eSENSE dsRNA reproducibly quantifies dsRNA contamination and provides critical information for sequence- and structure-based optimization to reduce impurities.
Contact useSENSE dsRNA provides actionable insights into the levels and sources of dsRNA by-products.
During in vitro transcription, abortive transcription and polymerase extension can lead to the generation of double-stranded RNA (dsRNA) contaminants. dsRNA in the final drug product can trigger innate immune responses and reduce translation, limiting therapeutic efficacy. Current methods for dsRNA detection have challenges with reproducibility and provide limited information for optimization. eSENSE dsRNA addresses these challenges by utilizing next-generation sequencing to reveal both the amount and the source of dsRNA contamination.
Contact usOverview
Use:
Quantify dsRNA, optimize RNA sequences
Typical species:
Viral, human
Typical samples:
IVT RNA
Measure dsRNA, reduce impurities
Ready to start your eSENSE dsRNA project? Contact us today.
Contact usPrepare libraries
Sequencing libraries are generated containing dsRNA contaminants
Sequence
Next-generation sequencing is performed
Analyze
Custom bioinformatics analyses determine the amount and source of dsRNA
dsRNA quantification and optimization
The following are some examples of the types of insights that eSENSE dsRNA can provide. eSENSE dsRNA is an exclusive assay within our eMERGE platform for RNA therapeutic characterization and optimization. Different analyses can be performed based on our partners’ needs.
Reproducibly measure dsRNA contamination
Antibody-based dot blots and ELISAs are typically used to quantify dsRNA but struggle with reproducibly issues and cannot measure short stretches of dsRNA that can trigger innate immune responses. eSENSE dsRNA quantification is highly correlated with antibody-based methods, while also capturing short stretches of dsRNA.
Quantification of dsRNA content in an RNA transcribed with unmodified bases or with N1-methylpseudouridine. Incorporation of base modifications reduces dsRNA content.
Identify where IVT runoff begins and ends
J2- and K1-based assays only identify that dsRNA is present. They don’t reveal where the polymerase runoff begins and ends, limiting their utility during sequence and manufacturing optimization. Through next-generation sequencing, eSENSE dsRNA reveals where dsRNA is located along the therapeutic without sequence bias.
The bottom line plot shows the amount of dsRNA at each base position along an RNA. The two sources of the dsRNA shown in the gray box are indicated by the red lines. The thickness and color of the line indicates the relative contribution of each base position to the generation of the dsRNA by-product.
Optimize sequences and structures to reduce dsRNA
Polymerase runoff can occur due to specific sequences and RNA secondary structures. We pair eSENSE dsRNA with our eSHAPE assay for structure profiling to identify specific structural features that contribute to dsRNA generation, providing actionable insights for RNA optimization.
eSHAPE was used to determine the secondary structure of an in vitro transcribed RNA. The bases highlighted in red interact, allowing the T7 polymerase extension to begin on the left and continue to the loop on the right.