Abstract: Cells regulate gene activity by post-transcriptional silencing of the genes using small double stranded RNAs (dsRNAs). This phenomenon is called RNA interference (RNAi). In mammalian cells, RNA duplexes, termed as short interfering RNAs (siRNAs), bind to cognate mRNA sequences and based on complementarity, repress translation or cleave mRNA strands. This gene-silencing pathway can be uti... read morelized to regulate various genes of interest. In particular, this method can be used to target genetic, and viral diseases and may help in cancer therapy. The anionic nature of siRNAs prevents them from associating with the plasma membrane. We have designed a new class of non-cationic phospholipid based delivery agents that make use of the increased acidity of the vesicles in the endocytic pathway and release cargo molecules into the cytoplasm. We have also shown that partially fluorinated hydrocarbon lipids are two to three times more efficient than their hydrocarbon counterparts. Using confocal fluorescence microscopy, we have shown that these agents can efficiently deliver highly fluorescently labeled ssDNA across the membrane into the cytoplasm. Our experiments show that these constructs traverse the plasma membrane via clathrin and/or caveolin dependent endocytic pathway. For the second part of the thesis, we have designed lipidated peptides that modulate G-protein coupled receptors (GPCRs), which form the largest superfamily of cell surface receptors. The have important physiological and pathophysiological roles including analgesia, appetite, inflammation, allodynia and spontaneous pain and peptide hormones modulate most of these functions. They are the most widely studied class of proteins and are targets for nearly 25% of the drugs currently available in the market. Despite of all the success in synthetic efforts to make peptides in large scale, there needs more studies to improve their proteolytic stability, rapid clearance. We have designed modified peptides that have sustained activity, longer circulation and improved proteolytic stability. We have tested our model on a variety of GPCRs in a platform approach, and the ligands showed enhanced activity in all of the tested GPCRs. We have also shown that these constructs are resistant to wash-off experiments. We are performing experiments to understand the biophysical properties of these constructs.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Chemistry.
Advisor: Krishna Kumar.
Committee: Elena Rybak-Akimova, Samuel Thomas, and Graham Jones.