Functional Nucleic Acid-based Fluorescent Probes For Cellular Metal Ions And RNAs Imaging

With the continuous innovation achieved in life science and technologies,human beings gain profounder understanding of the essence and regularities in life activities.At the same time,human beings are also facing challenges from the external world as well as themselves,such as the deterioration of the living environment,disease distress and so on.Therefore,the rapid and accurate detection of the targets related to life activities is of great significance in monitoring the state of the environment and achieving early diagnosis of diseases,which is also the central topic of modern biological analysis technology.Among the various strategies for biological analysis,the fluorescent probes have been extensively applied due to the advantages they exhibit including easy to fabricate,rapid response,high sensitivity and can be used for in situ imaging.Conventional fluorescent probes usually use proteins with the ability to recognize or catalyze,such as antibodies and enzymes,or organic small molecules that can bind or react with the target as identification units.Due to the restriction in preparations and applications,the conventional fluorescent probes can’t fully meet the requirements of modern analytical techniques.Fortunately,the discovery of functional nucleic acids（FNAs）has greatly expanded the application of fluorescent probes,which opened up a new way for the design of fluorescent probes.FNAs,which have been discovered and widely used,are divided into two main categories:the first category is known as aptamers,aptamers with antibody-like identification function,can specifically bind metal ions,small molecules,proteins,cells,tissues and even organs,which have been introduced as recognition units in the construction of FNA probes.Another category is named as DNAzyme,which can perform catalytic functions similar to protease with the assistance of cofactors（metal ions）.FNA probes have been widely concerned because of their advantages of simple sequence,easy design and synthesis,fast response rate and good selectivity.By reasonably designing the FNA sequences and combining the programmable features of DNA probes,researchers have developed a number of superior FNA fluorescent probes for the detection of targets in complex samples.The real-time monitoring of the concentration changes and the distribution states of biomolecules in living cells shows great significance in the researches of cellular physiological and pathological functions as well as the early diagnosis of diseases.Though the FNA probes have been extensively employed in the detection in vitro,their studies in living cell imaging is still in infancy.In order to further explore the potential of FNA probes in cellular study,this thesis develops multiple novel FNA fluorescent probes to image the cellular metal ions and RNAs,and explores their possibilities for disease diagnosis and treatment.The details are displayed as follows:（1）In Chapter 2,a cell membrane anchored FNA sensor was constructed to monitor the dynamic change of K⁺in the cellular microenvironment.This sensor employed a thrombin binding aptamer（TBA）as identification unit,a fluorescence resonance energy transfer（FRET）pair as the signal reporter,and a C₁₈ diacyllipid as the cell-membrane anchoring unit.Additionally,the sequence of traditional TBA-based sensor was optimized to achieve a better signal-to-background ratio.Owning to the superior sensitivity and selectivity as well as the rapid and revisable response of the probe,we realized the monitoring of K⁺in cellular microenvironment.（2）In recent years,genetically encoded fluorescent proteins have been used for metal ions detections by combining fluorescent proteins with metal-binding proteins or peptides.However,their applications are largely restricted to a limited number of metal ions due to the lack of available metal-binding proteins or peptides that can be fused to fluorescence proteins and the difficulty to transform the binding of metal ions into the change of fluorescent signal.To expand the number of metal ions that the genetically encoded fluorescent protein sensors can detect and to increase the impact of DNAzyme sensors in cellular biology,in Chapter 3,we report herein the use of an Mg²⁺-specific RNA-cleaving DNAzyme to regulate the expression of fluorescent proteins as a new class of ratiometric fluorescent sensors for metal ions.Specifically,we demonstrate the use of 10-23 DNAzyme to suppress the expression of Clover2,a mutant of the green fluorescent protein,by cleaving the mRNA of Clover2,while the expression of Ruby2,a mutant of the red fluorescent protein,is not affected.Since DNAzymes that are specific for a wide variety of metal ions,such as Mg²⁺,Na⁺,Cu²⁺,Zn²⁺,Pb²⁺,Hg²⁺,Ag⁺and UO₂²⁺,can be obtained through in vitro selection,and the results DNAzymes often share a similar secondary structure and reaction mechanism,the method described in this work can be applied to imaging many other metal ions and thus significantly expand the range of applying genetically-encoded fluorescent proteins,allowing this class of sensors to be even more powerful in providing deeper understanding of the roles of metal ions in biology.（3）Up to now,though the hybridization chain reaction（HCR）has been widely used for the ultrasensitive detection,its potential in cancer treatment has not been fully studied.In Chapter 4,we construct an intracellular HCR nanoprobe to selectively and sensitively recognize cancer cells in combination with amplified photodynamic therapy（PDT）.The fluorescent nanoprobe can identify the specific mRNA in cancer cells through DNA hybridization,with which the HCR amplification strategy will be activated to achieve highly sensitively cancer recognition.Simultaneously,the photosensitizer labeled on DNA sequences will also be activated,thus enabling the amplification of the PDT.Herein,this work takes the advantages of HCR to explore its feasibility in cancer treatment.（4）In Chapter 5,we constructed a metal-organic frameworks（MOFs）based nanosystem for the delivery and controllable release of nuclease,which can be used for intracellular miRNA amplified detection.The exonuclease III（Exo III）which has been widely used for signal amplification in extracellular detection was chosen as the target protein.It can be readily encapsulated in the MOFs through a one-pot synthesis,then the DNA sensor can also be adsorbed on the nanomaterial,by which both the Exo III and DNA sensor can be delivered to cells simultaneously.Then the nanoprobe will be decomposed in the cellular acidic environment to release both Exo III and DNA sensor.After the hybridization of the DNA sensor with the intracellular microRNA-21（miR-21）,the hybridized DNA sensor will be hydrolyzed by Exo III,thus realizing the signal amplification.This strategy is expected to provide a new alternative for enzyme-catalyzed DNA signal amplification in living cell imaging.