The Preparation And Application Of Magnetic Nanoparticle-Based Artificial Restriction Enzymes

The interaction of chemical molecules and biological ones, especially sequence specific recognition and site-specific cleavage of DNA have many applications such as in DNA sequence determinations, chromosome analyses, gene therapeutics, and recombinant DNA manipulation. So it is significant to develop sequence-specific DNA cleavage regent (artificial restriction enzymes) for the scission of nucleic acid with high activity and selectivity at normal condition. Meanwhile, with the nanoparticle pervading into the biological sciences, using the nanotechnology to study and resolve the problem of life sciences is becoming one of the most advanced fields. In this thesis, we had systematically studied these items and obtained several conclusions below.(l)Firstly, we prepared a novel artificial sequence-specific cleavage reagent, which consisted of peptide nucleic acid (PNA) as sequence-recognizing moiety and rare earth metal ions Cerium (IV) as "scissors". Subsequently, it was applied in the cleavage of 26-mer single-stranded DNA (ssDNA) target, which has 10-mer sequence complementary with PNA recognizer in the hybrids, under physiological conditions. Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) experiments indicated that the PNA-based site specific cleavage reagent could cleave the target ssDNA specifically.(2) Sequentially we prepared peptide nucleic acid-functionalized magnetic nanoparticles (PMNPs) by attaching the 4-pyridyldithiol-derivatived peptide nucleic acid (PNA) which were designed to recognize special gene to the 3-mercapropropyloxysilane coated magnetic nanoparticles (MNPs) via thiol-disulfide exchange reaction. Then the PMNPs were challenged with non-complementary and perfect-match DNA targets. The whole procedure mentioned above was monitored via surface-enhanced Raman scattering (SERS) method. The results showed that the PMNPs were able to efficiently hybridize with the perfect-match ssDNA target and showed no affinity towards non-complementary DNA.(3) Then we developed MNPs-based artificial sequence-specific cleavage reagents (MNPs-based artificial restriction enzymes), which consist of oligonucleotide (ODN) or PNA as sequence-recognizing moiety, the rare earth metal ion Ce(â…£)/EDTA complex as "scissors" for cleaving target DNA and MNPs as carrier. Subsequently, we investigated their ability to sequence-specific hydrolyze target single-stranded DNA (ssDNA) under physiological conditions. The experimental results revealed that MNPs-based artificial cleavage reagent could sequence-specifically hydrolyze the target ssDNA at the principal cleavage sites.