Castronovo, MatteoEssa, Lujin2024-07-042024-07-042024-03-23https://hdl.handle.net/20.500.14154/72484Achieving quantitative, multiplex protein imaging in biological samples remains a challenge towards developing quantitative biology. A promising approach to achieve this goal is by enhancing immunofluorescence optical imaging. A key roadblock, however, is the paucity of non-overlapping fluorescent dyes to use to orthogonally label each antibody. In this doctoral work, a method for imaging protein in a layer-by-layer fashion was proposed using a constant fluorescence channel and an antibody-conjugated DNA-programable fluorescent system, whose emission can be reversibly activated or deactivated by chemical inputs. The latter involves two nanostructure units: an antibody-conjugated “Encoder” and a “Decoder”, which reversibly bind the encoder and include a constant fluorescently labelled DNA molecule. Finally, Encoder-Decoder pairing involves the formation of a hybrid DNA-RNA duplex, which can be reversed by the action of RNase H enzyme, without Encoder alteration. In this study four pairs of DNA nanostructures were designed and optimised by varying a DNA tile model widely adopted in the literature to achieve (i) orthogonal Encoder-Decoder association, (ii) rapid Encoder-Decoder dissociation by RNase H action, and (iii) high thermal stability. The thermodynamic properties of nanostructure designs were numerically assessed using the NUPACK software package, while the reaction outputs were primarily analyzed by native gel electrophoresis, using near-infrared fluorescent dyes and near-IR laser scanning for enhanced sensitivity. A crucial step to ensure thermal stability was extending the DNA-DNA duplex region of 8 bp at one end in each nanostructure, which yielded a melting temperature of 70˚C. Furthermore, the Encoder unit was successfully be conjugated with a commercially available nanosecondary antibody using maleimide-thiol click chemistry and purified the product by anion exchange chromatography. Overall, this doctoral work provides the biophysical foundations of a novel imaging approach for enabling multiplexed immunofluorescence imaging by minimizing the chemical synthesis associated with the fluorescent dyes and offering a versatile solution that can be implemented with different imaging platforms.163enNanotecnologyDNA NanostructureThesis