ArticleThe Journal of biological chemistry
Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter.
Article in The Journal of biological chemistry. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed.
- Efficient production of a self-amplifying RNA vaccine sequence with the single subunit RNA polymerase fromRNA (New York, N.Y.) · 2026Article
- Fluorogenic RNA Aptamer-Based Activity Quantification of T7 RNA Polymerase.Biochemistry · 2026Article
- Fluorescence-Activated Cell Sorting Enables Rapid Screening of RNA Polymerase-Promoter Interaction in E. coli.Biochemistry · 2026Article
- A Modular Mechanistic In Silico Model for In Vitro Transcription Process Yield and Product Quality Prediction.Biotechnology and bioengineering · 2026Article
- High-throughput monitoring of free MgAnalytical and bioanalytical chemistry · 2026Article
- Active-learning-guided optimization of cell-free systems for genome-wide transcriptomic profiling reveals progressive layers of regulation.Nature communications · 2026Article
- Engineered T7 RNA polymerase to improve mRNA capping efficiency and reduce dsRNA generation duringMolecular therapy. Advances · 2026Article
- Employing molecular beacons to assess in vitro transcription with single-molecule resolution.Scientific reports · 2026Article
- Macromolecular crowding alters transcription: real-time measurements with SYBR Green II.Research square · 2026Article
- A nanopore-based HIV-1 reference epitranscriptome.Nucleic acids research · 2026Article
- Engineering Allosteric Transcription Factor-Based Biosensors: Advances and Prospects for Modern Food Contaminant Monitoring.Foods (Basel, Switzerland) · 2026Review
- Mechanism and reconstitution of circadian transcription in cyanobacteria.Nature structural & molecular biology · 2026Article
- Directed Evolution of T7 RNA Polymerase Minimizes dsRNA By-product and Enables High-Fidelity mRNA Synthesis for Demanding Therapeutic Applications.Research (Washington, D.C.) · 2026Article
- Heterogeneous template-dependent transcription dynamics of T7 RNAP revealed by single-molecule imaging.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Engineered chimeric T7 RNA polymerase improves salt tolerance and reduces dsRNA impurity generation during in vitro transcription of mRNA.Nucleic acids research · 2025Article
- Challenges in observing transcription-translation for bottom-up synthetic biology.QRB discovery · 2025Review
- Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.Chemical science · 2024Article
- Rapid protein evolution by few-shot learning with a protein language model.bioRxiv : the preprint server for biology · 2024Article
- Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.RNA (New York, N.Y.) · 2024Article
- Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA.ACS omega · 2024Article
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the "Broccoli" RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.