The Research On Conjugating And Purifying Peptide-oligonucleotide And The Application Of Functionalization Oligonucleotide

Oligonucleotides are a series of short-chain nucleotides, which have a strong biological activity. By using the Systematic Evolution of Ligands by Exponential Erichment ?SELEX? to filter out oligonucleotides, some of them could bind to proteins, small molecules, ions or cancer cells with high specificity and sentivity. These oligonucleotides can be used for a variety of biochemical sensing. Meanwhile, oligonucleotides can work as gene therapy or drugs proecursors. However, some inherent defects of oligonucleotides, including poor cell permeability, easy to be broken down and with weak force of target, impede their application. Whereas peptides have excellent characteristics of small molecular weight, adjustable electricity and enzymatic hydrolysis-resistant, conjugating peptides with oligonucleotides not only could enhance the cell permeability, but also improve the biological activity of oligonucleotides. In recent years, a variety of technologies of screening peptide, especially the development of phage display technology make the appearance of a large number of peptides with the functions of identification, detection and cure of cancer cells. These peptides give new ideas and methods to the diagnosis and treament of cancer. Biological molecules with bifunctions and drugs with synergistic effect can be obtained through effieciently connecting peptide with oligonucleotide. Therefore, the conjugation of peptides with oligonucleotides has a high practical value in biosensor and treatment. Based on the above reasons, the following works were carried out:?1? Using different strategies, different conjugates of peptide and oligonucleotide ?POCs? were synthesized. We optimized and improved the factors to increase the reaction efficiency during the process of synthesis, explored the key impact factors from theory and practice.Then we disscussed the advantages and disadvantages of these methods in the synthesis of POCs. Finally, we pointed out the scope and the most optimal reaction conditions of these techniques.?2? In order to find a rapid, efficient and standardized way to separate and purify POCs, high performance liquid chromatography ?HPLC?, ion exchange chromatography ?IEC?, polyacrylamide gel extraction, dialysis, ultrafiltration, digestion with nuclease were employed to purify POCs from the mixture of synthetics. We have discussed the features of these purification methods and improved the process of purification. Combing the fact with the advantages and disadvantages of these approachs, we analysed and discussed the problems encountered in the process of purification. Finally, according to the principle and the results of experiments we put forward proposals of suitable conditions and scope to these methods.?3? A double strand probe containing Pb²⁺ specific DNAzyme structure was designed. Such probe modified with a quenching group of BHQ1, which can quench the fluorescence of supercharged green fluorescence protein ?scGFP?, due to the electrostatic interaction of electropositive scGFP with electronegative DNA probe. Whereas, once Pb²⁺ is existed in the reaction solution, it can specific recognize and hydrolyze the substrate strand at the scissile ribonucleic acid adenosine ?rA?. The cleavage of substrate probe results a short single-stranded DNA with BHQ1 separated from the double strand probe adhered on scGFP, which can recover the fluorescence of scGFP. Therefore, the scGFP-DNAzyme based "turn-on" fluorescent strategy would be efficient to detection of Pb²⁺ ?even in real water samples?.