Detection Of Neurodegenerative Proteins And DNA Using Biological Nanopore At Single-Moleucle Level

The biopolymers including protein and DNAs are important for present-day life. The biopolymers own various conformations and interact with differnet molecules. Routine analytical methods just study the overall properity of materials rather than the interaction between different moleucles at single molecule level. Sensitive single-molecule detection (SMD) technique not ony qualitative observe single biopolymer but also provide structural information of every biopolymer. SMD could offer a novel analytical technique in deeper studying biopolymers with the problems that are hardly solved by traditional methods. Nanopore, as a efficient and radip single molecule detection method, has drawn scientist attation. α-hemolysin (α-HL) self-assembled on lipid bilayer to form a nano-scale pore, which could resistant high applid potential and ionic strength. The blockade events produced by translocation through α-HL are related to the conformational change, which remind us to detect pathogenesis-related proteins by using α-HL nanopore. From this point, we utilized thea-HL nanopore which contains a vestibule to explore the conformation change of the amyloid protein related to neurodegenerative diseases. By observing the aggregating change of amyloid protein at single-molecule level, we offered a novel method for the early diagnosis and drug screen for neurodegenerative disease. Furthermore, we developed a new biological nanopore based on SP1protein to observe the structural changes of single DNA inside the pore. Using SP1nanopore to investigate single molecules detection broaden the existing research areas from unsymmetrical biological nanopore to symmetrical biological nanopore, as well as establishing the theoretical model of the interaction between DNA and SP1nanopore. The detail research contents are as follows:1. Nanopore analysis of β-amyloid peptide aggregation transition induced by small moleculesβ-Amyloid42(Aβ42) is the predominant form of the amyloid peptide, which is found in the plaques of the brains of Alzheimer’s (AD) patients and is one of the most abundant components in amyloid aggregates. Information of the Aβ42aggregation states is essential for developing an understanding of the pathologic process of amyloidoses. Here, we used α-hemolysin (α-HL) pores to probe the different aggregation transition of Aβ42in the presence of β-cyclodextrin (β-CD), a promoter of Aβ42aggregations, and in the presence of Congo red (CR), an inhibitor of aggregations. Analyzing the characteristic transit duration times and blockade currents showed that β-CD and CR have opposite effects on the aggregation of Aβ42. Translocation events of the monomeric Aβ42peptide (i=25.47pA) were significantly lower in amplitude currents than protofilaments (i=96.25pA), and protofilaments were captured in the α-HL nanopore with a longer duration time. CR binds to Aβ42and its peptide fibrils by reducing the aggregated fibrils formation. In this process it is assumed CR interferes with intermolecular hydrogen bonding present in the aggregates. In contrast to CR, β-CD promotes the aggregation of Aβ42. These differences can readily be analyzed by monitoring the corresponding characteristic blockade events using a biological α-HL nanopore.2. Analysis of α-synuclein fibrillation using α-HL nanoporeThe fibrils procedure of α-synuclein is investigated in physiological condition by α-HL nanopore. The natively unfolded α-synuclein monomer will translocate through α-HL nanopore by applied potential. Changing the poteinal, a partially folded intermediate are involved in the critical early stage of the structural transformation which is monitored by captured inside the vestibule of α-HL nanopore with the capture current of20±1pA. Further blocking of the intermediate will produce block current of5±0.5pA, which revealed that the early-stage fibril of α-syn is affected by intramolecular electrostatic interaction. In addition, trehalose inhibited the fibriation by changing the surface hydrophobic interaction of A53T α-synuclein without any inhibition of WT α-synuclein. The result also proved that the interpeptide hydrophobic interactions in the elongation of A53T α-synuclein protofilaments cannot be greatly weakened by trehalose. The inhibitory effect of trehalose is partially nucleation specific, whereby formation of protofibril or nuclei is prevented. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.3. Single-molecule DNA detection using novel SP1protein nanoporeNanopore plays a central role in single-molecule analysis since the ionic current blockades they produce provide information about the identity, conformation and dynamic of target molecules. The SP1protein has remarkable stability under extreme environmental conditions and it exhibits a wider symmetrical channel constriction that is promising for DNA detection. Here, SP1protein as a new type of nanopore is primarily described and the functions of SP1nanopore were utilized to distinguish single-strand DNA at single-molecule level. It is found that the predominant effect on the blockage interaction originates from the quasi-rigid structures of the biopolymers. These are almost unaffected by the lengths of the polymer chains mainly in threading through the symmetrical SP1nanopore linearly. By comparing with α-HL nanopore, it is speculated that the geometry of SP1could slow down the translocating velocity of ssDNA. Thus, the SP1pore material has potential advantages may be used for future DNA sequencing applications. Non-vestibule structure results in ssDNAs entering the SP1nanopores without any vector constriction or direction followed by passing randomly. Using SP1nanopore to investigate single molecules detection broaden the existing research areas from unsymmetrical biological nanopore to symmetrical biological nanopore.