Electronic supplement to "Computational De Novo Design of Group II Introns Yields Highly Active Ribozymes"

PROJECT DESCRIPTION:Group II introns (G2Is) are large self-splicing ribozymes with emerging potential in biotechnological applications. Despite growing interest, their complexity has so far precluded efforts to design them from scratch. While computational approaches have enabled the design of small RNA catalysts, methods for engineering large ribozymes remain underdeveloped. Here we use the RNA inverse folding algorithm aRNAque to design G2Is de novo, yielding three novel self-splicing ribozymes with unusually stable structures. The designed intron Arq.I2 was revealed to be an unexpectedly proficient ribozyme, self-splicing at a rate comparable to the fastest known G2Is. While most G2Is are believed to be inactive in vivo in the absence of maturase proteins, we show that Arq.I2 self-splices in Escherichia coli cells. Our results demonstrate that highly active variants of large and complex ribozymes can be designed de novo with relative ease using existing inverse folding algorithms, paving the way for the design of bespoke ribozymes derived from G2Is for the development of biotechnological tools.This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Project-ID 364653263 - CRC 235 (H.M.), under Project-ID 521256690 - CRC 392 (H.M)DATASET DESCRIPTION:The dataset contains the sequence library of group II introns, the multi-sequence alignments (MSAs) of group II introns, pairwise alignments of the designed group II introns, as well as the data from the splicing time course kinetics experiments, and the splicing-dependent GFP expression experiments.Supplementary Data 1: Containes thean unaligned fasta file of the curated organellar IIB1 group II introns. Entry names mostly follow the standard group II intron nomenclature of the first 2-3 letters of the genus name, followed by the first 2-3 letters of the specific name, followed in some cases with an abbreviation of their host gene (e.g. "LSU" is the large subunit ribosomal RNA), followed by "I" for intron, and a number denoting the index of the intron, e.g. according to the order they appear in their host gene. The file can be opened with any text editor.Supplementary Data 2: Multiple sequences alignments (MSAs) generated by rMSA and RNA-MSM.zip: Contains fasta files of the MSAs generated by rMSA and RNA-MSM for a query consisting of the first three domains of P.li.LSU.I2. The fasta files can be opened with any text editor.Supplementary Data 3: Contains the python script for calculating the percent sequence identity of pairwise sequence alignments of the designed group II introns to P.li.LSU.I2, and the output fasta file from running the python script. Both files can be read with a text editor. The python script can be run using Python 3.9.5, and Biopython version 1.83.Supplementary Data 4: Contains both the raw gels scan .tif files, which can be opened with Fiji/ImageJ, as well as the extracted and processed datapoints .xlsx file, which can be opened with Excel or libre office. Additionally each folder contains README files explaining the folder contents.Supplementary Data 5: Contains raw images of agar plates scan .tif files, which can be opened with Fiji/ImageJ, as well as extracted and processed data .xlsx files, which can be opened with Excel or libre office. Contains .xlsx of RFU values of PURExpress fluorescence experiments. Contains MS data, which can be opened with software such as ProteoWizard (SeeMS).