The DuraScribe® T7 Transcription Kit* is an in vitro transcription kit that produces RNA - called DuraScript® RNA - that is completely resistant to RNase A digestion.
The DuraScribe T7 Transcription Kit includes DuraScribe T7 RNA Polymerase, an enhanced formulation of the Y639F mutant1 of T7 RNA Polymerase that efficiently incorporates 2´-Fluorine-CTP (2´-F-dCTP), 2´-Fluorine-UTP (2´-F-dUTP), ATP and GTP into RNase-resistant RNA transcripts called DuraScript RNA (Figure 1). The DuraScribe T7 RNA Polymerase uses the same T7 promoters as the wild-type T7 RNA Polymerase.
DuraScript RNA has demonstrated advantages for RNA aptamer synthesis, and ribozyme, RNA interference (RNAi) and antisense RNA studies.
Table 1. Yield of DuraScript® RNA from a DuraScribe® Kit reaction. One microgram of a 3-Kb DNA template was linearized at different sites and then transcribed in a DuraScribe T7 Transcription Kit reaction for 4 hours. The yield of DuraScript RNA produced from each template is shown in micrograms (µg) and in picomoles (pmol).
|Size of DuraScript RNA produced||DuraScript RNA Yield (µg)||DuraScript RNA Yield (pmol)|
|2600 nts||100 µg||116 pmol|
|1400 nts||58 µg||124 pmol|
|330 nts||18 µg||164 pmol|
|88 nts||9 µg||307 pmol|
Figure 1 (click to enlarge). The DuraScribe® T7 RNA Polymerase efficiently incorporates 2´-F-dCTP and 2´-F-dUTP into full-length DuraScript® RNA. The presence of the fluorine at the 2´-position of the 2´-F-dC and 2´-F-dU nucleotides prevents digestion by Rnase A.
Figure 2. Yield of RNA from a DuraScribe® T7 Transcription reaction. A standard reaction (4-6 hours) produced 40-60 µg of a 1.4-kb DuraScript® RNA.
Figure 3. DuraScript® RNA is resistant to Rnase A digestion. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were each incubated with 1 U of highly purified Rnase A for 30 minutes. The standard RNA transcript was completely degraded while the DuraScript RNA transcript remained intact. Lane M, size ladder; lane 1, 1.4-kb standard RNA transcript; lane 2, standard RNA after Rnase A treatment; lane 3, 1.4-kb DuraScript RNA; lane 4, DuraScript RNA after Rnase A treatment. Denaturing 1% agarose gel2.
Figure 4. DuraScript® RNA is completely resistant to "finger" nucleases. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were produced using sterile water or water that had been contaminated by exposure to the hands of a test subject. The standard RNA shows extensive degradation from "finger" nucleases in the contaminated water while the DuraScript RNA remains fully intact. M, Size ladder; Lane 1, Standard RNA transcript; Lane 2, Standard RNA after "finger" nuclease exposure; Lane 3, DuraScript RNA; Lane 4, DuraScript RNA after "finger" nuclease exposure. Denaturing 1% agarose gel2.
* Covered by issued and/or pending patents.