The purpose of this project is to test the possibility of developing TiO2 -biopolymer nanocomposites as new vehicles for biotechnology. We propose to use TiO2-DNA nanocomposites as inducible gene specific endonucleases, with allele-differentiating sequence specificity. This function of nanocomposites relies on (a) TiO2 nanoparticle dependent charge separation inducing DNA cleavage; and (b) sequence specificity of oligonucleotides attached to TiO2 nanoparticle.
The specific aims of this proposal are: 1. In vitro studies (outside cells) of the interaction between HSVtk specific nanocomposites and DNA and RNA specific for the HSVtk (Herpes Simplex Virus thymidine kinase) gene 2. Studies of the interaction between HSVtk specific nanocomposites and DNA specific for the HSVtk gene in cells in culture 3. Evaluation of mutations caused by interactions between HSVtk specific nanocomposites and HSVtk gene in cells in culture. We will study DNA scission following activation of single and multiple nanocomposites (with one or two different oligonucleotides attached per nanoparticle) annealed to the target sequence. The target sequence is Herpes simplex virus thymidine kinase gene (HSVtk). We chose this sequence to be our target because HSVtk acts as a suicide gene in cells treated with guanine analogues, and therefore mutagenesis of the HSVtk sequence provides cells with a selective advantage when guanine analogues are added into the cell medium. We believe that upon completion of these studies we will be able to link results of in vitro DNA scission assays with the spectra and frequency of mutations in cells in culture, use this knowledge to predict the behavior of nanocomposites inside cells, and associate the type of mutation that can be anticipated with different nanocomposite sequences and modes of nanocomposite activation.
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