Therapeutic Opportunities and Approaches to Sequence Control for Nucleic Acids

Author: Gethers, Matthew Leroy, III

Year: 2018

Degree: Dissertation (Ph.D.)

Advisors: Goddard, William A., III; Weiss, Paul S.

Committee Members: Tirrell, David A.; Rothemund, Paul W. K.; Goddard, William A., III; Weiss, Paul S.

Option: Bioengineering

DOI: 10.7907/WE1E-EZ49

Abstract

RNA interference (RNAi) is a powerful mechanism to regulate gene expression. A key feature of RNAi is its sequence specificity: a short interfering RNA (siRNA) assembles into the RNA induced silencing complex (RISC) and then targets cellular transcripts complementary to the siRNA for degradation. RNAi has been adapted for therapeutic applications, but is challenged by the need to identify unique target transcripts for each disease that are both effective and result in few off-target effects. This challenge could be eased if siRNAs could be activated only and specifically in diseased cells. If this were the case, rather than targeting a new transcript for each new disease, the same cellular housekeeping genes could be reused. Targeting housekeeping genes would result in greater potency, both effectively treating the disease and requiring less drug for treatment, alleviating problems associated with toxicity and delivery. A new class of nucleic acid therapeutics called conditional siRNAs (Cond-siRNA) is designed to act in this environment-specific manner. The first part of this thesis uses molecular dynamics simulations to understand the structure of Cond-siRNA and to suggest improvements in future designs.

Bioengineering like the work done in the development of Cond-siRNAs depends on the existence of tools that make work simple, fast, cheap, and reproducible. In the case of nucleic acids, de novo synthesis of custom constructs is a fundamental tool. While approaches to synthesis have improved immensely since their inception, increasing ambition demands increasingly powerful tools. As target constructs get longer, the synthesis can become intractably complicated, slowing the process, increasing costs, and making it less likely to be replicated by others. The source of complexity in nucleic acid synthesis is the inability to directly synthesize long fragments without errors. Finding a new means of sequence-controlled synthesis that results in fewer errors and perhaps allows for correction could address this challenge. The second part of this thesis looks at using graphene as a mask for patterning the deposition of molecules on a surface with an eye towards arranging and coupling reactants in a sequence-specific way.

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