Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit

Kool™ NC-45™ Universal RNA Polymerase Template

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Last modified: 2008-07-15 14:57:15

Kool™ RNA Polymerase Nanocircle Templates are small (28-150- nucleotide) circular single-stranded DNA (ssDNA) molecules. As observed in the laboratory of Dr. Eric Kool, these DNA nanocircles can be efficiently transcribed in vitro by a variety of DNA-dependent RNA polymerases (RNAPs) by rolling circle mechanism. Rolling circle transcription from nanocircle templates does not require canonical promoter sequences or primer. Bacterial RNAPs transcribe Kool™ templates in the absence of sigma or other transcription factors, permitting studies of RNA polymerization mechanisms and inhibitor effects on the core RNAP.

The Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit utilizes the Kool™ NC-45™ Template to screen inhibitors of rolling circle transcription by bacterial polymerases. Compounds can be screened for inhibition of E. coli RNAP or inhibitors can be assayed on an RNAP provided by the user. The high product-to-template ratio of rolling circle transcription allows a variety of detection methods in addition to following incorporation of radioactive nucleotides. End-point or real-time monitoring is possible with fluorescent dyes or with molecular beacons. The Kool™ NC-45™ Screening Kit uses SYBR® Green I Dye (Molecular Probes) for real-time detection of RNAP activity.

Figure 1. Real-Time Detection of Rolling Circle Transcription by E. coli RNAP in the Presence and Absence of Inhibitors. Two picomoles of Kool™ NC-45™ Template (3) was incubated at 37°C with 0.5 unit E.coli RNAP Core Enzyme, 0.5 mM NTP, and the inhibitors indicated above in 1X Reaction Buffer. SYBR® Green I dye (Molecular Probes) was added in a 1:15,000 final dilution. Reactions were monitored in an iCycler iQ™ Real-Time PCR Detection System (Bio-Rad). Strong inhibitions by Rifampicin, a known inhibitor of bacterial RNA Polymerase,4 and partial inhibition by Tagetin™5 can be detected, while α-amanitin, an inhibitor of eukaryotic RNA Polymerase II, has no effect.

Last modified: 2008-05-14 16:46:46

Applications

• Assay for DNA-dependent RNAP activity.
• Screen compounds for RNAP inhibitor activity.

Benefits

• A rapid, simple and sensitive method to assay RNAPs.
• RNAP activity can be assayed without a promoter sequence, or knowledge of the promoter sequence.
• Very high product-to-template ratio.
• Provides a rapid and simple method for assaying RNAP activity in real time without post-processing.
• Easy to multiplex and automate for high-throughput screening of RNAP enzymes or RNAP inhibitors.

Last modified: 2008-05-14 16:46:46

Protocols for: Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit

(catalogue number KNK49025 / KN411100)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46