Introduction to in vitro transcription
In vitro transcription (IVT) is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities . It is based on the engineering of a template that includes a bacteriophage promoter sequence upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques, structural studies, in biochemical and genetic studies, and as functional molecules.
Messenger RNA (mRNA) therapy has tremendous potential in the field of regenerative medicine, treatment of diseases and vaccination . mRNA uses the cell's natural translation machinery to make proteins. Synthetic mRNA allows for the transfection of cells with desired mRNAs to induce expression of target proteins under physiological conditions until it is completely degraded . Synthetic transcripts can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies using X-ray crystallography and nuclear magnetic resonance (NMR) . However, there are other uses for synthetic mRNA other than making mRNA to express protein in cells.
In vitro transcription processes
During IVT, DNA template, RNA polymerase, NTPs, RNase inhibitor, pyrophosphatase, and IVT buffer are combined to produce mRNA. In the first step, transcription, there are two types of DNA templates that can be used to promote cloning if they contain a double-stranded RNA polymerase promoter region in the correct orientation: a polymerase chain reaction (PCR) or a plasmid product. PCR allows conversion of any DNA fragment to a transcription template by appending the T7 (or SP6) promoter to the forward primer . When lesser amounts are needed and when templates are needed quickly, PCR products are the most convenient due to the flexibility in design of the template and the ease of its production . To ensure success, however, the RNA polymerase promoter must be found upstream in the sequence. Plasmid products have been found to have the ability to become biologically functional replicons that take on the same genetic properties and nucleotide base sequences from both parent DNA molecules present . To achieve the greatest transcription yields, the highest purity plasmid templated are needed and the DNA must be free of contaminating RNases, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), proteins, salts, and RNA .
The role of capping and capping technologies
Capping protects mRNA from phosphate and other nuclease attacks and helps to promote mRNA function. There are two main capping options to choose from when it comes to transcription: co-transcriptional capping and post-translational capping. Co-transcriptional capping involves the incorporation of a cap analog during transcription. The incorporation of the caps, with the trinucleotide cap being the most common, at the 5′-end by RNA polymerases with relaxed substrate specificity yields the respective 5′-capped mRNA . In co-transcriptional capping, the capping enzyme binds around the RNA exit tunnel of RNA polymerase II to ensure seamless RNA protection. There is one drawback of this approach—the theoretical capping efficiency of <100%—which is due to competition from the GTP for the starting nucleotide . Post-translational capping involves enzyme-based capping following the transcription reaction. The enzymes used in vitro originate from capping apparatuses of different eukaryotic organisms or viruses and can be produced recombinantly in E. coli. Enzymatic formation of cap0 comprises three consecutive reactions targeted to nascent 5′-triphosphorylated pre-mRNAs. First, a 5′-triphosphatase (TPase) hydrolyzes the γ-phosphate of pre-mRNA. Next, the β-phosphate of the resulting 5′-diphosphate end is coupled to GMP to form 5′–5′-linked Gppp-RNA . Finally, the cap structure is methylated at the N7-position by an RNA (guanine-N7) methyltransferase using S-adenosyl-L-methionine (SAM) as a co-substrate. In nature, the capping enzymes used in post-translational capping act co-transcriptionally once the transcript has reached a length of 20–30 nucleotides. These enzymes can be harnessed to produce capped RNA in vitro by adding them and their respective cosubstrates to the IVT reaction . It is important to note that IVT reactions need to be purified to remove unused nucleoside triphosphates (NTPs) for enzymatic capping to work, and that this type of capping requires more purification steps than co-translational capping.
There are three main generations of capping technologies that could be used during transcription, which are mCap analog, ARCA, and trinucleotide capping:
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 .
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 . 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 . ARCA can be inserted in the proper orientation, which reduces the possibility of synthesizing non-translational mRNA and improves translation efficiency.
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 .
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 .
RNA polymerases for in vitro transcription
RNA polymerases are enzymes that copy a DNA sequence into an RNA sequence. All eukaryotes have different polymerases which transcribe different types of genes. Some types include T7, T3, and SP6. T7 synthesizes RNA at a rate several times faster than E. coli RNA polymerase and it terminates transcription less frequently. It catalyzes the synthesis of RNA in the presence of a DNA template containing T7 phage promoter. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences . T3 is similar in structure to T7 but has been proven to have exclusive specialties that T7 cannot provide. For example, when researchers compared the nucleotide sequence of the region of T3 DNA that encodes T3 RNA polymerase with the corresponding T7 region, they found significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. They also found that the promoter for the T3 RNA polymerase is within a region that coincides with one of the clusters of amino acid substitutions between enzymes . Since it has been proven that T3 and T7 share an identical amount of amino acid residues, this might point to the reason for their differences . Finally, SP6 is a small, virulent bacteriophage which grows on Salmonella typhimurium LT2. It is like T7 but is genetically different in that it is a stable enzyme that is easily purified to homogeneity in good overall yield. It is used for the synthesis of RNA transcripts in the 5´→ 3´ direction from vectors containing the SP6 phage promoter. SP6 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI).(dC) as template. While RNA synthesis requires native SP6 RNA as template, DNAs from other bacteriophages including T3 and T7 are inert. Thus, SP6 RNA polymerase possesses a stringent promoter specificity that is alike but also distinct from that of the other phage RNA polymerases .
