In silico design of ligand triggered RNA switches

Abstract

This contribution sketches a work flow to design an RNA switch that is able to adapt two structural conformations in a ligand dependent way. A well characterized RNA aptamer, i. e., knowing its K d and adaptive structural features, is an essential ingredient of the described design process. We exemplify the principles using the well known theophylline aptamer throughout this work. The aptamer in its ligand-binding competent structure represents one structural conformation of the switch while an alternative fold that disrupts the binding competent structure forms the other conformation. To keep it simple we do not incorporate any regulatory mechanism to control transcription or translation. We elucidate a commonly used design process by explicitly dissecting and explaining the necessary steps in detail. We developed a novel objective function which methodically describes the function of this simple, ligand-triggered riboswitch and describe an extensive in silico analysis pipeline to evaluate important kinetic properties of the designed sequences. This protocol and the developed software can be easily extended or adapted to fit novel design scenarios and thus can serve as a template for future needs.

Evolving methods for rational de novo design of functional RNA molecules

“…These goals might be computationally too demanding to be iteratively calculated for each candidate sequence during the optimization. Examples are genome wide off-target searches for designed trans-RNAs or exhaustive kinetic folding experiments [77,57]. By ranking the solutions accordingly, it is possible to emphasize parts of the design objectives and choose candidates which fulfill various goals best, an approach which is similar to solving a posteriori multi-objective problem [78].…”

“…As design problems are usually very diverse, delivering a complete software package with specific design goals, sampling methods and optimization strategies is not very useful for biologically relevant applications. Thus, we think that computational tools for rational de novo design only succeed if they are built as software components which can be flexibly combined to solve a specific design task [51,57]. It should at least be possible to adapt the objectives, constraints and other prerequisites of the tools to real world scenarios in order to serve biologically meaningful applications.…”

“…Recent studies refrain from solving the inverse folding problem as the only design objective and thus relying solely on the close structure-to-function relationship of RNA. They rather specify the characteristics of their desired mechanistic model directly by including sequence-to-function measurements, crucial properties of the used components and the desired functional mechanism [53,54,55,56,57]. These models might still include structural features required at specific states, but should encompass much more than structure prediction at the thermodynamic equilibrium.…”

Efficient computation of co-transcriptional RNA-ligand interaction dynamics

“…The computational design and analysis of riboswitches focuses mainly on thermodynamic aspects of the full length regulatory element. These approaches ensure that the designed RNA element is optimal with respect to a cost function specifying multiple sequence, structure and energetic constraints [13,3]. However, RNA folding starts already during the transcription process under normal cellular conditions.…”

“…Computational predictions yield significant additional mechanistic insight, since the NMR experiment only captures the equilibrium structures for a number of transcriptional intermediates rather than the full co-transcriptional dynamics at the level of single nucleotide chain extension events. This allows not only to understand natural riboswitches on mechanistic level, but also perform systematic in silico screening and analysis of novel designs [3] prior to expensive experimental validation.…”