Analogues of the eukaryotic messenger RNA 5′ end (m⁷G cap) are useful tools for studying mRNA fate and serve as reagents for in vitro preparation of 5′ capped mRNAs. We designed a biotin-labeled dinucleotide cap analogue that can be incorporated into transcripts to produce 5′-capped and biotinylated mRNAs which retain their biological functionality and may be employed for biotin–(strept)avidin technologies.