PROGRAM | Chemical Engineering

By: Daniel Blackstock Chair: Wilfred Chen

ABSTRACT

Cellular systems have evolved a wide range of natural tools to receive specific inputs/signals and translate them into specified output responses (cellular functions). These tools are naturally occurring biological sensors and switches and they play a vital role in everyday cell survival and growth. By discovering and comprehending these natural tools, their performance principles can be translated into the creation of artificial sensors and switches. Inspired by the remarkable and extensive properties of biological machineries, we engineered unique sensors and switches with extensive functionality. We combined the properties of proteins and nucleic acids, producing protein-DNA conjugates, to create nucleic acid input mediated sensors and switches with protein activity outputs.

In objective one, we focused on remodeling a widely accepted nucleic acid based sensor, molecular beacons (MBs), for simplified construction. Centering our attention on the construction steps contributing the most to the creation time and costs, we engineered an alternative labeling method for simplified assembly. We replaced the synthetic dye and quencher and the chemical modification steps that are required for attachment with self-labeling (zinc finger)-fluorescent proteins (FPs) for MB assembly upon simple component mixing. The novel FP-MB design proved functional and tremendously simplified the MB production process for high throughput opportunities.

Our second objective was to improve upon the FP-MB design for increased specificity, sensitivity, and stability. To achieve improved specificity, we introduced a dual FP-MB system in which both FP-MBs must bind the same target for signaling to occur. Additionally, to achieve the stability and sensitivity goal, we replaced the affinity based labeling (zinc finger domains) with a mutant dehalogenase enzyme (HaloTag) for covalent attachment. This alteration resulted in a system with higher stability and lower background signaling for improved sensitivity (>20 fold enhancement).

Our final objective was to further utilize the combined powers of proteins and nucleic acids to create dynamic protein organization based on nucleic acid inputs. To achieve this goal, we applied the strand displacement and hybridization properties of DNA to actively control the spatial arrangement of proteins. We used the HaloTag-DNA linkage technology obtained in objective two for the creation of additional fluorescent protein-DNA conjugates. Using fluorescence resonance energy transfer (FRET) between FPs, we confirmed that strand displacement properties of DNA are conserved and can be exploited even when conjugated to proteins. Controlled protein assembly was executed for various design strategies including: single input, multi-input, reversible, and catalytic systems. The FRET based dynamic protein-DNA technology employed served as an excellent tool for modeling the behavior of protein assemblies controlled by nucleic acid circuits. This dynamic protein organization is the first of its kind and provides extensive opportunities for controlled regulation of protein co-localization outputs. Potentially, variations of the technology could be applied in cellular systems to create gene expression regulated (using RNA transcripts) enzymatic assemblies for cellular function programming.

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