Sensing Platforms Formed From Metal-organic Framework With Probe DNA And Their Fluorescence Detection Of Nucleic Acid Sequences

MicroRNAs(miRNAs)are a group of small(approximately 18-25 nucleotides),endogenous,non-protein-coding RNAs,which participate in gene regulation.As abnormal expressions of miRNA are usually related to various diseases,miRNA has emerged as a new type of biomarker.In addition,in the nocoding region of virus,it has high conserved nucleic acid sequences and such sequences keep unchanged during virus variation.Hence,the detection for miRNA,or high conserved nucleic acid sequences in the nocoding region of virus is beneficial to the early diagnostics and prognostics of corresponding diseases.Metal-organic frameworks(MOFs)are inorganic-organic materials synthesized by assembling metal ions with organic ligands in suitable solvents.In 2013,Chen and his co-workers had demonstrated that water-stable MOF can inteact with fluorescent labeled DNA(probe DNA,P-DNA)to form P-DNA@MOF sensing platform,which can be utilized for the detection of nucleic acid by fluorescence titration.However,MOFs for nucleic acid detection is still in the infant stage and few MOFs have been explored for the detection of DNA or RNA because of their poor water solubility and stability.Therefore,it is significant to construct water stable MOFs and develop sensing platforms based on these MOFs for detection of desease-related nucleic acid with sensitivity and specifility.MOFs assembled by zwitterionic carboxylate ligands containing methylene with metal ion process structural diversity and strong water stability characteristics,which can avoid MOFs collapsing in the fluorescence detection for nucleic acid in water.In this dissertation,this type of MOFs had been employed for the construction of sensing platform and their application for fluorescence detection for disease-related RNA sequences:Part 1:Three-dimensional MOF {[Cu(Cmdcp)(phen)(H2O)]2·9H2O}5(1)was employed to form P-DNA-1@1 and P-DNA-2@1 sensing platforms with FAM labeled P-DNA-1 and ROX labeled P-DNA-2,respectively.MOF 1 can bind to P-DNA-1 and P-DNA-2 and quench the fluorescence with high fluorescence quenching efficiencies(QE)of 75%and 95%,respectively.These two sensing platforms can rapidly detect conserved sequences(Ti)and miRNA-like fragment(T2)of EBOV and give detection limits of 63 pM and 206 pM,respectively.Furthermore,in order to overcome the shortcomings of time-consuming and complicated operation,P-DNA-1 and P-DNA-2 were applied to bind with MOF 1 at the same time and then form P-DNAs@1 sensing platform.P-DNAs@1 could detect Ti and T2 by synchronous fluorescence analysis with high sensitivity and give detection limits as low as 163 pM and 111 pM.It also exhibited high specificity that there were no cross reaction in the process of detection.Comparing these two methods,synchronous fluorescence analysis improves the detection efficiency by saving the time.Part 2:MOF can bind P-DNA through ?-? stacking,electrostatic or hydrogen-bonding interactions.In this part,we introduced a main ligand and an ancillary ligand with conjugated ? systems,that is 4,4'-bipyridine(bipy)and 1-(3,5-dicarboxybenzyl)-4,4'-bipyridiniumbromide(dcbb)for construction of MOF,with the aim to improve their ?-interactions with fluorescently-labeled probe DNA.A two-dimensional MOF[Cu(dcbb)(bipy)(H2O)]n(2)has been obtained and further used to construted sensing platforms of P-DNAs@2.Such platform can detect T3,T4 and T5,three conserved sequencs of Zika virus(ZIKV),by synchronous fluorescence approach.MOF 2 showed excellent fluorescence quenching properties to P-DNA-3(labeled by FAM),P-DNA-4(labeled by ROX)and P-DNA-5(labeled by Cy5)with the QE of 88%,96%and 80%,respectively.And P-DNAs@2 was able to detect T3,T4 and T5 rapidly with high sensitivity and high selectivity,showing detection limits of 564 pM,158 pM and 186 pM.Part 3:To ensure the practical application of P-DNA@MOF systems,we convert the detection objects from high conserved nucleic acid sequences in the nocoding region of virus to miRNA,which has approximately 18-25 nucleotides anda new type of biomarker related to various diseases.In this part,we synthesized five sensing platforms for the detection of five gastric cancer associated miRNAs.The sensing platforms are formed from a water-stable MOF {[Cu(dcbb)2(H2O)2]·10H2O}n(3,dcbb = 1-(3,5-dicarboxybenzyl)-4,4,-bipyri-diniumbromide),respectively with five carboxyfluorescein(FAM)labeled P-DNAs to form P-DNA-6@3?P-DNA-10@3 systems.MOF 3 can quench fluorescence of P-DNAs with the QE of 87%,94%,77%,95%and 96%,respectively.Each P-DNA@1 system is effective and reliable for the detection of its complementary target miRNA with the detection limits from 91 to 559 pM,and is not interfered by other four miRNA sequences.The results indicate that the sensing pltforms of P-DNA@MOF are applicable for the diction of multiplexed miRNA.Part 4:In order to further study the detection ability of the sensing platforms for the target miRNA at cellular level,we modified probe DNA as2'OMe-PS chimeras(mDNA),which could not only defend against exonuclease attack,but also possess high binding affinity toward target RNA.Herein,we employed a three-dimensional MOF {[Cu2(Cmdcp)2(4,4'-bipy)(H2O)2]·0.5H2O}n(4)to interact with three different type mDNAs which labeled by FAM,TAMRA and Cy5,respectively,and form P-mDNAs@4 sensing platform with the high QE of 89%,94%and 87%,respectively.Subsequently,P-mDNAs@4 was utilized to detect miR-21,miR-155 and miR-375,three abnormally expressed miRNAs associated to oral cancer,by synchronous fluorescence analysis.Compared to P-DNAs@4 sensing platform,P-mDNAs@4 was more rapid and sensitive in the synchronous detection of targeted miRNAs with lower detection limits of 229 pM,64 pM and 385 pM,mainly due to the strong affinity of mDNA to the corresponding target RNAs.Furthermore,we introduced P-mDNA@4 into UM1,HSC3 and SCC9 cells and successfully detected high expression of intracellular miR-21,miR-155,but not miR-375,which has low expression in intracellular.The results showed that all sensing platforms formed by MOFs 1-4 with the corresponding P-DNA or P-mDNA could rapidly detect corresponding target RNAs showing high sensitivity with the detection limits at picomolar level and hign selectivity without cross reaction among P-DNAs.The detection principle for fluorescence detection of target RNA by P-DNA@MOF had been illustrated preliminarily by calculation of binding constant,fluorescence anisotropy experiment and PAGE gel electrophoresis.