| The azobenzene-modified DNA is an oligonucleotide molecule with azobenzene moieties covalently linked to the DNA backbone via the d-threoninol bond. The reversible photoswitching capability of azobenzene allows optical irradiation (light) to control the hybridization of azobenzene-modified DNA. Meanwhile, gold nanoparticles functionalized with natural oligonucleotides show increased sensitivity in nucleic acid detection due to the DNA-directed biorecognition and nanoparticles' plasmonic effects. In the dissertation, I present my studies of surface functionalization of gold nanoparticles with azobenzene-modified oligonucleotides and the relevant photochemical characterization of the new system.;In chapter 2, I synthesize the photoswichable gold nanoparticle assemblies cross-linked with azobenzene-modified DNA. Beyond the classic DNA-directed assembly and sensing behaviors associated with DNA-modified nanoparticles, these particles exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces the dissociation of nanoparticle aggregates due to trans-to-cis isomerization of the azobenzene which destabilizes the DNA duplex. The assembly of nanoparticles is reversible upon exposure to blue light due to the reverse cis-to-trans isomerization of azobenzene-modified DNA. I further find that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and I demonstrate that photon dose can thus be used in place of temperature or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.;In chapter 3, I study the sequence dependence of the photoinduced isomerization quantum yield of azobenzene-modified DNA. Compared to the free azobenzene, the trans-to-cis isomerization quantum yield is decreased 3-fold (from 0.094+/-0.004 to 0.036+/-0.002) when the azobenzene is incorporated into single-strand DNA (ssDNA), and is further reduced 15-fold (to 0.0056+/-0.0008) for azobenzene incorporated into double-strand DNA (dsDNA). Quantum yield is also sensitive to the local sequence including both specific mismatches and the overall sequence-dependent melting temperature.;In chapter 4, I study hybridization and light-induced dehybridization of azobenzene-modified DNA bound to glass substrates with fluorescently-labeled oligonucleotide targets in solution. I show that fluorescent readout using a commercial array scanner is compatible with azobenzene-modified DNA capture sequences. In addition, I demonstrate that I can photoswitch azobenzene molecules on a surface in the presence of fluorophores and thus that I am able to control the dehybridization behavior of the immobilized azobenzene-modified DNA with its target sequence in solution. I further study the dehybridization of perfectly-matched target sequences and the single-base-mismatched sequences as a function of radiant fluence. I measure lower fluorescent signals for sequences with a single-base mismatch than that for perfectly-matched sequences, showing that mismatched sequences dehybridized more efficiently upon UV illumination. The extension of this photoinduced differential dehybridization phenomenon to surfaces in the presence of fluorophores indicates that optical DNA hybridization stringency is compatible with chip-based applications for heterogeneous assays. |
