Single-molecule Interactions On DNA Origami Based On AFM

Single-molecule detection can directly record the change of the characters ofmolecules by tracking the trajectories of single dynamic molecules, which contains awealth of dynamic information. At present, the detection methods mainly contain:first, electronic microscopy; second, single-molecule fluorescence microscopy; third,mechanical microscopy, for example, atomic force microscope (AFM). Comparingwith other methods, AFM possesses unique advantages, such as high-resolution,lable-free and keeping the activity of biological samples, which make it a strong toolto investigate single-molecule interactions. However, due to the feedback way-basedon force between tip and sample, the scanned samples need to meet two conditions:one, good adhesion abilities with substrate in order to keep stability in imagingprocedure; two, recognition of the specific reaction sites. DNA origami, in which along single-stranded DNA is folded into designed shapes by a large number of shorter“staple strands”, can form a wide variety of2D or3D nanoscale structures, which hasthe addressable property and can easily adsorb on substrate with the help of cationin buffer. Here, combing AFM with DNA origami, I studied the molecular interactionsat single-molecule level. The work mainly contains the following five parts.1. Developed a novel strategy for real-time and in situ monitoring single-moleculebinding reaction on functionalized DNA origami based on atomic forcemicroscope (AFM). Individual biomolecular binding events were recorded andthen single molecules bound to specifically functionalized DNA origami weresimply counts as a function of time to obtain a direct measure of the binding rate.2. Found an interesting phenomenon-“DNA threading”. By using an origami sheetattached with patterned biotinylated ssDNA tethers and monitoring streptavidinbinding with AFM imaging, we provide unambiguous evidence that the biotin ligands positioned on one side have indeed threaded through to the other side.This finding reveals a critical design feature that should provide newinterpretations to previous experiments and new opportunities for theconstruction of origami structures with new functional capabilities.3. Explored the influence of distance between two antigens on the antigen-antibodyinteractions. Then further detected the influence of temperature on thedissociation of monovalent and bivalent complexes respectively.4. Investigated the dependence of the tethering length on molecular capture eventsmonitored by AFM. The results show that the SA-biotin complexes are easilydetected with short tethered lengths, and that their morphological featuresclearly change with the tethering length.5. Investigated the orientation of adsorption of2D origami on substrate.