NTPs used in RNA synthesis
NTPs are molecules used in RNA synthesis as substrates that contain a nitrogenous base bound to a 5-carbon sugar (either ribose or deoxyribose), with three phosphate groups bound to the sugar . Natural nucleoside triphosphates include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), and uridine triphosphate (UTP). For PCR, deoxynucleotide triphosphates (dNTPs) are used to expand the growing DNA strand. dNTPs consist of four basic nucleotides: dATP, dGTP, dCTP, and dTTP. These four nucleotides are typically added to the PCR reaction in equimolar amounts for optimal base incorporation. dNTPs are limited in concentration inside cells because the enzyme that synthesizes deoxynucleotides from ribonucleotides—ribonucleotide reductase (RNR)—is synthesized and enzymatically activated as cells enter the S phase. Low levels and activity of RNR provide sufficient dNTPs for mitochondrial DNA synthesis and for DNA repair in noncycling cells and during the G1 phase of the cell-division cycle in proliferating cells. Researchers such as Franzolin et al. have found that dNTP destruction using sterile alpha motif and HD-domain containing protein 1 (SAMHD1) contributes to dNTP concentration control during the cell-division cycle of proliferating cells, which affects both DNA replication and cell-cycle progression .
Translation and addition of poly-A tail
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 . To 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 .
Methods for purification
Purification is the most crucial step in the production of synthetic mRNA due to the removal of toxic materials that would kill the cell if not removed . 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.
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 . 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 ideal 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 .
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, but 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 . Lastly, 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 .
Methods for transfection
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 using transient transfection. This type of transfection does not 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 .
RNA polymerases assist in the initiation and elongation during the IVT reaction. Some types include T7, T3, and SP6. T7 synthesizes RNA at a rate several times faster than E. coli RNA polymerase and it terminates transcription less frequently. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences . T3 is similar in structure to T7 but has been proven to have exclusive specialties that T7 cannot provide. For example, when researchers compared the nucleotide sequence of the region of T3 DNA that encodes T3 RNA polymerase with the corresponding T7 region, they found significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. They also found that the promoter for the T3 RNA polymerase is within a region that coincides with one of the clusters of amino acid substitutions between enzymes . Since it has been proven that T3 and T7 share an identical amount of amino acid residues, this might point to the reason for their differences . Finally, SP6 is a small, virulent bacteriophage which grows on Salmonella typhimurium LT2. It is like T7 but is genetically different in that it is a stable enzyme that is easily purified to homogeneity in good overall yield. SP6 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI).(dC) as template. While RNA synthesis requires native SP6 RNA as template, DNAs from other bacteriophages including T3 and T7 are inert. Thus, SP6 RNA polymerase possesses a stringent promoter specificity that is alike but also distinct from that of the other phage RNA polymerases .
A successful transfection reagent is MESSENGER Max, which is one of many Thermo Fisher Scientific products that can assist in making the transcription process smoother than ever before. It has up to five times the efficiency of DNA reagents in neurons and primary cell types. It is designed to transfect a higher amount of mRNA into neurons and a broad spectrum of difficult-to-transfect primary cells. This eliminates the need for electroporation or viruses and provides a >2-fold improvement in transfection efficiency compared to other lipid-based reagents. This product offers transfection efficiency in neurons and primary cell types, faster protein expression with no risk of genomic integration, and up to 10-times higher cleavage efficiency while also using the mRNA CRISPRs.
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 . LNPs have 4 ingredients: ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA; pegylated lipids that help stabilize the particle; and phospholipids and cholesterol molecules that contribute to the particle’s structure . 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 . 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 .
Thermo Fisher Scientific as a leader in in vitro research
Some other Thermo Fisher Scientific products and tools that can help supplement in vitro research and aide in transcription include MEGAscript T7 Transcription Kit, m7GpppG mCap available in 10 or 25 units, ARCA available in 10 or 100 units, and PolyA Tailing Kits (25 Rxn). They also offer a generation of bulk RNA for use as a research tool, including in situ hybridization probes, antisense/RNAi oligos, structural/functional studies, aptamers, ribosomes, and probes for northern blots and microarrays. For purification, Thermo Fisher Scientific offers the MEGAclear Clean-up Kit for the silica purification of mRNA, Dynabeads MyOne beads and Dynabeads Oligo (dT) beads, and POROS Oligo (dT)25 Affinity Resin. For transfection, Thermo Fisher Scientific also offers LNP coating, including ethanol, cholesterol, sucrose, tris, and salts.
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