An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry.
We welcome your comments at validation@mail.wwpdb.org 1 Overall quality at a glance i O The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 2.50 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based.

Metric
Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) O There are 6 unique types of molecules in this entry. The entry contains 7722 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.

Mol Chain Residues
Atoms ZeroOcc AltConf Trace  Full wwPDB X-ray Structure Validation Report 2PI4 3 Residue-property plots i O These plots are drawn for all protein, RNA and DNA chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.
The second graphic shows the sequence view annotated by issues in geometry and electron density.
Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.
All (873)  There are no ring outliers.
No monomer is involved in short contacts.
The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included. For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identied as an outlier by Mogul will be highlighted in the graph. For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identied by Mogul is calculated over all ring torsion angles. If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identied rings is also greater than 60 degrees, then that ring is considered an outlier.
The outliers are highlighted in purple. The color gray indicates Mogul did not nd sucient equivalents in the CSD to analyse the geometry. of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled`Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.
Mol Chain Analysed <RSRZ> #RSRZ>2 OWAB(Å 2 ) Q<0.9 The following is a graphical depiction of the model t to experimental electron density of all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included. Each t is shown from dierent orientation to approximate a three-dimensional view.
Electron density around GH3 A 902: