue regeneration scaffold.Therefore,this paper constructs a regenerable nanoreactor based on the regulation of DNA tweezers for sensitive detection of RNA.The thesis is mainly divided into the following four chapters:Chapter one is the introduction section,and it summarizes the concept,the detection significance and sensitive methods for RNA,as well as the scientific problems and main research content solved in this paper.In chapter two,a regenerated DNA circuit regulated by DNA tweezers is constructed.It is used for the sensitive detection of nucleic acid biomarkers mi RNA and uracil-DNA glycosylase(UDG).The DNA tweezers,which can be reversibly switched between open and closed states,are used as scaffolds to regulate the regeneration of DNA circuit.It is the first time to realize the reversible controllability of the detection process and the continuous and repeated detection of the target.It can detect cancer-related mi RNAs sensitively and selectively,with a detection limit of 8.7 f M,successfully realizing the quantitative analysis of mi RNAs in complex biological extracts.In addition,by changing the set strand and its complementary sequence of the tweezers,it can also be used for the sensitive and quantitative detection of cancer-related UDG in biological lysates.In Chapter three,a ratiometric regenerated nanoreactor regulated by DNA tweezers was constructed for the sensitive detection of viral RNA and heavy metal ions Hg²⁺.The target viral RNA is combined with the two ends of the DNA tweezers arm through base complementary pairing to close the originally open DNA tweezers,induce fluorescence resonance energy transfer,and quantify the viral RNA by the ratio of the fluorescent donor and acceptor signal changes,overcoming a single signal the response fluorescence method is susceptible to the problem of false positive signals caused by the internal environment and background fluorescence,which improves the accuracy and reliability of viral RNA detection,and the detection limit is 46.65 p M.The sensor can also be used for the sensitive and quantitative detection of heavy metal ions Hg²⁺.At the same time,the system can be recycled to achieve continuous and controllable target detection.Chapter four is a conclusion and expectation section.It mainly summarizes the research result of the paper.