In vitro transcription and the associated applications

Introduction

In vitro transcription, often abbreviated as IVT, is a fundamental technique in molecular biology with crucial applications in drug discovery. This process involves the synthesis of RNA molecules from a DNA template in a controlled laboratory environment, enabling the production of custom-designed RNA sequences. We will introduce you to the concept of in vitro transcription and how Integrated DNA Technologies (IDT) plays a pivotal role in this process, ultimately contributing to breakthroughs in drug discovery.

Principles of in vitro transcription

In vitro transcription is built upon the central dogma of molecular biology, which states that genetic information flows from DNA to RNA to protein. This process harnesses the natural enzymatic machinery present in cells to produce RNA molecules outside of their native cellular context. Key components of in vitro transcription include:

• DNA Template: A DNA molecule containing the sequence of interest serves as the template for RNA synthesis. This DNA template can be obtained through various methods, including synthetically sourced DNA from a vendor (such as gBlocks™ HiFi Gene Fragments) or polymerase chain reaction (PCR) amplification.
• RNA Polymerase: A specialized enzyme, typically T7, T3, or SP6 RNA polymerase, recognizes specific promoter sequences on the DNA template and catalyzes the synthesis of RNA complementary to the DNA strand.
• Ribonucleotides: The four ribonucleotide triphosphates (ATP, UTP, CTP, GTP) serve as the building blocks for RNA synthesis. The RNA polymerase adds these ribonucleotides one at a time, following the complementary base pairing rules with the DNA template.
• Transcription Buffer: A buffered solution provides the optimal conditions for RNA polymerase activity, including pH, ionic strength, and cofactors.
• Reaction Temperature: In vitro transcription is typically carried out at a controlled temperature (e.g., 37°C) to maintain enzymatic activity.

Applications in Drug Discovery

In vitro transcription plays a pivotal role in drug discovery, primarily by enabling the production of custom RNA molecules for various applications:

1. Functional Genomics: In vitro transcription can be used to synthesize RNA molecules that regulate targets like disease-related genes or pathways. These RNA molecules, such as small interfering siRNAs (siRNAs), are then introduced into cells to study their effects on cellular processes, aiding in the identification of potential drug targets.
2. RNA Therapeutics: RNA-based drugs, such as messenger RNA (mRNA) vaccines, use IVT to produce the desired RNA sequences. IVT ensures the controlled and efficient production of therapeutic RNA for clinical applications.
3. Target Validation: Researchers can synthesize RNA molecules that represent potential drug targets, allowing for in-depth validation studies to determine their suitability for drug development.
4. High-Throughput Screening: In vitro transcription is employed in high-throughput screening assays to test thousands of compounds for their effects on RNA targets. This is a crucial step in identifying small molecules that modulate RNA function.
5. RNA Structure-Function Studies: IVT is used to produce RNA constructs for structural and functional studies, such as determining RNA secondary and tertiary structures or investigating RNA-protein interactions.

Challenges and Future Directions

While in vitro transcription is a versatile technique, it does come with challenges, such as ensuring high yields of pure RNA and minimizing template-independent synthesis. Moreover, the field of RNA therapeutics continues to evolve, creating new opportunities and challenges for IVT-based approaches. Advancements in IVT technology, such as improved enzymes and buffers, will likely enhance the efficiency and scalability of RNA production.

One example of an advancement enabling the ease and scalability of in vitro transcription is IDT’s gBlocks HiFi Gene Fragments. With NGS-verified, clonal fragments from 1000–3000 bp, obtaining high quality template without the trouble of cloning and sequencing is vastly simplified. Using gBlocks HiFi gene fragments, researchers has been able to accelerate mRNA manufacturing through IVT [1,2].

In the future, as the understanding of RNA biology deepens, IVT will continue to be at the forefront of drug discovery efforts, contributing to the development of innovative therapies. The ability to generate custom RNA molecules for a wide range of applications makes IVT an indispensable tool for the advancement of drug discovery and the development of novel therapeutic strategies and IDT will be here to help.