In biotechnology, accurate and efficient RNA synthesis is a key element for many applications, from gene expression profile analysis to therapeutic purposes. The most frequently employed technique for synthesising RNA is T7 in vitro transcription. It utilizes the T7 RNA polymerase, perhaps the most effective enzyme in transcribing DNA into RNA in an out-of-cell method.
T7 RNA polymerase-directed transcription is now one of the most widely used techniques for investigators who want to investigate RNA in maintained settings, create RNA vaccines, or synthesize RNA-based drugs. In this blog, the reader will learn about T7 in vitro transcription, what is needed to prepare for transcription, the benefits of the technique, and its diverse usage in the field of biotechnology.
What is T7 in Vitro Transcription?
T7 in vitro transcription is a method of synthesizing RNA from a DNA template using the enzyme T7 RNA polymerase. The T7rnap is an enzyme which comes from a virus – the T7 bacteriophage – that infects bacteria. This polymerase has a promoter sequence recognition that targets the promoter T7 and the following DNA to be transcribed into RNA.
While in vivo transcription is performed within cells, in vitro transcription is performed in a test tube and this process is very well controlled that the researcher wants and the desired amount of RNA is produced. The primary benefit of T7 RNA polymerase is realized through its usability: it is much more efficient and more specific than traditional RNA polymerase enzymes. It copies a great deal of RNA from the DNA template within a short span of time, and therefore is suitable for the commercial production of RNA for use in diverse research and clinical investigation.
Components of T7 In Vitro Transcription
DNA Template with a T7 Promoter: One more restriction for the template is the presence of the T7 promoter – the sequence recognizable by T7 RNA polymerase. The T7 promoter, for example, is designed to be cloned upstream of the target gene or sequence to facilitate the binding of the T7-polymerase and begin transcription. It is often a linear plasmid, but other times it a circular plasmid depending on the needs of an experiment.
T7 RNA Polymerase: Which of the enzymes synthesizes the RNA from the DNA template? T7 RNA polymerase recognizes specifically the T7 promoter and synthesizes the template into a complementary RNA strand.
Nucleotide Triphosphates (NTPs): These are the nucleotides of RNA; the analogy are ATP, CTP, GTP, and UTP. These nucleotides are then utilized by the T7 RNA polymerase to synthesize the RNA strand as it continues to transcribe the DNA template.
Buffer System: The reaction mixture contains a buffer for maintaining the correct pH and other ionic conditions required for the proper functioning of the enzyme. Magnesium ions (Mg2+) can always be part of the design because they play an important role of maintaining the interactions between RNA polymerase and the nucleotides during transcription.
Ribonuclease Inhibitors: Although RNases (enzymes that degrade RNA) may be in the environment, their activity poses a great challenge to RNA synthesis. The synthesis of RNA is normally accompanied by the use of ribonuclease inhibitors in order to minimize chances of the newly synthesized RNA being broken down.
Pyrophosphatase: Pyrophosphate is synthesized throughout the transcription process as a side product and in substantial concentrations it may manage to hinder the activity of the polymerase. Pyrophosphatase may be used to further break pyrophosphate and improve the rate of RNA synthesis.
The T7 In Vitro Transcription Process
Template Preparation: The DNA template can be obtained either by releasing the plasmid of interest through digestion or by PCR amplifying the region of interest and checking that the sequence contains the T7 promoter upstream of the gene of interest.
In Vitro Transcription Reaction: The prepared DNA template is then incorporated with T7 RNA polymerase NTPs, a buffer and other optional addition like, RNase inhibitors and pyrophosphatase. The biochemicals are mixed for a prescribed time which is normally for 30 minutes to a few hours and at an ideal temperature usually about 37°C. During this incubation, the T7 RNA polymerase synthesizes the RNA on the DNA template by a process of transcribing.
RNA Purification: In order to study the transcription reaction more effectively it is imperative to purify the RNA to get rid of the DNA template, enzymes, and other reactants. The RNA extract is usually isolated using methods like phenol-chloroform extraction, purification through the silica column or through spin columns. Finally, when RNA purification is done, RNA can be further analyzed and used for other applications.
Conclusion
The T7 in vitro transcription is one of the widely used techniques in modern biotechnology for its speed, efficiency, and flexibility with RNA synthesis within a compartment. Not only in gene expression but even in therapeutic applications T7 RNA polymerase has made it possible to synthesize RNA with much more ease and in much more effective ways thus installing novel techniques and horizons for RNA in the field of medicine and many more.