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 mutant ¹ 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.

Applications

DuraScript RNA has demonstrated advantages for RNA aptamer synthesis, and ribozyme, RNA interference (RNAi) and antisense RNA studies.

Features

• DuraScript RNA is resistant to RNase A and DNase
• High Yields of DuraScript RNA
• T7 in vitro transcription reactions. DuraScribe T7 RNA Polymerase uses the same T7 promoters as the wild type T7 RNA Polymerase.
• Ideal for RNA aptamer studies. Numerous citations using the DuraScribe Kit with RNA aptamers
• SELEX-compatible. DuraScript RNA can be used in SELEX selection processes.
• Make long or short DuraScript RNA. Double-strand oligos, linearized plasmids, and PCR products with a T7 polymerase promoter can be transcribed.

Table 1. Yield of DuraScript RNA from a DuraScribe Kit reaction. One microgram of a 3-Kb DNA template was linearised 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 gel ².

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 gel ².

* Covered by issued and/or pending patents.

References

1. Sousa, R. and Padilla, R. (1995) EMBOJ. 14:18 4609-4621.
2. Molecular Cloning - A Laboratory Manual, Third Edition, 2001. CSHL Press. pp 7.27 - 7.34. J. Sambrook and D. Russell.