Design,Synthesis And Application Of Functional DNA Based On Functional Groups And Cell Penetrating Peptides

As a functional biomacromolecule,nucleic acid has been widely used in the field of biosensing and drug delivery.However,natural nucleic acid molecules have many limitations,such as easy degradation by nuclease,strong electronegativity and large molecular weight resulting in poor membrane permeability,which limit their biological functions.Chemical modification can help to improve the inherent defects of natural nucleic acid and broaden its application.In this thesis,given the diversity,modifiability and biocompatibility of DNA sequences,FAM labeled phosphorothioate modified DNA（PS-DNA）was selected as the basic skeleton to synthesize EMAmer probes through incorporating different types and/or numbers of positively charged functional groups at designated positions on nucleic acid scaffolds.Combining with monolayer molybdenum disulfide,We then constructed a chemical nose and tongue sensor platform（MoS₂-EMAmer）for pattern recognition analysis of target cells.Additioanlly,aiming for improving the transmembrane transport ability of DNA,we developed an innovative cell penetrating peptide（GLECPP）and constructed an GLECPP-DNA transport system based on the Michael addition reaction.The main research contents are as follows:1.PS-DNA was used as the basic scaffold,on which the position,number,and spatial distance of functional groups on the DNA scaffold can be accordingly regulated by adjusting the position,number,and spatial distance of PS.12 types of EMAmer probes were constructed through the nucleophilic substitution reaction given the strong nucleophilic reaction activity of PS-DNA,which provided a new method for the construction of non-specific sensing and recognition elements.In addition,we combined the established EMAmer probes and MoS₂ monolayer to construct a low-fluorescence background sensor platform with high reproducibility.Through pattern recognition method,we achieved rapid,convenient,sensitive and accurate identification of cancer cells and mixtures.The identification accuracy of 6 kinds of cells can reach 97%,holding great promise as a powerful means for cancer diagnosis.2.Based on the structure of classical cationic cell penetrating peptide R8,an innovative cell penetrating peptide（GLECPP）was designed by modifying R8.Compared with R8,the GLECPP structure comprises a hydrophobic core region（two tryptophan）flanked on each side by arginine-rich domains containing aminohexanoyl（X）andβ-alanine（B）spacers,and five histidines on each of the outermost sides of the sequence.Spacers are important for the optimal positioning of the charged arginine side chains,tryptophan can enhance interaction between GLECPP and cells and histidine can promote endosomal escape by taking advantage of the protonation of histidine in an acidic environment.In order to verify the rationality of GLECPP structure design,we compared the membrane penetration performance of GLECPP and R8.The results showed that compared with R8,GLECPP had higher membrane penetration efficiency（GLECPP was 5 times more efficient than R8 at a concentration of 10μM）and better lysosome escape ability（at a concentration of 2.5μM,the intracellular fluorescence of GLECPP was clearly visible and evenly distributed in the cytoplasm,but the fluorescence intensity of R8 is very weak）.GLECPP also had low cytotoxicity and good serum stability.In addition,in order to verify the cargo carrying capability of GLECPP,we coupled GLECPP and DNA through Michael addition reaction to form a drug delivery system.Compared with naked DNA,GLECPP-DNA drug delivery system has a higher membrane penetration efficiency,whereby GLECPP-DNA was uniformly distributed in cells with little cytotoxicity（87%survival rate at 10μM）at a concentration of 5μM.To sum up,with the aim to improve the chemical diversity of DNA and the permeability of cell membranes,the DNA skeleton was modified to construct MoS₂-EMAmer sensor array,which could provide a new approach for the construction of non-specific sensor libraries.Addtionally,the DNA-carrier delivery system was constructed through the functional modification of DNA and biological coupling reaction,which would provide a new perspective for the design and construction of nucleic acid drug delivery vectors.The above study is expected to provide a flexible and powerful platform in the field of biological analysis and drug delivery.