High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

Abstract

In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs. Graphical Abstract

High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates forMycobacterium tuberculosisRNA polymerase

“…Using sequence to predict transcriptional activity remains a challenge, as the context of the entire promoter must be considered when examining how sequence affects initiation kinetics (3). Using the aptamer-based assay, one could generate promoter libraries where sequence context is investigated by introducing sequence mutations in promoter regions of interest (41, 74, 80). Alternatively, one could design and measure basal and regulated transcription rates on large numbers of genomic promoter sequences.…”

“…Importantly, as the change in dye fluorescence requires the synthesis of a full-length RNA transcript containing the aptamer, the fluorescent readout is not complicated from short abortive products that may be generated during promoter escape. Using this approach, recent work has illustrated how the rates of in vitro reactions can be quantified in a fluorimeter cuvette with E. coli RNAP (40) and in a plate-reader format with T7 RNAP (41). Here, we follow up on these studies and provide a description of essential control experiments needed to clearly link the fluorescent signal with the transcription of a promoter-derived product.…”