1. Introduction of an autocatalytic hammerhead ribozyme sequence permits use of the optimal T7 promoter to enhance transcription levels.
Introduction of an autocatalytic hammerhead ribozyme sequence permits use of the optimal T7 promoter to enhance transcription levels. (A) Sequence of the Hh-Rbz (blue text) and its context within the plasmid carrying the viral antigenome (red text). The variable region is the reverse complement of the start of the antigenome, while the constant region is fixed, regardless of the sequence context. The sequence of the minimal and optimal T7 promoter is shown in black text. Dashed lines indicate the approximate primer binding regions of the qPCR primers used in panels B and C. (B) Transcription levels measured by qPCR were normalized to the minimal T7 promoter (no Hh-Rbz) condition. Bars represent the means of four replicates, and error bars indicate 1 standard deviation. Significance was evaluated by using a two-tailed unpaired t test. ****, P < (C) Quantification of cleavage efficiency measured by qPCR. Bars represent the means of four replicates, and error bars indicate 1 standard deviation.
Shannon M. Beaty et al. mSphere 2017; doi: /mSphere