Detection Of Single-base Change Based On Strand Displacement Reaction And Application Of Spherical Nucleic Acid For Intracellular Assay

Over the past few decades,the research and application of DNA is far beyond the scope of "biological genetic material".The development of DNA nanotechnology has attracted much attention.There mainly are two subfields in DNA nanotechnology:structural and dynamic DNA nanotechnology.The dynamic DNA nanotechnology,in particular the strand displacement reaction,has made great achievements in DNA logic gates,DNA molecular machine,and artificial DNA nanoactuators.On other hand,the DNA-functionalized gold nanoparticles are also emerging in biodetection.Particularly,these novel probes greatly facilitate intercellular assay.Due to the interests in these subjects,I mainly focus on the DNA strand displacement reaction and DNA-gold nanoparticle probes in the biodetection.The first part is on the four different DNA hybridization reactions:direct-linker-addition(DLA),hybridized-linker-addition(HLA),direct-complex-addition(DCA),and hybridized-complex-addition(HCA).In fact,the first two methods are traditional way to tune DNA hybridization.The other methods are based on the strand displacement reaction.The experiments were performed on the quartz crystal microbalance(QCM)using DNA-AuNP nanoconjugates as signal amplifiers.In the two first methods,the HLA is faster and have stronger signal intensity than DLA,because the pre-hybridization can enhance the rigidity strand and decrease conformational entropy.The two factors are beneficial to the DNA hybridization.In the strand-displacement-based strategies,two components are employed:catalyst strand and complex.The complex is pre-hybridized by linker strand and protector.The catalyst is a single strand which can displace the protector.Then,the DNA-AuNP can release the catalyst back into solution and the catalyst triggers another strand displacement again.Hence,the last two methods show strong signal intensity of QCM sensor and faster reaction rate.In the second part,we first designed a two-step DNA strand displacement reaction with random sequences for the unambiguous discrimination of a single-base change on the sensing platform of quartz crystal microbalance(QCM).Given that no study has been performed to investigate the design principle of the strand displacement fueled by DNA-gold nanoparticle conjugate(DNA-AuNP)on gold-electrode surface,we use DNA-AuNP as a probe to achieve the discrimination;moreover the probe can release the target sequence back into the solution for the target sequence to circularly initiate the DNA strand displacement,and this reaction circuit can improve the sensitivity.After optimizing and testing the detection strategy,it has been successfully applied to detect mutation of a realistic sequence(p53 gene fragment)associated with human cancer,thereby indicating that this method has potential applicability in general.In addition,we also show some differences in the design between strand displacement in solution and that on gold-electrode surface.The third section focuses on the intracellular assay by using DNA-AuNP.Spherical nucleic acid(SNA)nanoconjugates are powerful tools for intracellular assay.Especially,by using highly effective fluorescence quenching capability of gold particle core,SNA labeled with fluorophore can visualize,track,and quantify targets in living cells.However,sometimes false positive signal may emerge,such as the instability of thermodynamics or signaling properties which can be eliminated by careful design of oligonucleotide shell.The major challenge is the measurable fluorescence leakage caused by intracellular degradation.To avoid this fundamentally,the site and behavior of intracellular degradation need to be investigated.Herein,we mainly describe degradation events happening to SNAs in MCF-7 cell.We find that degradation is specific to the 5' region of oligonucleotide.More interestingly,only the first nucleotide of 5' terminal is excised from DNA strand and residual portion,3' and internal nucleotide,remains stably attached to gold particle surface.This unusual degradation action totally differs from previous studies that foreign oligonucleotides would be randomly digested into tiny fragments or single nucleotide in intracellular environment.Importantly and helpfully,these findings offer a route to avoid degradation-caused false positive signal that one can choose a secure region for labeling fluorophore along DNA strand.For instance,in this work,fluorophore can be tagged at 3' terminal or internal nucleotide of oligonucleotide,except 5' terminus which is specially targeted by nuclease degradation in MCF-7 cell.The final section aims at microRNA(miRNA)delivery by using SNA nanocarriers.MiRNAs are a class of endogenous,noncoding small RNAs.Recently,vast studies have shown that miRNAs are very promising anticancer reagents,because they play an important role in downregulating the gene expression by mRNA degradation or translational repression.In particular,microRNA-34a(miR-34a)is one of the best-studied cancer suppressors.It has been exhibited that overexpression of miR-34a can inhibit proliferation,migration,and invasion in various cancer cells,including breast cancer which remains a major cause of cancer death for women.However,some biopharmaceutical problems,such as rapid degradation by nuclease,poor cellular uptake,limit the development and application of miRNA biotherapy.Chemical modification and transfection reagents can increase miRNA lifetime and cellular uptake,but also may affect the function of miRNA,induce unwanted toxicity and immune response.Fortunately,SNA nanoconjugates,due to their unique properties,offer a route to extremely weaken these challenges.Typically,a SNA nanostructure consists of an AuNP core and an oligonucleotide shell.The AuNP core is used as scaffold and the oligonucleotides are anchored on the surface of AuNP core by AuNP-S bond.SNA nanoconjugates are capable of autonomous cell entry without assistance of transfection reagents,have stronger nuclease resistance than linear counterparts and do not elicit observable cytotoxicity or immune response.Hence,we believe SNA can be an ideal nanocarrier for miRNA delivery and releasing in living cell.In this work,we deliver and release miR-34a in MCF-7 cell line by means of two sets of SNA nanocarriers:R-F SNA and miR-34a SNA.This strategy contains a two-step strand displacement reaction:the survivin mRNA transcript firstly swaps out an intermediate strand(R-F strand)from the R-F SNA,and then the free R-F strand invades the miR-34a SNA to release the miR-34a mimics.Therefore,this design rationale allows concurrence of mRNA depletion and miRNA overexpression.Validated by quantitative RT-PCR,the expression of miR-34a is significantly elevated and survivin mRNA level is reduced by-77%.Meanwhile,the released miR-34a largely reduces cell viability and induces apoptosis of MCF-7 cancer cell.These measurements indicate the high-efficiency and availability of this SNA-based delivery system.