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RiboMAX™ Systems: The Many Uses for Large Scale In Vitro Transcription

Leah Cronan

Promega Corporation
Publication Date 09/2021; tpub_211

Abstract

This article describes recent advancements in RNA research made possible using the RiboMAX™ Large Scale RNA Production Systems and RiboMAX™ Express Large-Scale RNA Production Systems for in vitro transcription.

Introduction

Our Riboprobe® Systems are great for preparing microgram amounts of single-stranded RNA transcripts with radiolabeled ribonucleotides, such as RNA probes. But what if you need large amounts of non-radiolabeled RNA that is biologically active? Our variety of RiboMAX™ Systems have you covered.

The RiboMAX™ Large Scale RNA Production Systems are in vitro transcription systems designed for consistent and scalable production of large amounts of RNA, while the RiboMAX™ Express Systems offer the same benefits in a shorter amount of time. You can generate milligram amounts of high-quality RNA transcripts up to 27kb using these kits. All RiboMAX™, RiboMAX™ Express and Riboprobe® Systems are compatible with a variety of templates including plasmids or cloning vectors, PCR products, synthetic oligonucleotides, and cDNA.

ribomax in vitro transcription 15460mb-w
Figure 1. How the RiboMax™ Systems work.

The high-quality RNA transcripts produced from RiboMAX™ Systems can be used directly for in vitro or in vivo translation. This means that you can make DNA sequence modifications, quickly transcribe DNA into RNA, and the RNA can then be used in translation studies to determine effects of the DNA modification. Similarly, a DNA sequence encoding a protein of interest can be quickly transcribed into mRNA, which can be packaged in a delivery system of choice and then used in therapeutic or vaccination studies.

The RiboMAX™ Systems are also compatible for use with modified nucleotides and cap analogs, and can be used to create labeled RNA probes. It’s important to determine which in vitro transcription system is best for your application. See Table 1 to compare the features of our various in vitro transcription systems.

Table 1. Comparison of Riboprobe®, RiboMAX™ and RiboMAX™ Express Systems.

  Riboprobe® Systems RiboMAX™ Large Scale RNA Production Systems T7 RiboMAX™ Express Large Scale RNA Production System T7 RiboMAX™ Express RNAi System
Cat.#
P1420, P1430, P1440
P1280, P1300
P1320
P1700
Radiolabeling
Included Icon
not-available
not-available
not-available
T3 Promoter
Included Icon
not-available
not-available
not-available
SP6 Promoter
Included Icon
Included Icon
not-available
not-available
T7 Promoter
not-available
Included Icon
Included Icon
Included Icon
Premixed rNTPs
Included Icon
not-available
Included Icon
Included Icon
Compatible with Modified Nucleotides
Included Icon
Included Icon
Included Icon
Included Icon
Time
1 hour
2–4 hours
30 minutes
30 minutes
RNA Yield (per 1ml reaction) Low (100–250µg) Medium (2–5mg) High (5–8.5mg) High (5–8.5mg)

RNA:Protein Interaction in Human Disease

The mRNA in an organism is regulated through RNA editing and splicing. Errors in these processes can lead to altered protein expression or mutations in the RNA, causing neurological disorders, cancer or other maladies. To understand these processes, it is often required to study the physical interaction between protein and RNA through methods that use labeled RNA probes, such as the RNA electrophoretic mobility shift assay (REMSA).

In one example, Tang et al. used the RiboMAX™ Large Scale RNA Production System to produce labeled RNA probes to study the crosstalk between RNA editing and splicing machineries, and their role in cancer (1). They used the labeled RNA probes to confirm the interaction between protein and RNA by REMSA and in vitro UV crosslinking assays.

RNA:Protein Interaction in Plant Development

Grafting is a horticultural technique in which a bud or shoot of one plant is joined with the rootstock of another plant to cause phenotypical changes, such as taste or disease-resistance. This effect is partly due to long-distance transportation of mRNAs across the graft union to regulate plant growth. The movement of these mRNAs is facilitated by their binding to RNA-binding proteins and formation of ribonucleoprotein (RNP) complexes.

