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Maximizing transcription of nucleic acids with efficient T7 promoters

Authors

  • T. Conrad
  • I. Plumbom
  • M. Alcobendas
  • R. Vidal
  • S. Sauer

Journal

  • Communications Biology

Citation

  • Commun Biol 3 (1): 439

Abstract

  • In vitro transcription using T7 bacteriophage polymerase is widely used in molecular biology. Here, we use 5'RACE-Seq to screen a randomized initially transcribed region of the T7 promoter for cross-talk with transcriptional activity. We reveal that sequences from position +4 to +8 downstream of the transcription start site affect T7 promoter activity over a 5-fold range, and identify promoter variants with significantly enhanced transcriptional output that increase the yield of in vitro transcription reactions across a wide range of template concentrations. We furthermore introduce CEL-Seq(+) , which uses an optimized T7 promoter to amplify cDNA for single-cell RNA-Sequencing. CEL-Seq(+) facilitates scRNA-Seq library preparation, and substantially increases library complexity and the number of expressed genes detected per cell, highlighting a particular value of optimized T7 promoters in bioanalytical applications.


DOI

doi:10.1038/s42003-020-01167-x