Construction Of Activatable DNA Probes Based On Hairpin-contained I-Motif And Their Application In Tumor Cell Imaging And Killing

It is of great significance to develop highly sensitive and highly specific activatable theranostics probes for achieving precise diagnosis and efficient treatment of tumors.As an important tumor marker,the extracellular acidic microenvironment of tumors provides new ideas for the construction of activatable theranostics strategy.At present,a series of p H-sensitive organic small molecules,polymers,functionalized nanomaterials and DNA elements（such as A-motif,triplex DNA,and i-motif,etc.）have been widely used for the activatable imaging and treatment research of tumors.Among them,i-motif is a DNA fragment rich in cytosine nucleotides,which can be folded into a tetramer structure by forming C:C⁺base pairs in an acidic environment.It can be used as an ideal molecular tool for the construction of activatable theranostics probes in response to acidic microenvironment.However,most of the currently reported diagnosis and treatment systems based on i-motif presented a relatively wide p H response range,or their response range were within the non-physiological p H,making it difficult to sensitively respond to small p H changes outside the tumor cells.Recently,some works have focused on optimizing the base composition or structural design of related functional domain in the i-motif sequence to improve sensitivity and narrow the p H response interval.Particularly,hairpin-contained i-motif greatly improves its sensitivity to small p H variations due to the formation of hairpin structure,which provid es a new opportunity for the design of acid-sensitive theranostics probes.In this thesis,by using hairpin-contained i-motif as the acid-responsive element,a series of specific activatable theranostics probes with capacity of sensitively respond to small p H changes in tumor cells were constructed by introducing other functional nucleic acid molecules such as aptamer,antisense oligonucleotide（ASO）and molecular beacon（MB）,based on the sensitive acid-induced allosteric property of hairpin-contained i-motif,as well as the homogeneity of functional nucleic acids,resulting in the specific imaging and killing research of human liver cancer cells,colon cancer cells and breast cancer cells.The detailed description is listed as follows:1.Construction of fluorescent ratiometric probe based on hairpin-contained i-motif in response to small p H variations and imaging of tumor cellsIn order to construct a specific activatable theranostics probe that can respond sensitively to small p H changes of tumor cells,this chapter first explorered whether the DNA probe containing hairpin i-motif possesses the ability to respond to the small p H changes of tumor cells.A facile ratiometric fluorescent probe was cleverly designed by using an intramolecular hairpin-contained i-motif as p H-sensitive element（I-strand）,labeled with Rhodamine Green and BHQ2 at their 5′-and 3′-ends,respectively,and a complementary strand（C-strand）labeled with Rhodamine Red at its 5′-end.At alkaline p H,both I-strand and C-strand hybridized into a rigid duplex（I-C）,which holded the Rhodamine Red and the BHQ2 in close proximity.A s a result,the fluorescence emission of the Rhodamine Red was strongly suppressed,while the Rhodamine Green was in a“signal on”state.However,the slightly acidic p H enforced the I-strand to form an intramolecular i-motif and initiated the dehybridizat ion of I-C duplex,leading to Rhodamine Red in a“signal on”state and a decreased fluorescence of Rhodamine Green.The ratio of Rhodamine Green to Rhodamine Red(F _{542 nm}/F_597nm)could be used as a signal output mode.The results showed that an almost 70-fold change in the signal-to-background ratio was observed in the physiological p H range（6.50-7.40）,possessing efficient anti-interference ability,fast response,and reversible p H responsiveness.Furthermore,the SMMC-7721 cells were used as model to explorer the feasibility of I-C probe for imaging small p H changes.The results showed that the I-C probe could successfully achive the high-resolution imaging of small p H changes on the tumor cells,which laid a foundation for the construction of acid-activated theranostics probes for imaging and killing of tumor cells.2.Engineering a facile aptamer“molecule-doctor”with hairpin-contained i-motif enables low-background fluorescence imaging and killing of cancer cellsThe work in the previous chapter has confirmed that the construction of DNA probes with hairpin-contained i-motif as the acid-responsive element could achieve a highly sensitive response to small p H changes in tumor cells.However,we hope that it can possess specificity in the application of tumor diagnosis and treatment.Herein,by utilizing a hairpin-contained i-motif as the acid responsive element to be complementary with a tumor-targeted aptamer and combining with the principle of DNA double-stranded GC bases for doxorubicin（Dox）intercal ation,we subtly engineered a p H and membrane receptor dual-activatable DNA“molecule-doctor”（denoted as p H-Apt-MD）for bispecific imaging and killing of HCT116 cells.Specifically,the p H-Apt-MD consisted of two DNA strands,where the Apt-sgc8c was labeled with AF488 and Cy3 at its 5′-and 3′-end,respectively.The I-strand,a hairpin-contained i-motif complementary to Apt-sgc8c strand partially,labeled with a BHQ2 in the middle,thus generating Cy3 with quenched fluorescence and only AF488 emitted fluorescence.The double helix region of p H-Apt-MD was designed rich in GC bases,providing sites for doxorubicin（Dox）intercalation.Once