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Maximizing transcription of nucleic acids with efficient T7 promoters.

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.

SUBMITTER: Conrad T

PROVIDER: S-EPMC7429497 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Maximizing transcription of nucleic acids with efficient T7 promoters.

Conrad Thomas T Plumbom Izabela I Alcobendas Maria M Vidal Ramon R Sauer Sascha S

Communications biology 20200814 1

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 transcr ...[more]

PMID: 32796901

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