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RNAiFold2T: Constraint Programming design of thermo-IRES switches.

Juan Antonio Garcia-Martin1, Ivan Dotu2, Javier Fernandez-Chamorro3

  • 1Biology Department, Boston College, Chestnut Hill, MA 02467, USA.

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Engineered RNA thermometers (RNATs) enable precise gene regulation. A new computational tool, RNAiFold2T, designs functional thermoswitches and thermo-IRES elements, advancing synthetic biology applications.

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Area of Science:

  • Computational Biology
  • Molecular Biology
  • Synthetic Biology

Background:

  • RNA thermometers (RNATs) are temperature-sensitive RNA elements that regulate gene expression.
  • Engineering RNATs is crucial for applications in biosensors and conditional gene regulation.
  • Solving the 2-temperature inverse folding problem is key for RNAT engineering.

Purpose of the Study:

  • To introduce RNAiFold2T, a novel computational tool for solving the 2-temperature inverse folding problem.
  • To enable the rational design of engineered RNA thermoswitches and thermo-IRES elements.

Main Methods:

  • Development of Constraint Programming (CP) and Large Neighborhood Search (LNS) algorithms implemented in RNAiFold2T.
  • Benchmarking RNAiFold2T against existing inverse folding programs.
  • Rational design and experimental validation of two thermosensor internal ribosome entry site (thermo-IRES) elements.

Main Results:

  • RNAiFold2T generates two orders of magnitude more solutions than existing methods, facilitating extensive exploration of RNA sequence space.
  • Two rationally designed thermo-IRES elements demonstrated a ~50% increase in translation efficiency at 42°C compared to 30°C.
  • This represents the first purely computational design of functional RNA thermoswitches and thermo-IRES elements.

Conclusions:

  • RNAiFold2T is a powerful tool for the computational design of functional RNA thermoswitches.
  • Engineered thermo-IRES elements can be effectively designed using computational approaches for temperature-dependent gene regulation.
  • The developed RNAiFold2T software significantly advances the field of synthetic biology and RNA-based genetic control.