# Evolving methods for rational de novo design of functional RNA molecules

## TL;DR

This review discusses computational methods for the de novo design of functional RNA molecules, emphasizing the process from biological target analysis to experimental validation, and highlights open questions in the field.

## Contribution

It provides a comprehensive dissection of existing computational approaches for RNA design and identifies open questions for each step of the design process.

## Key findings

- Key steps include target analysis, component selection, constrained sampling, optimization, and in silico analysis.
- Experimental validation is crucial for confirming design objectives and refining models.
- Open questions remain in each step of the computational design process.

## Abstract

Artificial RNA molecules with novel functionality have many applications in synthetic biology, pharmacy and white biotechnology. The de novo design of such devices using computational methods and prediction tools is a resource-efficient alternative to experimental screening and selection pipelines. In this review, we describe methods common to many such computational approaches, thoroughly dissect these methods and highlight open questions for the individual steps. Initially, it is essential to investigate the biological target system, the regulatory mechanism that will be exploited, as well as the desired components in order to define design objectives. Subsequent computational design is needed to combine the selected components and to obtain novel functionality. This process can usually be split into constrained sequence sampling, the formulation of an optimization problem and an in silico analysis to narrow down the number of candidates with respect to secondary goals. Finally, experimental analysis is important to check whether the defined design objectives are indeed met in the target environment and detailed characterization experiments should be performed to improve the mechanistic models and detect missing design requirements.