encountering target cells,the p H-Apt-MD disassembled due to the specific recognition of aptamer and conformation change of i-motif,with activated fluorescence resonance energy transfer（FRET）signals between AF488 and Cy3,accompanied by Dox release in situ,thus resulting in specific imaging and killing of cancer cells.The results showed that p H-Apt-MD presented a narrow p H response range（p H 6.0-6.8）with a transition midpoint（p H^T）of 6.50±0.04.Furthermore,the dual-responsive FRET signal of p H-Apt-MD was successfully achieved on the HCT116 cells surface with ultralow background and enhanced imaging contrast,as well as accurate drug release and cell killing to target cells in an acidic microenvironment.As a dual-responsive activatable theranostics probe,p H-Apt-MD holds the advantages of facile design,narrow response interval and strong specificity.3.Hairpin-contained i-motif-based in situ bipedal hybridization chain reaction for specific activatable imaging of cancer cells and enhanced delivery of antisense oligonucleotidesIn the chapter three,we successfully achieved p H and membrane receptor dual-activatable imaging and killing of cancer cells with significantly improved specificity.However,the sensitivity also needs to be considered in further application.Therefore,in order to improve the imaging sensitivity and expand the application of hairpin-contained i-motif in the field of tumor activatable theranostics,we herein designed a hairpin-contained i-motif-initiated in situ bipedal hybridization chain reaction（p H-Apt-Bi HCR）amplification strategy for specific imaging of MCF-7 cells and enhanced gene delivery of ASOs by using hairpin-contained i-motif as p H-sensitive element and aptamer as specific target recognition element,and integration of ASOs with gene silencing function.Specifically,three DNA single strands were used to self-assemble into a“Y-type”DNA scaffold（p H-Apt-Y6）with p H response performance and aptamer target recognition ability.When encountering target cells,the aptamer fragment on the p H-Apt-Y6 probe could bind to cell surface receptors specifically.At the same time,the i-motif sequence tended to form an intact intramolecular hairpin-contained i-motif structure and falled down from p H-Apt-Y6due to the low extracellular p H,thus leading to the exposure of triggers.Furthermore,under the copresence of H1 and H2,the p H-Apt-Bi HCR assembly could be realized on both sides of the“Y-type”DNA scaffold.Using this strategy,the p H-Apt-Bi HCR not only contained repeated FRET units for activatable target tumor cells imaging with high contrast but also arrayed with plenty of ASOs as interference molecules for cells inhibition.The results showed that p H-Apt-Bi HCR presented an excellent response ability within a narrow p H range（6.0-7.0）with a transition midpoint（p H^T）of 6.44±0.06.Furthermore,the ASOs arrayed p H-Apt-Bi HCR exhibited improved internalization efficiency and enhanced gene silencing to MCF-7 cells compared to single ASO alone.This p H-Apt-Bi HCR strategy possesses bi-specificity,high sensitivity and high loading effect of antisense oligonucleotide s,which is expected to be applied in precision imaging and gene therapy of tumors.4.Hairpin-contained i-motif-driven in situ multivalent tile assembly for improved visualization of TK1 m RNA and chemo-gene killing of cancer cellsIn the last two chapters,we solved the specificity of acid-activated theranostics probes with hairpin-contained i-motif by introducing aptamer as specific target recognition element.However,the monovalent aptamer-based activatable theranostics probes may have problems such as insufficient affinity and low killing efficacy in the application.Therefore,we herein introduced multivalent aptamer recognition and chemo-gene combined killing mode,by taking advantages of the design flexibility and acid-induced allosteric properties of intermolecular hairpin-contained i-motif structure,and combined with MB,an aptamer-targeted and p H-driven in situ multivalent tile assembly strategy was presented for improved visualization of TK1m RNA and chemo-gene killing of MCF-7 cells.Specifically,the p H-Apt-TM,a p H-responsive and aptamer functionalized tile motif that formed by the self-assembly of four custom-designed DNA single strands,was functionalized with split i-motif fragments overhanging,possessing p H-responsiveness and Dox loading capacity.Furthermore,the p H-Apt-TM was loaded with a self-quenched MB that can specifically recognize intracellular TK1 m RNA.When encountering target cells,the cells could recruit p H-Apt-TM through the interaction between aptamer and ligands on the cell surface.Meanwhile,the acidic extracellular p H gathered p H-Apt-TM into a multivalent hand-in-hand DNA tile assembly（HDTA）on the cells surface owing to the split i-motif fragments formed an intact intermolecular hairpin-contained i-motif.Compared to p H-Apt-TM,systematic studies revealed that HDTA exhibited enhanced recognition,efficient cellular uptake,and sensitive responsiveness to intracellular TK1 m RNA.The hybridization of HDTA and TK1 m RNA resulted in down-regulation of TK1 m RNA in cytoplasm and induced apoptosis of MCF-7 cells.Moreover,using Dox as a chemotherapeutic model,specific drug delivery and improved cell killing were achieved to target tumor cells.The HDTA strategy not only introduced monovalent aptamer on the probe through p H-driven DNA Tile assembly,improving the recognition affinity,but also the combination of chemo-gene killing was realized by Dox loading and the introduction of antisense sequence s,which enhanced the killing efficiency.