|According to the Human Development Index, it is estimated that all cancer cases will rise from 12.7 million cases in 2008 to 22.2 million by 2030. Traditional therapies, including surgery, radiation therapy, and chemotherapy, have difficulty in specifically selecting malignant tissues for destruction. To overcome these limitations, nucleic acid-based therapies, such as short interfering RNAs (siRNAs), have been developed to aid in the regulation of proteins. SiRNAs serve as substrates for the highly conserved RNA interference (RNAi) pathway, which gives rise to their high degree of mRNA target specificity and their catalytic nature. However, the full potential of these therapeutic drugs will not be attained unless better methodologies are developed to enhance target delivery and to enhance resistance to ribonuclease-mediated degradation. The focus of this study was to chemically modify the sense strand of an siRNA duplex with a triazole backbone unit and pyrimidine-modified hydrophobic substituents in an attempt to improve its pharmacokinetic and pharmacodynamic properties. This was accomplished by linking together uracil and cytosine monomers with the Cu(I)-assisted Huisgen 1,3-dipolar cycloaddition reaction to create a cytosine-triazole-uracil-iodine dimer (CtUI). The dimer was further modified at the C5 position of uracil with various lipophilic moieties including cholesterol and phenylacetylene utilizing the Sonogashira cross-coupling reaction. Subsequent reactions were performed on the dimer to give rise to a 4,4’-dimethoxytrityl (DMT)-protected phosphoramidite building block, which allowed for its site-specific incorporation into an oligonucleotide using solid-phase synthesis. Once the modified strand was annealed to its counterpart, circular dichroism measurements revealed that modified siRNA duplexes retained their standard A-form helix. Thermodynamic profiling of the siRNA library illustrated that internally modified siRNAs were greatly destabilizing while, external placements were slightly destabilizing. Furthermore, the lipophilic moieties protruding off of the uracil base did not have a profound effect on the stability of the duplex. Results from cell-based assays indicated that that triazole-modified siRNAs bearing hydrophobic groups were compatible with the RNA induced silencing complex (RISC) and effectively silenced reporter gene, firefly luciferase, and an endogenous gene, BCL-2, in a dose-dependent manner. Further exploration of the effect of the hydrophobic moieties in a carrier-free assay revealed that the added cholesterol group was able to enhance the delivery of native siRNAs. To the best of my knowledge, this is the first time that the effect of a non-ionic linkage unit derivatized with a base-modified hydrophobic group has been explored within siRNAs. The following study demonstrated the compatibility of these novel constructs within the RNAi pathway and helped to better understand the structure function relationships involved with RISC.