in vitro Transcription and Translation

Modified Nucleotides in IVT: Small Changes, Big Impact 

Posted on by Anna Bennett

In our final blog post on double-stranded RNA (dsRNA), we turn our attention to the chemical building blocks of mRNA therapeutics—modified nucleotides. These seemingly minor changes to the RNA sequence play a crucial role in the success of mRNA-based vaccines and treatments. However, they also introduce complexities in accurately detecting and quantifying unwanted dsRNA byproducts— key steps in ensuring the therapeutic efficacy of your mRNA product. 

What Are Modified Nucleotides? 

Modified nucleotides are ribonucleotides containing chemically altered nucleosides — like specialty ingredients swapped into a classic recipe to improve taste and nutrition. Just as a chef might use a lactose-free milk or gluten-free flour to make a dish easier to digest without changing its core structure, scientists use chemically altered nucleosides during in vitro transcription (IVT) to improve how mRNA therapies perform. These modifications replace their natural counterparts (e.g., uridine or cytidine) in the final RNA product. Their incorporation improves the performance and safety of mRNA therapeutics in several ways: 

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dsRNA QC Considerations: How I Learned to Stop Worrying and Love my IVT Reactions

Posted on by Anna Bennett

As mRNA therapeutics continue to expand across clinical pipelines, one persistent challenge remains for developers: reducing double-stranded RNA (dsRNA) contaminants that can compromise safety and efficacy. These unintended byproducts of in vitro transcription (IVT) can trigger unwanted immune responses and reduce the potency of the final product. Developers must prioritize dsRNA detection and control as essential steps in the process. In our previous blog post we offered a high-level discussion of what is double-stranded RNA (dsRNA), its biological function, and importance of detection in a therapeutic context.  Here, we’ll take a closer look at origins of dsRNA contamination, quality control measures, and improvement strategies.

Large-scale production of single-stranded RNA (ssRNA) for mRNA-based therapeutics is primarily done through in vitro transcription (IVT), an enzymatic process designed to generate high-yield, functional mRNA transcripts from a DNA template. This process uses purified RNA polymerase enzymes, such as T7, that recognize specific promoter sequences in the DNA template, generating the RNA transcripts of interest. However, IVT reactions also generate unwanted dsRNA byproduct. Below, we delve into some of the major quality control (QC) considerations and strategies to reduce dsRNA byproducts.

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mRNA Vaccine Manufacturing: Responding Effectively to a Global Pandemic

Posted on by Ken Doyle

We’ve learned a few important lessons from the COVID-19 pandemic.

Perhaps the most significant one is the importance of an early and rapid global response to the initial outbreak. A coordinated response—including widespread use of masks and other personal protective equipment (PPE), travel restrictions, lockdowns and social distancing—could save lives and reduce long-term health effects (1). Widespread availability of effective vaccines goes hand in hand with these measures.

New Boosters to Fight Omicron

Last month, Pfizer/BioNTech announced the US Food and Drug Administration (FDA) had granted emergency use authorization (EUA) for a new adapted-bivalent COVID-19 booster vaccine for individuals 12 years and older. This vaccine combines mRNA encoding the wild-type Spike protein from the original vaccine with another mRNA encoding the Spike protein of the Omicron BA.4/BA.5 subvariants. Moderna also announced FDA EUA for its new Omicron-targeting COVID-19 booster vaccine. The Omicron variant of SARS-CoV-2 shows multiple mutations across its subvariants, and it is currently the dominant SARS-CoV-2 variant of concern across the world.

Genomic epidemiology of SARS-CoV-2 with subsampling focused globally over the past 6 months. This phylogenetic tree shows evolutionary relationships of SARS-CoV-2 viruses from the ongoing COVID-19 pandemic. Image from Nextstrain.org; generated September 20, 2022

Booster doses of vaccines have become a way of life, both due to declining effectiveness of the original vaccines especially in older adults (2), and the rapid mutation rate of SARS-CoV-2 (3). Clinical data for the new Pfizer/BioNTech booster vaccine showed superior effectiveness in eliciting an immune response against Omicron BA.1 compared to the original vaccine. Previously, Moderna published interim results from an ongoing phase 2-3 clinical trial, showing that the new bivalent booster vaccine elicited a superior neutralizing antibody response against Omicron, compared to its original COVID-19 vaccine (4).

