Correction: Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap® Analog by In Vitro Transcription

Henderson, Jordana M.; Ujita, Andrew; Hill, Elizabeth M.; Yousif‐Rosales, Sally; Smith, Cory; Ko, Nicholas; McReynolds, Taylor; Cabral, Charles R.; Escamilla-Powers, Julienne R.; Houston, Michael E.

doi:10.1002/cpz1.336

Cited by 41 publications

(65 citation statements)

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“…CleanCap AG is used for large-scale manufacturing of the BioNTech BNT162b2 SARS-Cov 2 vaccine, demonstrating its scalability and wide utility [35]. We observed ~97% capping for T7-68 reactions using 1mM CleanCap AG, comparable to the efficiency we observed for 4mM CleanCap AG with the WT T7 RNAP, and consistent with the literature value of 94% [18]. The difference between T7-68 and the WT polymerase were even greater when compared in the same reaction buffer, indicating that the improved capping efficiency is an inherent characteristic of T7-68.…”

Section: Discussionsupporting

confidence: 90%

“…Using the recommended conditions for co-transcriptional capping with WT T7 RNA polymerase and CleanCap AG (Buffer C, 4mM CleanCap AG), we observed 97% capping efficiency on the luciferase mRNA (Fig 4B ), consistent with the 94% value reported from the literature [18]. Maximizing capping efficiency to ~95% or greater is important to avoid the RIG-I mediated response to uncapped 5' triphosphate RNA [19].…”

Section: Capping Efficiency With a Trinucleotide Analogsupporting

confidence: 87%

“…CleanCap AG (m7GpppAmG), has several useful features for co-transcriptional capping. It is selectively incorporated over GTP, achieving 94% or higher capping efficiency when used with WT T7 RNAP [18]. The analog also has a 2'-O-methyl modification, yielding a native Cap-1 structure, without the requirement of a separate 2'-O-methyltransferase reaction [5].…”

Section: Discussionmentioning

confidence: 99%

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An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Miller

Alvizo

Chng

et al. 2022

Preprint

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Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5-prime 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

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Section: Discussionsupporting

confidence: 90%

Section: Capping Efficiency With a Trinucleotide Analogsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Miller

Alvizo

Chng

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…The most striking one is the RNA capping strategy: although Pfizer-BioNTech has opted for a cotranscriptional capping using the recently developed CleanCap system, Moderna has adopted an enzymatic capping approach. This last option requires extensive intermediate purification steps, impacting overall recovery and RNA integrity, but yields almost 100% capped RNA, even for hard-to-cap structures [62]. Second, in vitro…”

Section: Knowledge Assessment Of Product-process Interactionsmentioning

confidence: 99%

Quality by Design for enabling RNA platform production processes

Daniel¹,

Kis²,

Kontoravdi³

et al. 2022

Trends in Biotechnology

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“…Currently, the cap structure can be incorporated into an in vitro transcript by two means: 1) incorporation of a chemically synthesized cap structure such as the anti-reverse cap analog (ARCA) or capped dinucleotides (Henderson et al 2021) during transcription, or 2) post-transcriptional capping using a mRNA capping enzyme. These approaches have well-established methods that are readily adaptable and scalable (Fuchs et al 2016; Vaidyanathan et al 2018).…”

Section: Introductionmentioning

confidence: 99%

RNase H-based analysis of synthetic mRNA 5’ cap incorporation

Chan

Whipple

Dai

et al. 2022

Preprint

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Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5′ cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5′ capping is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5′ end of the kilobase-long synthetic mRNA molecules using RNase H or a DNAzyme, 5′ cleavage products enrichment, and LC-MS intact mass analysis. In this communication, we describe 1) our results on the sequence preference of RNase H cleavage and a framework to design site-specific RNA cleavage; 2) a method to fluorescently label the RNase H cleavage fragments for readouts such as easily accessible urea-PAGE or high throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5′ cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

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Correction: Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap® Analog by In Vitro Transcription

Cited by 41 publications

References 0 publications

An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Quality by Design for enabling RNA platform production processes

RNase H-based analysis of synthetic mRNA 5’ cap incorporation

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