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.
*Covered by issued and/or pending patents.