| The sequence-specific detection of DNA hybridization has attracted considerable interest in a wide range of areas including molecular diagnostics, environmental monitoring, and antibioterrorism. Therefore, there is a continuing demand for sensitive and selective DNA probes. Many kinds of DNA probes have been developed in recent years through various molecular-engineering strategies to meet this demand. Amount these DNA probes, MBs have attract much attentions due in part to their stability, unique functionality and molecular specificity.The conventional MBs are single-stranded oligonucleotide probes that possess a stem-and-loop structure. The loop portion of an MB is complementary to a target single-stranded DNA, while the stem is formed by 5 to 7 bp from two complementary arm sequences that are on either end of the MB. A fluorophore is attached to the end of one arm, while a quencher is attached to the end of the other arm. The stem maintains a close proximity of the two moieties, causing fluorescence to be quenched by fluorescence resonance energy transfer (FRET). When an MB hybridizes with its complementary DNA (cDNA), the MB undergoes a spontaneous conformational reorganization with the opening of the stem, leading to a fluorescence restoration. The unique target recognition and signal transduction capabilities of MBs have led to their application in many biochemical and biological assays including quantitative PCR, protein-DNA interactions, multiplex genetic analysis, and the detection of mRNA in living cells. As part of a general program aimed at developing free-labeled MBs, we want to investigate whether the non-covalently bind small fluorescence molecules can be a signal molecules for MBs.The paper includes two parts, review and study.The first chapter is part of the overview section to describe the principle of molecular beacons, application and research. Focuses on the structure of molecular beacons, we also introduced domestic and foreign non-labeled DNA testing technology, explained the background of this thesis, and to study the meaning, purpose and research content.The second chapter is research report:First, we synthesit the fluorescent small molecules AMND (2-amino-7-methyl-1,8-naphthyridine) as it has been reported that AMND can bind to cytosine bases specifically.And then, a free-labeled molecular beacon (MB) based on non-covalent binding of a fluorescence molecule,2-amino-7-methyl-1,8-naphthyridin-(AMND), to the intentional gap site in the stem moiety of a hairpin DNA has been developed. When the free-labeled MB was under a closed state, a gap site was formed in the stem moiety of a hairpin DNA. The fluorescence of the small fluorescence molecule was significantly quenched by binding to the unpaired base at the gap site through hydrogen bond, and thus the free-labeled MB shows almost no fluorescence. Upon hybridization with a complementary target ss-DNA (cDNA), the free-labeled MB undergoes a conformational change to take an open state, resulting in an effective fluorescence enhancement owing to a release of the small fluorescence molecules from the gap site. Fluorescence titration and circular dichroism (CD) spectra were used to demonstrate that the binding of AMND to cytosine base at gap site was strong and do not induce a significant conformational change of the hairpin DNA. Under the optimal conditions, the fluorescence intensity of the free-labeled MB increased with an increase of the concentration of cDNA and a detection limit of 9×10-11 M cDNA was achieved. Single mismatched target ss-DNA can be effectively discriminated from complementary target ss-DNA. There may be some advantages in utilizing this type of free-labeled MB over more-traditional ones. First, both ends of the free-labeled MB can be left free to introduce other useful functionalities. Second, our free-labeled MB synthesis is relatively simple and inexpensive because no label is required.The third part is colorimetric detection method for Hg2+ ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Mercury is a widespread pollutant with distinct toxicological profiles, and it exists in a variety of different forms (metallic, ionic, and as part of organic and inorganic salts and complexes). Solvated mercuric ion (Hg2+), one of the most stable inorganic forms of mercury, is a caustic and carcinogenic material with high cellular toxicity. Here, we report a simple and sensitive colorimetric detection method for Hg2+ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Complementary DNA strands with T-T mismatches could effectively protect AuNPs from salt induced aggregation. While in the presence of Hg2+ions T-Hg2+-T coordination chemistry leads to the formation of DNA duplexes, and AuNPs are less well protected thus aggregate at the same salt concentration, accompanying by color change from red to blue. Employing duplex oligonucleotides with T-T mismatches in the sensing system, a sensitive linear range for Hg2+ ions from 0.05 to 2μM and a detection limit of 17 nM are obtained. |
PDF Full Text Request