In Vitro Transcription of Modified RNAs

RNAs containing a variety of terminal and internal modifications can be produced using bacteriophage polymerases often with a few simple adjustments to standard transcription protocols. RNAs containing a single phosphate or a cap structure at their 5′ ends can readily be generated either co-transcriptionally or through enzymatic treatments of transcription products. Likewise, a variety of modified bases, including fluorescent or biotinylated species, can be effectively incorporated co-transcriptionally. The key to effective co-transcriptional incorporation lies in determining the efficiency of incorporation of modified base relative to its standard counterpart. Finally, an approach to place a poly(A) tail at the exact 3′ end of a desired transcription product is presented. Collectively, these protocols allow one to synthesize RNAs with a variety of modifications to serve as versatile molecules to analyze biological questions.