Fast and Modular Biomolecular Circuits Using Spatial Organization

"Biological information processing hubs" ranging from brain to cells to enzyme cascades extensively use spatial organization to process massively parallel molecular instructions and accurately respond to external and internal stimuli. Similarly, human-engineered systems ranging from ancient irrigation networks to modern semiconductor circuits have extensively used spatial organization to steer flux along intended pathways and minimize undesired interactions. In the quest of building artificial nanosystems driven by the fundamental concepts of natural design, DNA has emerged as an incredibly powerful biomaterial due to its biocompatibility, polymeric mechanical properties and predictable sequence programmability. DNA-based nanostructures can potentially detect intracellular or extracellular components like RNAs, proteins or small biological molecules and activate specific bioprocesses (e.g. RNA interference) upon successful detection. Furthermore, these nanodevices can perform molecular computation using one or more intracellular components as input signals and generate an output signal (e.g. a readout or bioprocess activation). During my Ph.D. I have been working on developing a novel class of such DNA-based molecular circuits for molecular computation and molecular imaging using spatial organization as the fundamental design principle.;In my thesis, I start with presenting a brief overview and key advancements in the field of DNA Nanotechnology (Chapter 1). Here, I have also presented different DNA-based nanosystems that use spatial organization.;For the major part of my Ph.D. I have worked on the design and development of an integrated architecture for building fast and modular molecular circuits on a DNA origami scaffold using spatial localization as a basic design principle (Chapter 2). These spatially-localized DNA-based circuits exhibit significantly faster circuit operation and extensive reuse of circuit components with minimal interference due to spatial localization of circuit components. These nanoscale circuit boards can potentially be used for multiplexed molecular sensing in living cells.;The fundamental challenges for detecting intracellular components in living cells with nucleic acid nanodevices include their in vivo stability, efficiency, and control over intracellular localization.We have worked on these challenges and used nucleic acid strand displacement probes to detect intracellular mRNAs in living mammalian cells (Chapter 3). Using the categorically redesigned probes, we performed mRNA quantification and real-time mRNA visualization studies. The findings from this study will act as fundamental guidelines for building complex DNA-based molecular systems which can operate with intracellular components in living mammalian cells.