The present invention relates to kits and methods for efficiently generating 5′ capped
RNA having a modified cap
nucleotide and for use of such modified-
nucleotide-capped
RNA molecules. The invention is used to obtain novel compositions of such modified-
nucleotide-capped
RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping
enzyme system, such as poxvirus capping
enzyme. The present invention finds use for
in vitro production of 5′-capped RNA having a modified cap nucleotide and for
in vitro or
in vivo production of polypeptides by
in vitro or
in vivo translation of such modified-nucleotide-capped RNA for a variety of research, therapeutic, and commercial applications. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, such as RNA synthesized in vitro or obtained from a biological source, including prokaryotic mRNA that is in a mixture with other prokaryotic and / or eukaryotic nucleic acids. The method for capturing modified-nucleotide-capped RNA also provides methods and kits for obtaining only type-specific or condition-specific modified-nucleotide-capped RNA by cap-dependent subtraction of that portion of the captured modified-nucleotide-capped RNA in cells of one type or condition that is the same as RNA in cells of another type or condition. The invention further provides methods and kits for using a capping
enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate with detectable dye or enzyme moieties.