Agents and Methods for Modifying the 5' Cap of RNA

Simple SummaryContent extracted from patent full text and abstract with AI.

This invention provides novel enzymes (specifically, modified versions of the Giardia lamblia trimethylguanosine synthase 2) and methods to selectively modify the 5' cap structure of RNA molecules. By mutating the enzyme at a specific amino acid position, the enzyme is enabled to use synthetic S-adenosylmethionine (AdoMet) analogs as cofactors. These modified enzymes can then transfer various chemical groups to the N2 position of the guanosine in the RNA cap, allowing subsequent chemical modifications such as labeling or immobilization of RNAs. This enables selective tagging, isolation, and study of capped RNAs such as mRNA and specific types of non-coding RNA.

Use CasesContent extracted from patent full text and abstract with AI.

• Selective labeling and tracking of capped messenger RNAs (mRNAs) in biological samples for transcriptomics or gene expression analysis.
• Highly specific isolation of mRNAs (including those with short or no poly(A) tails) from biological mixtures for downstream applications like sequencing, array analysis, or biomarker discovery.
• Development of advanced molecular biology kits for RNA purification based on cap structure modifications, enabling more stringent washing and higher purity RNAs.
• Site-specific attachment of chemical groups, fluorophores, or affinity tags (e.g., biotin) to mRNAs for imaging, detection, pull-down assays, or live-cell studies.
• Functional studies requiring manipulation or immobilization of specific RNA species (e.g., for drug screening, RNA-protein interaction assays, or structural biology).
• Novel biosensors or diagnostic assays utilizing covalent RNA modification at the 5' cap for enhanced specificity.

BenefitsContent extracted from patent full text and abstract with AI.

• Enables direct and highly specific chemical modification of the RNA 5' cap, independent of the poly(A) tail.
• Allows the use of diverse chemical cofactors, vastly expanding the types of groups that can be transferred to RNA and downstream modification strategies.
• Improves selectivity and robustness of RNA isolation by enabling covalent, stable attachment to surfaces or beads, allowing for more stringent purification protocols.
• Supports a wide range of downstream applications such as fluorescent labeling, affinity capture, or chemical functionalization of RNAs.
• Applicable to a broad spectrum of capped RNA molecules, including mRNAs and important non-coding RNAs, facilitating more comprehensive transcriptome analyses.
• The mutated enzymes retain or improve upon the activity and stability of wild-type enzymes, making them practical for laboratory and commercial use.

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Patent Abstract

The invention provides an agent and method for modifying the 5' cap of RNA, for example for the purposes of isolation and analysis. According to one aspect the invention provides modified enzymes, namely modified trimethylguanosine synthases 2 from Giardia lamblia (GlaTGS2), the enzymatic activity of which is changed such that as compared to wild type enzymes the former can use AdoMet analogues better as cofactors.