T7-RNA-Polymerase-Plus-and-SP6-RNA-Polymerase-Plus-From-Ambion

SP6 RNA Polymerase encoded by bacteriophage SP6 is remarkably similar in structure and activity to the T7 RNA polymerase encoded by bacteriophage T7. The major difference between T7 and SP6 RNA Polymerases is that each is highly specific for its own promoter sequence. SP6 RNA Polymerase is used for the synthesis of RNA transcripts in the 5´→ 3´ direction from vectors containing the SP6 phage promoter, while T7 RNA Polymerase catalyzes the synthesis of RNA in the presence of a DNA template containing T7 phage promoter. T7 RNA Polymerase-Plus™ and SP6 RNA Polymerase-Plus™ by Ambion are recombinant enzymes that are encoded by DNA sequences having single-base active-site mutations in the respective T7 or SP6 RNA Polymerase gene. These active-site mutations enable the corresponding T7 or SP6 Polymerase to incorporate 2’-deoxyribonucleoside triphosphates (dNTPs) into full-length transcripts much more efficiently than the corresponding wild-type enzymes, while retaining the same catalytic activity for incorporation of canonical NTPs and the same high promoter specificity as the respective wild-type T7 or SP6 RNA Polymerase. In addition, these enzymes incorporate ³²P, ³⁵S, and ³H for the synthesis of high specific activity radioactively-labeled RNA probes. Other applications include the generation of RNA for in vitro translation, biologically active mRNA and/or preparative quantities of defined length RNA by run off transcription. Both enzymes come in three convenient sizes, including the highly concentrated (200 units/ul) product.

An important added value to the T7 RNA Polymerase-Plus™ and SP6 RNA Polymerase-Plus™ products is SUPERase•In™. This is a proprietary RNase inhibitor manufactured by Ambion, offering broad-spectrum protection against enzymatic degradation of the synthesized RNA. While ribonuclease inhibitor protein has historically been added to in vitro transcription reactions for this purpose, SUPERase•In™ is a much more advanced protector since it inhibits a broader range of RNases, including RNase T1 and RNase 1 as well as RNase A-type enzymes (RNases A, B, and C). It is active over a broad pH and temperature range (pH 5.5-8.5; 4-65°C).

I successfully used both enzymes to synthesize large amounts of unlabeled RNA molecules to be further used for gel-shift assays. I amplified a 1 kb piece of human mitochondrial a 12S rRNA molecule by PCR and cloned it using a blunt-end cloning vector. After positive clones were identified, plasmids containing the correct insert were extracted, linearized and the resultant DNA was used as a template for RNA synthesis. Depending on the direction of the cloned insert in the plasmid, either T7 RNA Polymerase-Plus™ or SP6 RNA Polymerase-Plus™ was used to ensure the correct 5´→ 3´ direction of the RNA product. 2 ug of plasmid DNA in the transcription reaction yielded 40 ug of synthesized RNA. Agarose gel analysis of the synthesized RNA showed it to be of the expected size.

On a technical note, it is important to know that Ambion calculates enzyme activity differently from other manufacturers. All calculations use linearized rather than supercoiled templates, thus one Ambion unit actually contains more polymerase activity than a unit calculated by the other manufacturers.

In summary, I am very satisfied with the reliable performance and large product yields of T7 RNA Polymerase-Plus™ and SP6 RNA Polymerase-Plus™ products from Ambion and highly recommend them.

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Yelena Bykhovskaya, PhD
Research Scientist I
Molecular Hematology Laboratory
Cedars-Sinai Medical Center
United States