PH Responsive Nucleic Acid Nanoprobes For Hypoxia And Intracellular Survivin MRNA Imaging Analysis

Cancer,as one of the deadliest diseases worldwide,has high mortality and incidence.According to recent reports,the main reason of high cancer mortality is tumor recurrence and metastasis.Tumors can be divided into tumor cells and microenvironment,understand the connection between the tumor cell microenvironment with tumor cell metabolism which makes great significance for diagnosis and treatment of cancer.In recent years,scientists have combined functional nucleic acids,nanotechnology and life medicine to provide a reliable tool for the early diagnosis of cancer.Functional n ucleic acids have high specific target recognition abilities,nanomaterials are used as new carriers simultaneously,and the combined nucleic acid fluorescent nanoprobes owned the advantages of strong specificity and high sensitivity.They are widely used in cancer early imaging and diagnosis.Based on the interconnection between cancer cells with the tumor microenvironment and the nucleic acid fluorescent nanoprobes' advantages,this paper design two kinds of pH-responsive nucleic acid fluorescent nanoprob es,which not only achieved high selectivity of azoreductases detection in lysosome under hypoxic conditions,but also realized the intracellular pH and survivin m RNA imaging analysis.The research contents are as follows:1.pH-regulated and target-activated fluorescent nanoprobes for lysosomal azoreductase imaging under hypoxiaIn this chapter,we constructed a pH-regulated and target-activated fluorescent nanoprobe for monitoring changes of lysosomal azoreductase under hypoxia.At First,i-motif DNA sequence was fluorescently labeled with donor(TAMRA)and acceptor(Cy5)at two ends,then extended with a polyT sequence and modified an azobenzene group to form sequence P1.SH-CD was modified on the surface of AuNPs to form AuNPs-CD,the P1 could be conjugated onto SH-CD through host–guest interactions to form the fluorescent nanoprobe.Upon the nanoprobes entering into lysosomes through endocytosis,P1 folded into a closed i-motif structure under acidic pH,so TAMRA and Cy5 are closed to each other;when the cell under hypoxia conditions,azoreductase reduced the azobenzene to amino group,the folded P1 could be liberated from AuNPs,thus activating an enhanced fluorescence resonance energy transfer(FRET)signal in the living cell.This signal is determ ined by the acidic pH of lysosome and azoreductase,indicating that the fluorescent nanoprobe can selectively monitor the change of azoreductase in lysosome.2.Triplex-functionalized DNA tetrahedral nanoprobe for imaging of intracellular pH and survivin mRNAIn this chapter,we combine DNA tetrahedron and pH-responsive DNA triplex structure to design sensitive fluorescent probes.First,two single-stranded DNA sequences whose 5' termini were extended with extended a TA/CG pairing DNA hairpin(loop modified BHQ1),and the 3' ends of the other two single-stranded DNA sequences extended the same sequence.Four single-stranded DNA sequences were annealed together to fabricate expected DNA tetrahedron(contain small hairpins on four vertices).The ends of H1,H2(labeled Cy3 and Cy5,respectively)extended with T/C-rich sequences.In the acidic extracellular pH in tumor cells,H1,H2 were assembled with DNA tetrahedron through form DNA triplex and maintained stable.When the nanoprobe entered the cell,the high intracellular pH causes H1,H2 to be released from the tetrahedron,and the Cy5 fluorescence signal is restored.The presence of m RNA triggered the hybrid chain reaction of H1 and H2 caused the Cy3 and Cy5 close to each other,and then activated the fluorescence resonance energy transfer(FRET)signals.Therefore,we can attain two different fluorescence intensities of Cy5 under different excitation wavelengths which correspond to intracellular pH and survivin mRNA expression,respectively.