Synthesizing RNA in the laboratory is critical to many techniques, including analytical, biochemical, and molecular biology studies. In vitro transcription (IVT) is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence—from short oligonucleotides to several kilobases, in μg to mg quantities. Generating IVT mRNA involves engineering a template with a bacteriophage promoter sequence upstream of the sequence of interest, followed by transcription using the corresponding RNA polymerase. This comprehensive overview will describe:


Introduction to in vitro transcription

By using reaction components such as buffer systems, ribonucleotide triphosphates (NTPs), DNA templates, and phage RNA polymerases, researchers can efficiently produce high-quality RNA. This overview will guide you through the key aspects of IVT, including the necessary components, synthesis processes, delivery methods, scaling techniques, and various applications, providing a comprehensive understanding of this critical tool for discovery.

In vitro transcription combines a DNA template, RNA polymerase, and nucleotides to form IVT mRNA.


Components needed for in vitro transcription reactions

In vitro transcription requires a purified linear DNA template containing a promoter, NTPs, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase to produce mRNA. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.


Buffer systems

The purpose of the buffer system in in vitro transcription (IVT) is to create an optimal environment for the transcription reaction to occur. The addition of the buffer system to the IVT reaction serves to:

  • Maintain an optimal pH: The buffer helps ensure that the pH remains within a range that is conducive to the activity of the RNA polymerase enzyme.
  • Provide essential ions: Ions such as magnesium (Mg²⁺) and potassium (K⁺), are critical for the enzymatic activity and stability of the transcription process.
  • Support ionic strength: Maintaining the overall ionic strength of the reaction mixture is important for the proper folding and function of key enzymes.


    • Ribonucleotide triphosphates (NTPs)

    Ribonucleotide triphosphates (NTPs) are crucial for the transcription process, influencing both the efficiency, and quality of mRNA production as the:

  • Building blocks for RNA synthesis: The NTPs (ATP, CTP, GTP, and UTP) are essential substrates used by RNA polymerase to synthesize the RNA strand. They are incorporated into the growing RNA chain, helping ensure the correct sequence is formed based on the DNA template.
  • Energy source: The high-energy phosphate bonds in NTPs provide the necessary energy for the polymerization process. Optimal NTP concentrations ensure efficient transcription.
  • Determininer of yield and quality: The concentration of NTPs directly impacts the yield and quality of the synthesized RNA.


    • DNA template

    The DNA template in an in vitro transcription (IVT) reaction provides the sequence information that guides the synthesis of the RNA molecule through the following key functions:

    1. Sequence blueprint: The DNA template contains the nucleotide sequence that will be transcribed into RNA. The RNA polymerase reads the DNA template and synthesizes an RNA strand that is complementary to the DNA sequence.
    2. Promoter recognition: The DNA template typically includes a promoter region, which is a specific sequence recognized by the RNA polymerase. This promoter is crucial for initiating transcription at the correct location on the DNA template.
    3. Directionality: The DNA template determines the direction of transcription. RNA polymerase moves along the DNA template strand in the 3' to 5' direction, synthesizing the RNA strand in the 5' to 3' direction.

    Jump to IVT Templates to learn more about plasmid, PCR, oligonucleotide, and cDNA templates.


    Phage RNA polymerase

    RNA polymerases are enzymes that copy a DNA sequence into an RNA sequence, assisting in the initiation, and elongation during the IVT reaction. All eukaryotes have different polymerases which transcribe different types of genes. Some types includeT7, T3, and SP6:

  • T7 RNA polymerase rapidly and selectively transcribes from its own promoter, with faster synthesis, and less termination than E. coli RNA polymerase.
  • T3 RNA polymerase resembles T7 structurally but differs functionally due to distinct promoter interactions linked to specific amino acid substitutions.
  • SP6 RNA polymerase is a stable, highly specific enzyme that transcribes from its own promoter and does not recognize T3 or T7 DNA.


    • IVT templates

    To facilitate promoter recognition, DNA templates for in vitro transcription must contain a double-stranded RNA polymerase promoter region in the correct orientation. These templates include plasmids, PCR products, oligonucleotides, and cDNA:

    Plasmids

    Many common plasmid cloning vectors include phage polymerase promoters. They often contain two distinct promoters, one on each side of the multiple cloning site, allowing transcription of either strand of an inserted sequence. Some vectors contain all three phage polymerase promoters in tandem (on the same side of the multiple cloning site), allowing any of the three polymerases, SP6, T7, or T3 to be used.

