IVT Enzymes for RNA Synthesis

The T7 RNA polymerase, sometimes referred to as T7 RNA Pol or T7 RNAP, is widely considered to be the gold standard enzyme for the transcription of RNA from a DNA template. Let’s take a look at the basics of this handy enzyme, including the T7 RNA polymerase promoter sequence, its molecular weight and structure, and how it works.

Let’s explore the function of T7 RNA polymerase. T7 RNA polymerase has two conformational states. In the unstable initiation complex (IC), the NTD of the polymerase binds to its 23 base pair promoter sequence in DNA, then proceeds to unwind the DNA duplex. Here it begins what is known as abortive cycling, producing short, transcripts of two to 10 nucleotides.

After the transcripts have reached more than 10 nucleotides in length, the promoter-binding residues rearrange, and the promoter region is released. T7 RNA polymerase then changes to its stable conformation – the elongation complex (EC). Highly active, the EC can produce hundreds of copies of full-length RNA transcripts. The polymerase will terminate transcription either when it reaches the end of a linearized template, or when it recognizes a specific T7 RNA polymerase termination sequence .

At 883 amino acids in length , the T7 RNA polymerase molecular weight is approximately 99 kilodaltons (kDa). The T7 RNA polymerase structure includes two domains: the amino-terminal domain (NTD) and a carboxy-terminal domain (CTD).

The T7 RNA polymerase promoter sequence is 5′ TAATACGACTCACTATAG 3′, which it recognizes with very high specificity. RNA synthesis occurs from 5’ – 3’, beginning at the final G of the T7 RNA polymerase promoter sequence.

Synthego’s T7 RNA polymerase is produced with stringent control measures to ensure high quality and purity. A sample from each batch is subjected to several tests, including an in vitro transcription assay for functionality, and E. coli DNA contamination and RNase contamination assays for purity. With customizable concentrations and formulations, we provide solutions that seamlessly integrate into your workflows, and costs are significantly reduced when you order in bulk. More importantly, a recent study demonstrated that Synthego’s T7 RNA polymerase enzyme outperforms many commercially available equivalents when applied in in vitro transcription assays.

When working with RNA for any application, using the best RNase inhibitor is crucial to protect your samples from degradation. Because it is typically a single-stranded molecule, RNA is inherently unstable compared to double-stranded DNA, making proper sample storage of the utmost importance.

However, even perfect storage conditions at -80°C can’t protect your RNA samples from degrading, because it won’t stop RNase contamination from destroying them. Let’s discuss RNase contamination, how it occurs, and how using an RNase inhibitor can protect your samples.

The mammalian RNase inhibitor weighs in at 50-kDa and is 460 amino acids long . It has a curious structure, with alternating α-helices and β-strands that form the shape of a horseshoe. These repeating structural units contain leucine-rich repeats, which are commonly found in proteins that are involved in protein-protein interactions.

Synthego’s RNase inhibitor offers high performance and quality, maximizing your RNA yield and preventing degradation. Produced with high purity and analyzed via SDS polyacrylamide gel electrophoresis, a sample from each batch for RNase inhibitor is tested by incubation with an RNA template to ensure no RNase activity can be observed. As with our T7 RNA polymerase, our RNase inhibitor can be provided in customized concentrations and formulations, so that you can effortlessly integrate this enzyme into your methods. For bulk orders we significantly lower costs, making RNase inhibitor a cost-effective addition to your laboratory toolkit.

There is a wide range of T7 RNA polymerase uses, all of which benefit from the addition of RNase inhibitor. However, RNase inhibitor can also be used in techniques where T7 RNA polymerase is not required. Let’s take a closer look at how we can use the T7 RNA polymerase and RNase inhibitor enzymes.

In the post-pandemic era, mRNA vaccines have taken center stage. mRNA molecules used in vaccines require five key components: a 5’ cap, a 5’ untranslated region, an open reading frame, a 3’ untranslated region, and a 3’ poly-A tail. Using T7 RNA polymerase in vitro transcription, researchers can produce large quantities of the desired mRNA sequence in short timeframes and prevent degradation of the end product by incorporating the RNase inhibitor enzyme. Additionally, in vitro transcribed mRNA can be used in other biotherapeutics, such as cell therapies, for the transient expression of required proteins.

T7 RNA polymerase in vitro transcription is a common method to produce RNA probes for applications like in situ hybridization (ISH). ISH uses complementary RNA probes to determine the spatial localization of specific RNA transcripts within cells and tissues. To generate probes, the target sequence is cloned into a plasmid vector along with the T7 RNA polymerase promoter sequence; this can be inserted in either orientation depending on whether sense or antisense RNA probes are required.  

After linearizing the plasmid, in vitro transcription using T7 RNA polymerase and RNase inhibitor can be performed. The in vitro transcription reaction can include labeled nucleotides to enable detection later on. The resulting RNA probes are then purified before use in downstream ISH experiments. The use of RNase inhibitor is key to ensure the integrity of the purified RNA probes and the success of ISH.

in vitro transcription with T7 RNA polymerase and RNase inhibitor can similarly be used to synthesize RNA aptamers: short, single-stranded RNA oligonucleotides that work similarly to antibodies, binding to specific target sequences with high affinity. RNA aptamers have therapeutic and diagnostic applications in cancer and infectious disease, among others.

At Synthego, we produce high-quality synthetic guide RNAs (gRNAs) for CRISPR editing. However, researchers often require in vitro transcribed guides, including prime editing guide RNAs (pegRNAs), highly modified guides, and DNA oligos that are longer than 55 nucleotides (longmers). For example, in prime editing experiments, both the prime editors and the pegRNAs can be delivered to cells or tissues as in vitro transcribed mRNAs.

In these cases, T7 RNA polymerase and RNase inhibitor are the best enzymes to produce high fidelity RNA guides for efficient editing through in vitro transcription. in vitro transcribed gRNAs and pegRNAs are often used in functional genomics studies to explore the links between genes and phenotypes. Similarly, in vitro transcription with T7 RNA polymerase and RNase inhibitor can also be used to produce short interfering RNAs (siRNAs) for RNA interference experiments in functional genomics.

in vitro transcribed RNA molecules are often used to study the structure and function of RNA, for example using nuclear magnetic resonance (NMR) or X-ray crystallography. By exploring the three-dimensional structure of RNA, researchers can elucidate the complex molecular biology of cells.

in vitro transcription using T7 RNA polymerase is the most common method to produce RNA molecules for structural studies because NMR and X-ray crystallography require large amounts RNA at high purity. Chemical modifications such as 2’-OH groups can also be added during in vitro transcription to make the resulting RNA more stable. In addition, the use of RNase inhibitor is paramount in structural studies to avoid RNase contamination degrading the samples.