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In Vitro Transcription: Common Causes of Reaction Failure

Posted on by Kelly Grooms
FemaleWhiteLab-AAES001042, In Vitro Transcription

A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies (X-ray crystallography and NMR). Synthesized RNA transcripts are also used for studying cellular RNA functionality in processes such as splicing, RNA processing, intracellular transport, viral infectivity and translation.

Problems in the transcription reaction can result in complete failure (i.e., no transcript generated) or in transcripts that are the incorrect size (i.e., shorter or longer than expected). Below is a discussion of the most common causes of in vitro transcription problems.

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Will This Kit Work with My Sample Type?

Posted on by Joliene Lindholm

Whether you are working with cells, tissues or blood—making sure you use the correct assay system is critical for success.

In Technical Services, we frequently answer questions about whether a kit will work with a particular type of sample. An easy way to find out if other researchers have already tested your sample type of interest is to search a citation database such as Pub Med for the name of the kit and your specific sample type. We also have a searchable peer-reviewed citations database on our web site for papers that specifically cite use of our products. And on many of our product pages, you can find a list of papers that cite use of those products. In Technical Services, we are happy to help you in this search and let you know if scientists here at Promega have tested a particular application or sample type. This information provides a good starting point to optimize your own experiments.

One common question is “can the Caspase-Glo® Assays be used with tissue homogenates?” While Promega has not tested the Caspase-Glo® Assays with tissue homogenates, scientists outside of Promega have used the assays with tissue homogenates with success. As with almost all of our kits, Resources are provided on the catalog page including a list of Citations. As an example, here is a link to the Citations for the Caspase-Glo® 3/7 Assay Systems. We also have an article highlighting a citation on detecting caspase activities in mouse liver. A variety of lysis buffers have been used to make tissue homogenates for this application. To avoid nonspecific protein degradation, it is useful to include a protease inhibitor cocktail in the lysis buffer. The use of protease inhibitors doesn’t usually affect our assay chemistries. Additionally, many commercially available protease inhibitor sets can be used that do not contain caspase inhibitors. It is important to consider the specificity of the kit being used and include proper controls to ensure that the luciferase reaction is performing as expected. For more information on citations and example protocols, feel free to contact us here at Technical Services and we can help get you started with your sample type.

High-Yield Cell-Free Protein Expression: Prokaryotic Based

Posted on by Gary Kobs

S30 E coli high yield extract schematicMany applications require amounts of protein that cannot be obtained using a eukaryotic cell-free expression system. As an alternative, a prokaryotic system can be used when this need arises. The E. coli S30 T7 High-Yield Protein Expression System is designed to express up to 500μg/ml of protein in 1 hour from plasmid vectors containing a T7 promoter and a ribosome binding site. The protein expression system provides an extract that contains T7 RNA polymerase for transcription and is deficient in OmpT endoproteinase and lon protease activity. All other necessary components in the system are optimized for protein expression. This results in greater stability and enhanced expression of target proteins.The following references highlight the use of this system for a variety of unique applications:

Loh, E. et al. (2011) An unstructured 5′-coding region of the prfA mRNA is required for efficient translation. Nuc. Acids. Res. (online) Examines the effect of upstream codon sequence/length on the correct ribosome binding and translation initiation of the pfrA protein.

Mitsuhashi, H. et al. (2010) Specific phosphorylation of Ser458 of A-type lamins in LMNA-associated myopathy patients. J. Cell. Sci. 123, 3893–900 By creating a series of mutations in the protein lamin A, Akt1 phosphorylation sites were determined.

Halvorsen, E. et al. (2011) Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis. Microbiology 157, 387–97. S30 High Yield System was used to characterize the inhibitory effect of Txe toxin on protein expression.

Mo, P. et al. (2010) MDM2 mediates ubiquitination and degradation of activating transcription factor 3. J. Biol. Chem. 285, 26908–15. By using in vitro pull down experiments the researchers characterized the binding of AFT3 to MDM2 leading to the proteolysis of AFT3 system by ubiquitination.

Optimized Protein Expression: Flexi Rabbit Reticulocyte Lysate

Posted on by Gary Kobs
A protein chain being produced from a ribosome.

mRNAs commonly exhibit differing salt requirements for optimal translation. Small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi® Rabbit Reticulocyte Lysate System allows translation reactions to be optimized for a wide range of parameters, including
Mg2+ and K+ concentrations and the choice of adding DTT. To help optimize Mg2+ for a specific message, the endogenous Mg2+ concentration of each lysate batch is stated in the product information included with this product.

