最新产品
Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit
Kool™ NC-45™ RNAP Activity & Inhibitor Screening Kit
货号
KNK49025
规格
25 Rxns
存储温度
-20℃
产品详情
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.
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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. |
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Benefits
References
*Covered by issued and/or pending patents. |
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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). |
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