Assessing the Quality of Cotranscriptional Folding Simulations

Abstract

Structural changes in RNAs are an important contributor to controlling gene expression not only at the post-transcriptional stage but also during transcription. A subclass of riboswitches and RNA thermometers located in the 5' region of the primary transcript regulates the downstream functional unit -usually an ORF -through premature termination of transcription. Such elements not only occur naturally but they are also attractive devices in synthetic biology. The possibility to design such riboswitches or RNA thermometers is thus of considerable practical interest. Since these functional RNA elements act already during transcription, it is important to model and understand the dynamics of folding and, in particular, the formation of intermediate structures concurrently with transcription. Cotranscriptional folding simulations are therefore an important step to verify the functionality of design constructs before conducting expensive and labour-intensive wet lab experiments. For RNAs, full-fledged molecular dynamics simulations are far beyond practical reach both because of the size of the molecules and the time scales of interest. Even at the simplified level of secondary structures further approximations are necessary. The BarMap approach is based on representing the secondary structure landscape

Beyond Plug and Pray: Context Sensitivity andin silicoDesign of Artificial Neomycin Riboswitches

“…This allows to efficiently simulate the folding dynamics. To determine the energy thresholds for the enumeration of structures with RNAsubopt in the individual landscapes, we used the quality scores for both the enumeration and the simulation provided by BarMap-QA [36]. We set a simulation stop time of 1 000 au (arbitrary time units) for BarMap.…”

“…Candidate riboswitches were screened to satisfy the following criteria: (i) the leader sequence does not interfere with the formation of the aptamer's binding pocket, (ii) the binding pocket is significantly populated during the transcription of the spacer and the 5' part of the terminator, and (iii) the terminator hairpin dominates the structure ensemble as soon as it is fully transcribed. For a precise description of the preparation steps, quality metrics, and the post-processing, we refer to [36]. To allow for easy reproduction, the entire simulation pipeline was packaged into a self-contained and publicly available Docker image.…”