    Plasmid vectors used as transcription templates should be linearized by restriction enzyme digestion. To produce RNA transcripts of a defined length the restriction site does not need to be unique, but the promoter should remain adjacent to the transcription template. It is not necessary to purify the promoter-insert sequence. Restriction enzyme digestion should be followed by purification since contaminants in the digestion reaction may inhibit transcription.

    PCR products

    PCR products can also function as templates for transcription. A promoter can be added to the PCR product by including the promoter sequence at the 5' end of either the forward or reverse PCR primer. These bases become double-stranded promoter sequence during the PCR reaction.

    Oligonucleotides

    Two oligonucleotides can also be used to create short transcription templates. Two complementary oligonucleotides containing a phage promoter sequence are simply annealed to make a double-stranded DNA template. Only part of the DNA template—the -17 to +1 bases of the RNA polymerase promoter—needs to be double-stranded. It may be more economical, therefore, to synthesize one short and one long oligonucleotide, generating an asymmetric hybrid (see Minimal Sequence Requirements).

    cDNA

    A more recent use of in vitro transcription is in aRNA amplification reactions. For these reactions, transcription templates are generated from RNA by using an Invitrogen oligo(dT)-T7 promoter primer during reverse transcription. The cDNA is converted to a double-stranded transcription template by a second-strand synthesis reaction.

    The choice of template depends on the application, the amount of RNA needed, and whether sense or antisense transcripts are needed.

    • Antisense transcripts: Used as probes for hybridization to mRNA (e.g., Northern blots, in situ hybridizations, and nuclease protection assays).
    • Sense transcripts: Used for expression, structural, or functional studies, or for constructing a standard curve for RNA quantitation.

    The +1 G of the RNA polymerase promoter sequence is the first base incorporated into the transcription product. For sense RNA, the 5' end of the coding strand must be adjacent to or just downstream of the +1 G. For antisense RNA, the 5' end of the noncoding strand must be adjacent to the +1 G.


    Key processes to synthesize IVT mRNA

    The reaction processes that occur in in vitro transcription including purification, capping, transcriptional labeling, and poly-A tail addition play a vital role in enhancing the stability, translation efficiency, and overall quality of the mRNA. By understanding and implementing these key processes, researchers can optimize their IVT reactions and produce mRNA suitable for a wide range of applications.


    Purification

    Purification is the most crucial step in the production of synthetic because it removes toxic materials that could kill the cell. [18] These materials include extra NPTs, enzymes, degraded DNA, etc. There are four possible methods to remove the toxic materials: oligodeoxythymidylic acid-cellulose, lithium chloride, ammonium acetate, and spin columns.

    1. Oligo(dT)-cellulose

    Oligodeoxythymidylic acid-cellulose, or Oligo(dT)-cellulose, has been used extensively for the isolation of poly-A RNA from a variety of sources. A stand-out product for this process is the POROST Oligo (dT)25 Affinity Resin, which separates mRNA from components of the transcription reaction process, such as enzymes and plasmid DNA. Nonspecific binding occurs when Oligo(dT) is used to analyze or prepare poly-A RNA, such as nonpolyadenylated nucleic acid binding and elution as well as “tight” nonspecific binding when poly-A RNA fails to elute, thus dissociating A-T bonds.[19]

    2. Lithium chloride

    Lithium chloride is efficient at precipitating RNA molecules of at least 100 nucleotides, but does not efficiently precipitate DNA, tRNA and other small RNA fragments, most proteins, and nucleotides—making it an excellent choice for the purification of mRNAs following IVT, or for the purification of ribosomal RNA. However, lithium chloride may not be as effective with low concentrations (<400 μg x mL), so dilute RNAs may be more efficiently precipitated by ethanol and salt.[20]

    3. Ammonium acetate

    Ammonium acetate is efficient for ethanol precipitating small and larger RNAs, but does not precipitate nucleotides, making it a good candidate for the purification of RNAs after reactions. However, because it also precipitates proteins, phenol/chloroform extractions are usually performed first. Also, ammonium acetate can inhibit T4 polynucleotide kinase, so it is not a viable choice for purifying RNAs that will be phosphorylated following purification.[21]

    4. Spin columns

    Spin column purification with silica resin is the preferred method since silica allows for easy binding, washing, and elution of nucleic acids in the purification process.[22]


    Capping

    Capping protects mRNA from phosphate and other nuclease attacks and helps to promote mRNA function. There are three main generations of capping technologies that could be used during transcription, which are mCap analog, ARCA, and trinucleotide capping.

