Studies On Controlled Drug Release And Foodborne Pathogens Detection Using Triplex Nucleic Acid With High Stability

Triplex nucleic acid probe is a duplex nucleic acid structure formed by embedding the third oligonucleotide chain into the duplex nucleic acid groove with Hoogsteen hydrogen bond on the basis of Watson-Crick duplex nucleic acid.It has the advantages of easy preparation,low synthesis cost,flexibility in structure and design,and has been widely used in intelligent drug delivery,biochemical sensing and other fields.However,the triplex nucleic acid structure is highly acid dependent,so it still faces the challenge of insufficient stability when applied in practical complex systems.Based on this,this paper proposes a new strategy to stabilize the triplex structure by introducing a duplex stem,which breaks through the limitations of the existing triplex nucleic acid stabilization strategy that requires adjustment of the base composition or chemical nature of the triplex region.On this basis,the functionalized Au@Pt NP drug delivery system was constructed by using the highly stable triplex nucleic acid developed above,which was used to study the controlled release of miR-559.A free-labeled colorimetric probe was constructed for the detection of Escherichia coli.The specific work is as follows:(1)By introducing the duplex stem and optimizing the length of the duplex stem,it was found that when the duplex stem contained 5 bp,the stability of the clamp triplex strand was improved the most.Compared with the traditional clamp triplex nucleic acid(CTS),the pH stability range of the obtained double stalk stable clamp triplex nucleic acid(CTSH5)was 1.22 pH units wider,showing significantly improved anti-interference ability in fetal bovine serum,cell lysates and other complex biological systems.In addition,we found that CTSH5 was much less sensitive to base mismatch in the triple helix region and changes in loop length than CTS,breaking the restriction of the highly conserved composition of intermediate chain bases and the restriction of loop length on the design and application scope of triplex nucleic acids.Fluorescence experiments showed that the triplex nucleic acids stabilization strategy with duplex stem was universal and improved the stability of triplex nucleic acids with different T-A·T ratios.This study provides a new idea for the construction of stable triplex nucleic acids probe,and is expected to expand and deepen the application dimension and depth of triplex nucleic acids by increasing the flexibility of triplex nucleic acids design.(2)miRNA therapy has shown great potential in the treatment of multi-gene malignant diseases such as tumors due to its "one-click multi-target" effect.However,most of the currently reported tumor therapeutic strategies for delivering tumor suppressive miRNA are deficient in uncontrolled drug activity and release site,which may lead to off-target toxic and side effects.In addition to the target-induced allosterism,the pincer-shaped triplex nucleic acid can realize the separation of recognition unit and signal output unit,which provides the possibility for controlled delivery of tumor suppressive miRNA analogue-like.Based on this,we constructed a highly stable triplex nucleic acid functionalized Au@Pt NP drug delivery system(Au@Pt-CTSH5)for the controlled release of miR-559 in glioma cells by using the duplex stem stabilized triplex nucleic acid constructed in the previous chapter as a molecular switch to block the activity of tumor suppressive miRNA.After endocytosis of the drug delivery system to target glioma cells,the ring part of the triplex molecular switch can specifically hybridize with miR-155,which is highly expressed in the cytoplasm,to induce conformational changes of the triplex molecular switch and then disintegrate,thus promoting the in situ release of miR-559,which is enclosed in the triplex structure,and realizing its intervention on downstream mRNA expression.Then achieve the goal of gene therapy.Confocal laser experiments showed that Au@Pt-CTSH5 could enter cells within 4 h,and the triplex nucleic acid switch could be specifically switched on in target cell U251.Cytotoxicity test(MTT)showed that Au@Pt-CTSH5 had obvious killing effect on glioma cells,and the killing rate was about 40%.This study breaks through the limitations of traditional triplex nucleic acids,which are difficult to deliver miRNA and other functional nucleic acids with fixed sequences due to the highly conservative composition of intermediate chain bases,and is expected to provide a new idea for the development of accurate and controllable drug delivery system.(3)Triplex nucleic acids can realize the separation of signal recognition unit and signal output unit,which has obvious advantages in signal transduction,and is widely used to construct molecular probes to carry out biochemical sensing research.However,the triplex nucleic acids probe is not stable enough in complex system,and it is easy to lead to false positive and other problems.Based on this,in this chapter,an enzyme-free colorimetric sensor based on high stability triplex nucleic acid probe was constructed with triplex nucleic acids(THMS)as the recognition unit,G-quadruplex as the signal transduction unit,and Escherichia coli(E.coli)as the model target,which was used for the rapid detection of food-borne microorganism Escherichia coli.The ring of the probe was designed as an antisense base sequence of E.coli ssDNA,and G-quadruplex changes occurred after binding to E.coli ssDNA,resulting in the disintegration of the triplex and the release of the G-quadruplex.The ribozyme chain then binds hemin to form a complex with catalytic activity,which catalyzes the oxidation of TMB by H2O2.The result shows this method has good accuracy and high sensitivity in the detection of E.coli ssDNA,the detection limit is as lower as 3.03 nM,and the spiked recovery in milk is 83.28-109.33%,showing a good application prospect in the detection of Escherichia coli in food.