Rapid Automated Optimization of Riboswitch Function for Synthetic Biology

Open Access
Huso, Walker D
Area of Honors:
Chemical Engineering
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Howard M Salis, Thesis Supervisor
  • Ali Borhan, Honors Advisor
  • Riboswitch
  • riboswitches
  • aptamer
  • aptamers
  • DNT
  • dinitrotoluene
  • TNT
  • trinitrotoluene
  • in vivo
  • sensor
  • automated
  • biophysical
Riboswitches are ligand dependent regulatory RNA’s that can sense a specific ligand in the cellular environment and mediate a specific gene expression response to the detected ligand. Although significant progress has been made in identifying a variety of riboswitches that are directly coupled to activation, it is not clear how riboswitch sequences near the ligand binding site control the physics of riboswitch switching and translational activation. A better understanding of the biophysical principles governing switching would support the rational design of novel riboswitches for synthetic biology and could have transformative applications in remediating the environment, providing new medical therapies and diagnostics, or in developing new sensitive biosensors. We developed a biophysical model and an automated design method to convert aptamers into functional riboswitches. To validate our approach, we utilized 7 different aptamers to design and characterize 62 novel synthetic riboswitches with highly divergent sequences and structures. We validated predicted function with actual function. As part of this effort, we describe the design and characterization of riboswitches with specificity for dinitrotoluene (DNT). We compared the effects of sequence optimization before and after the aptamer domain for the DNT ligand and characterized the activation ratios of DNT riboswitch variants using a red fluorescent protein reporter. By comparing activation of DNT riboswitch variants and the activation of other riboswitch variants, a biophysical model was validated that explains the basis for novel riboswitch functional optimization. Finally, a predictive online tool was created that can rapidly optimize sequences of any individual riboswitch and can automatically incorporate functional activity considerations into riboswitch design.