This invention describes an optimized, constitutive active decapping complex from S. pombe for efficient transcription start site (TSS) mapping.
Changes in 5’ untranslated regions (UTRs) can have profound effects on a gene’s translational efficiency, resulting in both qualitative and quantitative differences in expressed proteins. Therefore, mapping the 5’ UTRs in organisms ranging from single cell organisms to humans has rewritten our understanding of genetic plasticity and its impact on various human diseases including cancer.
RNA-seq methods are constantly being innovated to reproducibly map TSS. Most of these methods rely on the enzymatic removal of m7G cap structure and subsequent ligation of an adaptor sequence to the reactive 5’ monophosphate. Though bacterial RNA 5’ Pyrophosphohydrolase (RppH) is currently being used for this reaction, its specific activity is directed towards triphosphorylated, uncapped bacterial RNAs, and not eukayotic 5’ capped RNAs.
The decapping complex discovered by scientists at UCSF has the following advantages:
This invention describes an IPTG inducible single plasmid expression system designed to purify a constitutively active S. pombe decapping complex using standard production and purification methodologies. The expressed active decapping enzyme is catalytically more efficient (Vmax/Km) than RppH in multiple turnover conditions for 5’ m7G cap removal from eukaryotic RNA. The end product suitability for downstream cloning applications has been verified using splinted-ligation procedure. Therefore, this active “super” decapping complex can be used for both small-scale and large-scale sequencing methods for efficient TSS mapping.
To develop & commercialize the technology as a specific and highly efficient eukaryotic alternative to commercially available bacterial decapping enzymes.
Proof of Concept
Under a CDA/ NDA