Design On Sensitive Fluorescence Anisotropy DNA Probes And Their Applications

Due to their versatility, sensitivity and quantitative capabilities, the development of sensitive and selective nucleic acid probes has become a very active research field in recent years. Fluorescence anisotropy(FA) is also called fluorescence polarization(FP), which is a promising choice for fluorescence sensing based on fast, accurate and sensitive signal reporting. Moreover, as a ratiometric approach, it is closely related to its rotational relaxation time, which, in turn, depends on its molecular volume(molecular weight) under constant temperature and solution viscosity. Furthermore, FA is insensitive to fluorescence fluctuation and photobleaching, and it can be directly used in complex biological environment. During the past few decades, FA assays have been successfully employed in many research areas, such as protein-protein interaction, protein-DNA interaction and immunoassays for drug discovery, diagnosis, food analysis, and environmental monitoring. However, one of the important requirements for a workable FA probe is that the molecule being measured should induce a significant change in molecular mass before and after recognition. Thus, the FA method has been limited to macromolecules such as proteins, and this approach is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. To address this problem, with elegant design based on the unique conformational flexibility of aptamers, some aptamer-based FA probes for small-molecule detection have recently been proposed using competitive displacement, induced-fit binding, and mass amplification strategies. In this paper, we developed a range of novel mass amplified fluorescence anisotropy strategies, and applied to analysis of small molecules, including adenosine triphosphate(ATP), adenosine, Nicotinamide Adenine Dinucleotide(NAD). The main contents are as follows:(1) By introducing streptavidin as a molar mass amplifier, was used in a hybridization chain reaction(HCR) to construct a target-triggered cyclic assembly of DNA-protein hybrid nanowires for highly sensitive detection of small molecules by fluorescence anisotropy. In this assay, one blocking DNA strand could be released by target-aptamer recognition, and then it served as an initiator to trigger enzyme-free autonomous cross-opening of hairpin probes via HCR to form a DNA nanowire for further assembly of streptavidin. Using adenosine triphosphate(ATP) as the model small molecule, this novel dual-amplified, aptamer-based FA assay afforded high sensitivity with a detection limit of 100 n M, which is much lower than that of the disassembly approach without HCR amplification or the assembly strategy without streptavidin. Moreover, because of its resistance to environmental interferences, this FA assay has been successfully applied for direct detection of 0.5 ?M ATP in complex biological samples, including cell media, human urine, and human serum, demonstrating its practicality in real complex biological systems.(in chapter 2)(2) Combining target-triggered enzymatic cleavage protection and the extraordinarily strong interaction between biotin and streptavidin, we proposed a novel mass amplification strategy for the sensitive detection of small molecules in homogeneous solution through fluorescence polarization. Our design makes it possible to use FP as a signal transduction mechanism to construct aptamer probes against small molecules. In contrast to simple target-triggered enzymatic cleavage protection or protein amplification, the combination of target-triggered enzymatic cleavage protection and mass amplification through the use of streptavidin resulted in significant change of mass and, hence, sensitive FP assay with a LOD of 500 n M for adenosine. Moreover, by simply replacing the aptamer with other aptamer sequences, the detection of various targets can be accomplished, such as tyrosine and cocaine. These unique properties of this proposed strategy will enable the development of a new class of probes for rapid, sensitive, and selective detection of small molecules by means of FP in complex biological samples.(in chapter 3)(3) To develop sensing systems for ATP and NAD with high selectivity, we turned our attention to DNA ligases, as there are two kinds of DNA ligases which specifically employ ATP and NAD as cofactors, respectively, and their catalytic activities are cofactor-dependent. Combining with the extraordinarily strong interaction between biotin and streptavidin, we employed a novel mass amplification strategy for the sensitive detection of small molecules through fluorescence anisotropy. In the presence of ATP or NAD, the DNA ligase catalyzed the ligation reaction and mass amplification through the use of streptavidin resulted in significant change of mass, resulting in detection limits of 50 n M and 10 n M for ATP and NAD, respectively, much lower than those of previously reported FA biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, this work shows significantly high selectivity toward the target cofactor(ATP or NAD), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel mass amplified fluorescence anisotropy method may find a broad spectrum of applications in both environmental and biomedical fields.(in chapter 4)...