    There are three cap structures in total: 0, 1, and 2. Cap-0 is essential for efficient translation of the mRNA that carries the cap, while cap-1 is important in evading the cellular innate immune response in vivo. In humans, cap-0 and cap-1 methylations are present on all mRNA molecules, while about half of the capped poly(A) molecules contain a 2′-O-ribose methylated residue on the second transcribed nucleotide. This sequence, also called cap-2, is required alongside cap-1 for spliceosomal E-complex formation and, consequently, for efficient pre-mRNA splicing.[12]

    Structural formula of a first-generation mCap analog used in the production of IVT mRNA

    1. mCap analog capping involves the cap interacting with different binding proteins, which initiates the factors and elements of ribosomes. The drawback of mCap analog is that the 3′ OH of either the G or m7G can serve as the initiating nucleophile for transcriptional elongation leading to the synthesis of two isomeric RNAs of either forward or reverse form in equal proportions depending upon the ionic conditions of the transcription reaction. The reverse form of capped mRNA is not recognized during translation, the second step of transcription, and only forward sequences will be translated. Therefore, only 50% of capped transcripts are translatable.[9]

    Structural formula of Anti-Reverse Cap Analogs (ARCA) used during in vitro transcription

    2. Anti-Reverse Cap Analogs (ARCA) are used during IVT for the generation of capped transcripts and have been found to promote cap-dependent translation more effectively than previous cap analogs.[10] It’s used to prevents the likelihood that synthetic mRNAs will have the cap-0 structure described below in the reverse orientation. Since there are no isomeric mRNAs present, all capped transcripts are translatable.[10] ARCA can be inserted in the proper orientation, which reduces the possibility of synthesizing non-translational mRNA and improves translation efficiency.

    Structural formula of a third-generation trinucleotide cap analog used to cap IVT mRNA

    3. Finally, trinucleotides have been shown to have remarkable higher yields and a higher capping efficiency. They have been found to outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. There have been limitations found with the cap 0 structure discussed below, but this has been effectively addressed by the use of a trinucleotide cap analog. Co-transcriptional capping with m7GpppNmN-derived trinucleotide generates a cap 1 structure, in which the first transcribed nucleotide has the 2′-O-methyl group. Shanmugasundaram et al. found that mRNAs capped with nicotinamide-containing trinucleotide cap analogs 128a and 128b are resistant to deNADing enzymes such as NudC, Nudt12, and DXO. The synthetic trinucleotide cap analog is currently used to make the IVT mRNA transcripts of Pfizer-BioNTech’s mRNA vaccine for the novel COVID-19 virus.[11]


    Co-transcriptional capping vs. post-transcriptional capping

     Co-transcriptional cappingPost-transcriptional capping
    Process

     

    		Incorporation of a cap analog during transcriptionEnzyme-based capping following the transcription reaction
    MechanismRNA polymerases with relaxed substrate specificity incorporate caps at the 5′-endEnzymatic formation of cap0 involves three consecutive reactions targeting nascent pre-mRNAs
    Capping enzymeCaps bind around the RNA exit tunnel of RNA polymerase IIEnzymes from eukaryotic organisms or viruses, produced recombinantly in E. coli.
    StepsSingle step during transcription
    • Hydrolysis of γ-phosphate by 5′-triphosphatase (TPase)
    • Coupling of β-phosphate to GMP to form Gppp-RNA
    • Methylation at the N7-position by RNA (guanine-N7) methyltransferase
    EfficiencyTheoretical capping efficiency of <100% due to competition from GTP for the starting nucleotide

