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.
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA.
Stephanie L
SL
Moon
Jeffrey
J
Wilusz
D001483:Base Sequence; D005821:Genetic Techniques; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011061:Poly A