Protocol

Recombinant and In Vitro RNA Synthesis

Volume 941 of the series Methods in Molecular Biology pp 171-180

Date:

In Vitro Transcription of Modified RNAs

  • Stephanie L. MoonAffiliated withDepartment of Microbiology, Immunology and Pathology, Colorado State University
  • , Jeffrey WiluszAffiliated withDepartment of Microbiology, Immunology and Pathology, Colorado State University Email author 

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Abstract

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.

Key words

Transcription Capping 5′ Monophosphate Fluorescent nucleotides Poly(A) tail Modified bases