    High-efficiency

    PurificationFewer purification steps requiredRequires removal of unused NTPs and additional purification steps
    ApplicationSeamless RNA protection during transcriptionProduces capped RNA in vitro by adding enzymes and cosubstrates to the IVT reaction


    Transcriptional labeling

    Transcriptional labeling involves incorporating labeled nucleotides into the RNA during the transcription process. This is typically achieved by including modified nucleotides such as fluorescent or radioactive nucleotides in the IVT reaction. The labeled RNA can then be used for various downstream applications, including tracking RNA localization, studying RNA-protein interactions, and monitoring RNA stability. Thermo Fisher Scientific offers a range of labeled nucleotides and labeling kits to facilitate transcriptional labeling in IVT reactions, helping ensure high efficiency and specificity for diverse research needs.


    Poly-A tail addition and translation

    During translation, which is the second major step in gene expression, the mRNA is "read" according to the genetic code, which relates the DNA sequence to the amino acid sequence in proteins. Each group of three bases in mRNA constitutes a codon, and each codon specifies a particular amino acid. The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein.[13] To help ensure molecules are stabilized and degradation is prevented, the poly-A tail is the key element. Poly-A tails are non-templated additions of adenosines at the 3’ end of most eukaryotic messenger RNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly-A site and then polyadenylated before being exported to the cytoplasm. In the cytoplasm, poly-A tails play pivotal roles in the translation and stability of the mRNA.[13]


    Delivery methods

    Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. mRNA is directly delivered and expressed in the cytoplasm, so it does not require crossing the nuclear membrane. After delivery, mRNA can immediately be translated into a protein in the cytoplasm usingtransient transfection, which doesn’t require integrating nucleic acids into the host cell genome. Nucleic acids may be transfected in the form of a plasmid or as oligonucleotides. Therefore, transgene expression will eventually be lost as host cells replicate.[15]


    Lipid nanoparticles (LNPs)

    Lipid nanoparticles, or LNPs, have been found to be appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA using therapeutic proteins.[25] LNPs have 4 ingredients:

    • Ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA
    • Pegylated lipids that help stabilize the particle
    • Phospholipids that contribute to the particle’s structure
    • Cholesterol molecules that contribute to the particle’s structure.[26]

    Researchers have found that lipid nanoparticle-mRNA formulations based on zwitterionic ionizable lipids can escape the endosome, leading to efficient protein expression and genome editing in vivo. Also, in addition to functioning as a delivery component, lipids can have therapeutic effects synergistic with mRNA-encoded proteins.[27] LNPs are composed of a few helper lipids which come in many shapes—for example, cylindrical-shaped lipid phosphatidylcholine can provide greater bilayer stability, which is important for in vivo application of LNPs.[28]

    MESSENGER Max reagent, a Thermo Fisher Scientific product, significantly enhances the transcription process. It boasts up to five times the efficiency of DNA reagents in neurons and primary cells, transfecting more mRNA into these challenging cell types without the need for electroporation or viruses. This reagent offers over a two-fold improvement in transfection efficiency compared to other lipid-based options, faster protein expression without genomic integration risks, and up to ten times higher cleavage efficiency using mRNA CRISPRs.

    Also see: mRNA Delivery Technology |Selecting a RNAi Strategy |Guidelines for RNA Transfection


    Scaling IVT reactions

    In vitro transcription reactions can be divided into two types: conventional and large scale. Conventional reactions are typically used for synthesizing radiolabeled RNA probes or for incorporating modified nucleotides into transcripts. Large-scale reactions, which generate >100 µg RNA per reaction, are useful for structural and expression studies, as well as for aRNA amplification.

    See additional applications for each scale


    Conventional reactions

    Conventional reaction conditions, such as those used in the Invitrogen MAXIscript Kit, use relatively low nucleotide concentrations (0.5 mM each). Higher nucleotide concentrations are not necessary since, in these reactions, the low concentration of radiolabeled or modified nucleotide present effectively limits the total yield of the reaction.

    The total concentration of the limiting nucleotide (labeled/modified and unlabeled) should be at least 3 µM for efficient synthesis of full-length RNA transcripts of <400 nt (more will be needed to synthesize longer transcripts).

