Light-activated biomacromolecules

A dissertation in Pharmaceutical sciences and ChemistryIncludes bibliographical references (p 292-303)Biomacromolecules, mainly nucleic acids and proteins, are involved in every cellular process and therefore their aberrant level or function is linked with most of the diseases. Controlling these macromolecules using light can therefore be immediately applied towards a range of biomedical fields including bioengineering, biotechnology, biochemistry, medicinal chemistry, diagnostics, therapeutics, and so on. In this dissertation, multiple approaches to create the photoactivable macromolecules and their applications in biomedical field are discussed. In the first part of this dissertation, optimization of light activated RNA interference (LARI), a technique developed earlier in our lab, is discussed. By modifying nucleic acid with various new photolabile groups, RNA interference (RNAi) was brought under the control of light. Using these novel caged macromolecules, gene expression was patterned in cell monolayers, demonstrating the potential of LARI in controlling the spacing, timing and extent of gene expression. In second part of the dissertation, insulin, a protein, was modified with new photolabile groups and then crosslinked to a solid matrix to create an insoluble photoactivable depot (PAD). Insulin with various degrees of photolabile modifications were purified and studied for their photokinetics. Furthermore, using this new approach, the release of protein from a matrix system was precisely controlled using light. This new photoactivable insulin depot could load several weeks worth of insulin in the volume of a single injection. Therefore, this could potentially solve the problem of multiple insulin injections, administered by millions of diabetics everyday. Furthermore, the amount of insulin released from the depot can be tightly controlled using light. In future, this technique can potentially be coupled with non-invasive continuous glucose monitoring devices for automatic detection and control of blood sugar level in patients. Finally, this PAD approach could be applied to other macromolecules, hormones and drugs. Furthermore, we utilized a universal photolabile crosslinking reagent, developed for insulin, for site-specific end labeling of different types of nucleic acids with these groups. Using these groups, we can temporarily attach other molecules like fluorophore, intercalator, drugs, proteins, etc. on to nucleic acids. Finally, these groups can be photocleaved to regenerate the native nucleic acids.Introduction: Light activated RNA interference -- Various approaches to achieve LARI -- Introduction: Insulin photo activated depot -- Construction of insulin photo activated depot -- A universal reversible reagent for site-specific end labeling of nucleic acids -- Summary and conclusions -- Reference