Superior 5' homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter

Transcription from the commonly used GTP- initiating T7 class III promoter phi6.5 frequently produces heterogeneous RNA at both 3' and 5' ends. We demonstrate here that RNA transcripts from the T7 class II promoter phi2.5 have superior 5' homogeneity over those from the phi6.5 promoter, with comparable total RNA yields. The overall homogeneity of RNA transcripts is improved to different degrees depending on RNA sequences, although transcription under phi2.5 does not affect the 3' heterogeneity of RNA. In combination with 3' RNA trimming by DNAzymes or ribozymes, this ATP- initiated transcription system based on the T7 phi2.5 promoter can provide excellent quality of RNA for applications requiring a high degree of RNA size homogeneity.