Kool™ NC-45™ Universal RNA Polymerase Template
Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit

Applications

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

Kool™ Universal RNA Polymerase Templates are small (28- to 150-nucleotide) circular 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 (RNAP) by a rolling-circle mechanism.1-3 Rolling-circle transcription from nanocircle templates does not require canonical promoter sequences or a primer. Bacterial RNAPs transcribe Kool templates in the absence of sigma factors, permitting studies of RNA polymerization mechanisms and inhibitor effects on the core RNAP.

The Kool NC-45™ RNAP Activity & Inhibitor Screening Kit* uses the Kool NC-45 Template (a 45-nucleotide circular ssDNA) to screen inhibitors of rolling-circle transcription by bacterial polymerases.1,2 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 (Fig. 1). End-point or real-time monitoring is possible with fluorescent dyes or with molecular beacons.3 The Kool NC-45 Screening Kit uses a dye that binds the RNA for real-time detection of RNA 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 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. A fluorescent dye was added and 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, and partial inhibition by Tagetin™ Inhibitor can be detected, while α-amanitin, an inhibitor of eukaryotic RNA Polymerase II, has no effect.

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 postprocessing.
  • Easy to multiplex and automate for high-throughput screening of RNAP enzymes or RNAP inhibitors.

References

  1. Daubendiek S.L. and Kool, E.T. (1997) Nature Biotechnol. 15, 273.
  2. Ohmichi, T. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 54.
  3. Marras, S.A. et al. (2004) Nucl. Acids Res. 32, e72.

*Covered by issued and/or pending patents.

 Figure 2. Rolling circle transcription of Kool™ Nanocircle-45™ Template. 1.5 pmole of Kool™ Nanocircle-45 Template was incubated for 1 hour at 37°C in 1x Reaction Buffer containing 0.5 mM NTP & the following RNAP: 1: E. coli RNAP Holoenzyme; 2: E. coli RNAP Core Enzyme; 3: S. aureus RNAP; 4: Thermus RNAP; 5: T7 RNAP; 6: T3 RNAP; 7: SP6 RNAP; 8: MiniV™ RNAP. Agarose gel was stained with SYBR® Gold Dye (Molecular Probes).