    A 3 µM concentration of radiolabeled rNTP can be obtained by adding 5 µL of a 800 Ci/mmol, 10 mCi/mL (or 12.5 µM) solution of [α-32P] NTP. Higher specific activity labeled rNTPs are available but are provided at a much lower stock molar concentration (e.g. the 3000 Ci/mmol, 10 mCi/mL has a stock concentration of only 3.3 µM). Without the addition of unlabeled NTP, it is impossible to achieve the final minimum 3 µM reaction concentration.

    Because limiting nucleotide concentration can result in premature termination of transcription, there is a trade-off between synthesis of high specific activity (or extensively modified) transcripts and full-length transcripts. Diluting the limiting radiolabeled or modified nucleotide with unlabeled nucleotide proportionally lowers the specific activity (or extent of modification) of the transcript but yields more full-length transcript. To make very high specific activity or extensively modified transcripts one should limit or omit any unlabeled limiting nucleotide present.


    Large-scale synthesis

    Large-scale in vitro transcription reactions can produce up to 120–180 µg RNA per microgram template in a 20 µL reaction. Invitrogen MEGAscript technology allows the phage RNA polymerases to remain active at high nucleotide concentrations that would ordinarily inhibit the enzyme. Yields from these large-scale reactions are typically 10 to 50 times higher than those possible with conventional transcription reactions (without any limiting nucleotide). Reaction conditions (e.g., the type of nucleotide salt, type and concentration of salt in the transcription buffer, enzyme concentration and pH) are all optimized not only for each polymerase but for the entire set of components. Only under these conditions can you achieve optimal yields.

    Ordering information

    Order IVT products

    Order IVT mRNA kits through Thermo Fisher Scientific. Most IVT mRNA kits and products above are available in multiple options based on your anticipated number of reactions.

    Catalog # Name Size Price (USD) Qty
    AM1334 MEGAscript™ T7 Transcription Kit, 40 Reactions Each
    406.78

    Special offer

    Online exclusive

    473.00
    Save 66.22 (14%)
    AM1345 mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit Each
    543.52

    Special offer

    Online exclusive

    632.00
    Save 88.48 (14%)
    AM1344 mMESSAGE mMACHINE™ T7 Transcription Kit Each
    567.60

    Special offer

    Online exclusive

    660.00
    Save 92.40 (14%)
    LMRNA001 Lipofectamine™ MessengerMAX™ Transfection Reagent, 0.1 mL Each
    110.88

    Special offer

    Online exclusive

    126.00
    Save 15.12 (12%)
    AM1908 MEGAclear™ Transcription Clean-Up Kit Each
    164.26

    Special offer

    Online exclusive

    191.00
    Save 26.74 (14%)
    AM8052 Cap Analog (m7G(5')ppp(5')G), 100 A254 Units Each
    1,302.00
    AM1350 Poly(A) Tailing Kit Each
    304.11

    Special offer

    Online exclusive

    327.00
    Save 22.89 (7%)
    AM8045 ARCA (Anti-Reverse Cap Analog) Each
    323.64

    Special offer

    Online exclusive

    348.00
    Save 24.36 (7%)
    A57622 MegaScript™ T7 Transcription Kit Plus, 50 reactions Each
    598.92

    Special offer

    Online exclusive

    644.00
    Save 45.08 (7%)
    A57620 mMESSAGE mMACHINE™ T7 mRNA Kit with CleanCap™ Reagent AG, 50 reactions Each
    1,793.10

    Special offer

    Online exclusive

    2,085.00
    Save 291.90 (14%)


    In vitro transcription assays by application

    Scale of reactionApplicationTechniques/Methods
    Conventions/small-scale transcriptionProbe generation and RNA modification
    • generating radiolabeled and nonisotopically labeled RNA probes for blot hybridizations
    • nuclease protection assays
    • incorporation of modified nucleotides into transcripts
    Large-scale transcriptionaRNA amplification
    • microarray analysis
    • RNA-Seq
    • QPCR
    • functional studies
    Expression studies
    • microinjection
    • infection with viral transcripts
    • in vitro translation
    Structural analysis
    • protein-RNA binding studies
    • X-ray crystallography
    • nuclear magnetic resonance (NMR)
    Mechanistic studies
    • ribosome analyses

     


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