Study Of Biological Weak Interactions At Single-Molecule Level By Nanopore Technology

Studies of biological weak interatctions at single-molecule level investigate and regulate the properties of individual molecules including their conformational dynamics, structures and functions. Since single-molecule detection provides detailed information on the individual molecules, it will further expand to profoundly understanding of life science, nanotechnology and related interdisciplinary studies. In this dissertation, an a-hemolysin (a-HL) protein channel has been used as an ultra-sensitive sensor for single-molecule detection of biological weak interactions. By using this approach, real-time detections of the spontaneous and target induced conformational changes of individual biomolecules have been achieved. After introducing biological weak interactions between antibody and oligonucleotides into nanopore analysis, the conformational entropic barrier for DNA transloeation was reduced. More importantly, a para-sulfonate calix[4]arene functionalized nanopore which is based on host-guest interactions has been constructed. This nanopore system could be used to regulate the individual motions of molecule shuttles. Details of the studies in this dissertation are summarized as follows:1. Study of the Transloeation Behavior of Individual Peptide-Oligonucleotide ConjugatesThe unique three-segment current traces could be considered as a strong and direct evidence for the translocation of self-assembly peptide-oligonucleotide conjugate through an α-HL nanopore. The individual monomer could be identified in the solution of peptide-oligonucleotide conjugate. Our findings show that an unfolding process accompanys with the entire transloeation of a collagen-like peptide scaffolded bioconjugates. Moreover, the models of transolocation behavior for self-assembly peptide-oligonucleotide conjugate and its monomer have been established, respectively.2. Single-Molecule Study of an ATP-Binding Aptamer and Its Conformational ChangesA single a-HL biological nanopore is employed to sense the spontaneous conformations of an ATP-binding aptamcr which are linear form and folded form,Each conformation of the ATP-binding aptamer produces a unique duration time and a characteristic signal. More importantly, the frequency of transloeation events can be readyly used to distinguish the conformational changes of ATP-binding aptamer induced by ATP or complementary ssDNA. By measuring the duration of each characteristic signals, the nanopore methods for discrimination of binding abilities of ATP-binding DNA aptamer towards target molecules has been developed at single-molecule level.3. Enhanced Transloeation of Oligonucleotides through a Nanopore by Binding with AntibodyThe transloeation time of poly(dT)45through an a-HL nanopore was reduced by introduce the weak interactions between antibody and oligonucleotides into nanopore system. The Fab appears to behave as a rudder whieh significantly decreases the energy barrier for poly(dT)45 translocation. A more rigid or extended conformation of poly(dT)45would reduce the time required to find the entrance of the narrow constriction in the nanopore. The entropic barriers which are originated from linearize the DNA strand are the dominant contributions to the entire energy barrier. Therefore, this method significantly increases the efficiency of nanopore analysis.4. A Stimuli-Responsive Nanopore and Its ApplicationsAn artificial gating mechanisms of nanopore triggered by the para-sulfonate calix[4]arene (SC4)-based host-guest interactions have been developed. By the virtue of this novel a-HL: SC4system, the functionalized a-HL has achieved to be commanded by both ligand molecule and photo-stimulation at the single-molecule level. The application of this stimuli-responsive nanopore system has been extended to the real-time study of light-induced molecular machine at single-molecule level.