Design, Synthesis And Investigation Of Locked Nucleic Acid Molecular Beacons

As a new class of nucleotide analogues, locked nucleic acids (LNA) have many advantages such as high binding affinity, excellent base mismatch discrimination capability and nuclease resistance. Other advantages include non-toxicity and convenience to synthesize using conventional phosphoramidite chemistry. Based on these advantages, LNA has been widely used in antisense therapy, single nucleotide polymorphism (SNP) detection and so on. In this thesis, the major goal is to design more effective molecular beacons (MB) with locked nucleic acid. The research work of this dissertation is summarized as follows:(1) Design, synthesis and investigation of locked nucleic acid molecular beacons (LNA-MB) with high nuclease resistance.A novel kind of MBs with LNA end-modification was synthesized and their nuclease resistance, melting temerature, and hybridization kinetics were investigated by fluorometry and HPLC. It was found that these new LNA-MBs had not been digested after a long reaction time with DNase I, which indicated that they had high nuclease resistance. The LNA-MBs still kept their hairpin structure even at 90℃, which showed their high thermodynamic stability. In addition, since the background fluorescent signal of the MBs was much lower than conventional MBs, the singal to background ratio (SBR) increased significantly, while their single-base mismatch discrimination capability did not decrease. These findings could be beneficial for the application of LNA-MB in complex biological systems.(2) Design, synthesis and investigation of LNA molecular beacons with high single-base mismatch discrimination capability.A novel kind of LNA-MBs with the mismatch site or nearby replaced with one or three LNAs were synthesized, and their single-base mismatch discrimination capability, melting temerature, hybridization kinetics and SBR were investigated by fluorometry. It was found that the LNA-MBs with only the mismatch site replaced with LNA had the highest single-base mismatch discrimination capability, and it would not improve when the number of LNAs increased. In addition, the SBR of these LNA-MBs improved with the increase of the number of LNAs at the mismatch site. The melting temperature of these MBs showed that their higher efficiency was caused by the affinity and specificity of LNA. In conclusion, this is a universal method to improve the performance of molecular beacons and it can be used to detect SNP and can be applied in samples with low-abundant target.(3) Design, synthesis and investigation of mercury(II) specific LNA molecular beacons.A nove kind of mercury(II) specific LNA-MB was synthesized and its specific interaction with mercury(II) ions was investigated by fluorometry. It was found that the LNA sequence that contain T-T base pair can also form T-Hg2+-T duplex in the presence of mercury(II) ions. The assay could determine mercury(II) in the range of 25-150 nM with a detection limit of 14.7 nM, which is comparable with conventional MBs.