The following references utilize the features of Flexi Rabbit Reticulocyte Lysate System to investigate certain parameters of translation:

Vallejos, M. et al. (2010)The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation. Nucl Acids Res. 38, 618–32. Identification of internal ribosomal ribosomal entry site in the 5’ untranslated region of the mouse mammary tumor virus mRNA.

Spriggs, K. et al. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucl. Acids. Res. 17, 5881–93. Identification of a functional internal ribosome entry site in the human insulin receptor mRNA.

Powell, M. et al. (2008) Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein. RNA 14, 2394–06. Analysis of the mRNA signals involved in the expression of influenza B virus BM2 protein.

Sato, V. et al. (2007) Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Vir. 81, 11569–76. Charaterized the effect of the measles virus N protein binding to the translation initiation factor eIF3-p40 on the expression of various proteins in rabbit reticulocyte lysate.

Hirao, K. et al. (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J. Biol. Chem. 281, 9650–58. The EDEM3 protein was expressed in the presence of canine microsomal membranes to establish that co-translational translocation occurs into the endoplasmic reticulum.

Shenvi, C. et al. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations–implications for the study of ribosome dynamics. RNA 11, 1898–08. Characterization of ribosome dynamics under different ionic conditions.

Nair, A. et al. (2005) Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J. Biol. Chem. 280, 42809–16. Examined the affect of luteinizing hormone receptor mRNA binding protein on transltional suppression of luteinizing hormone receptor RNA.

Screening for Protein Activity Using Cell-Free Expression

Posted on by Gary Kobs

The analysis of functional protein typically requires lengthy laborious cell based protein expression that can be complicated by the lack of stability or solubility of the purified protein. Cell free protein expression eliminates the requirement for cell culture thus providing quick access to the protein of interest (1).

The HaloTag® Technology provides efficient, covalent and oriented protein immobilization of the fusion protein to solid surfaces (2).

A recent publication demonstrated the feasibility of using cell free expression and the HaloTag technology to express and capture a fusion protein for the rapid screening of protein kinase activity (3). The catalytic subunit of human cAMP dependent protein kinase was expressed in a variety of cell free expression formats as a HaloTag fusion protein. The immobilized cPKA fusion protein was assayed directly on magnetic beads in the active form and was shown to be inhibited by known PKA inhibitory compounds.

Therefore this unique combination of protein expression and capture technologies can greatly facilitate the process of activity screening and characterization of potential inhibitors

References
ResearchBlogging.org

  1. Zhao, K.Q. et al. (2007) Functional protein expression from a DNA based wheat germ cell-free system. J. Struc. Funct. Genomics. 8, 199-208.
  2. Los, G.V. and Wood, K. (2007) The HaloTag: A novel technology for cell imaging and protein analysis. Meth. Mol. Biol. 356, 195-208
  3. Leippe DM, Zhao KQ, Hsiao K, & Slater MR (2010). Cell-free expression of protein kinase a for rapid activity assays. Analytical chemistry insights, 5, 25-36 PMID: 20520741

Protease K Protection Assay: Cell Free Expression Application

Posted on by Gary Kobs

Microsomal vesicles are used to study cotranslational and initial posttranslational processing of proteins. Processing events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the transcription/translation of the appropriate DNA in the TNT® Lysate Systems when used with microsomal membranes.

The most general assay for translocation makes use of the protection afforded the translocated domain by the lipid bilayer of the microsomal membrane. In this assay protein domains are judged to be translocated if they are observed to be protected from exogenously added protease. To confirm that protection is due to the lipid bilayer addition of 0.1% non-ionic detergent (such as Triton® X-100) solubilizes the membrane and restores susceptibility to the protease.

Are you looking for proteases to use in your research?
Explore our portfolio of proteases today.

Many proteases have proven useful for monitoring translocation in this fashion including Protease K or Trypsin.

The following are examples illustrating this application:

  1. Minn, I. et al. (2009) SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans. Mol. Biol. Cell. 20, 4586–95.
  2. Padhan, K. et al. (2007) Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J.Gen.Virol. 88, 3067–77.
  3. Tews, B.A. et al. (2007) The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix. J.Biol.Chem. 282, 32730–41.
  4. Pidasheva, S. et al. (2005) Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia. Hum. Mol. Gen. 14, 1679–90.
  5. Smith, D. et al. (2002) Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation. J. Immun. 169, 99–107.

Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein

Posted on by Gary Kobs
Wheat Germ Extract for high-yield protein expression

Cell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1).

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