To study these RNP complexes in more detail, Wang et al. used the T7 RiboMAX™ Express Large Scale RNA Production System to produce biotin-labeled mRNA to perform REMSAs with RNA binding proteins (RBPs) of interest (2). They concluded that the stability of the RNP complex increases when mRNA binds more than one RNP, and that different combinations of RNPs also affect stability.

mRNA Therapeutics for Leigh Syndrome

Leigh syndrome is an inherited neurodegenerative condition that results in the loss of mental and motor skills, often causing death at just a few years of age. There are many genetic causes for this disease, one of which is a mutation in mitochondrial DNA. Yamada et al. used the RiboMAX™ Large Scale RNA Production System to study the potential use of mRNA as a therapeutic for mitochondrial DNA-associated Leigh syndrome (3). They showed that in vitro-transcribed mRNA encapsulated in a liposome-based carrier reduced mutant RNA in the mitochondria of diseased cells.

Guide RNA for CRISPR Gene Editing

To perform CRISPR/Cas9 gene editing, a guide RNA (gRNA) is needed to “guide” the Cas protein to the desired DNA modification site. In vitro transcription is becoming a common choice for creation of these guide RNAs. If desired, you can easily clone the DNA template into a PCR cloning vector, such as pGEM ®-T Easy, or any other vector of choice.

Bai et al. used the T7 RiboMAX™ Express Large Scale RNA Production System to create long gRNAs to test a new CRISPR/Cas9-mediated genome editing method for introducing precise mutations in zebrafish (4). The new method, which uses a long single-stranded DNA template, introduces accurate point mutations more efficiently and can be useful for creating human disease models in zebrafish.

cDNA Library Preparation

Complementary DNA (cDNA) libraries can be used to study gene expression and protein interactions, allowing you to search for new genes. They are most often used to study eukaryotic genes and proteins in a prokaryotic host. To prepare a cDNA library, you need to start with mRNA. One way to obtain this mRNA is through in vitro transcription of an organism or a specific gene of interest. These mRNAs are then reverse-transcribed to create the cDNA library. In vitro transcription is beneficial over other methods because it allows you to select the specific segments or sequences of the genome of interest.

Fujimori et al. developed a high-throughput cell-free mRNA display method combined with next-generation sequencing (NGS) for exploring protein interactome networks (5). The cDNA library used for the cell-free method was prepared with an mRNA library generated by the RiboMAX™ Large Scale RNA Production System (SP6).

Viral RNA Structure and Function

Four of the six known coronaviruses are sources of common cold infections around the world. Among them, the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) viruses have been known to cause severe, sometimes deadly illness (6).

With the help of the T7 RiboMAX™ Express Large Scale RNA Production System, Madhugiri et al. were able to transcribe a large amount of high-quality human coronavirus RNA for use in RNA structure probing with primer extension (7). This allowed them to analyze and visualize RNA secondary structures. Through bioinformatics and in vivo experiments performed with mutated viral DNA, they were able to determine that three stem-loop structures are required for viral replication. They also concluded that these structures are highly conserved across genera.

Development of RNAi Therapeutics

White Spot Syndrome Virus (WSSV) is considered the most serious infectious threat to the shrimp aquaculture industry. This virus is highly virulent and pathogenic, with a potential 100% mortality rate in shrimps such as tiger prawn and Atlantic white shrimp. Currently, there is no treatment for this virus. A WSSV non-structural protein, VP9, is suspected to be involved in viral genome replication, production of viral particles, and inhibition of host cell functions. However, the function of VP9 in vivo was not well understood.

Alenton et al. used RNAi to study host-pathogen molecular interactions in shrimp infected with WSSV (8). RNAi involves the use of double-stranded RNA (dsRNA) to silence expression of the corresponding gene. The T7 RiboMAX™ Express Large Scale RNA Production System was used to create dsRNA for VP9 and control target genes. They found that silencing of VP9 increased the viral clearance and overall survival rates in shrimp.

Viral RNA for Inoculation Studies

According to the CDC, hepatitis C virus (HCV) infection is a contributing factor in 50% of liver cancer cases in the United States (9). HCV is an oncogenic RNA virus that can be screened for and is largely treatable with antivirals. Many oncogenic DNA viruses have been shown to inactivate the p53 tumor suppressor protein, but how HCV affects p53 was unknown.

Mitchell et al. used the T7 RiboMAX™ Express Large Scale RNA Production System to transcribe genome-length HCV RNA from replication-incompetent control plasmids (10). The transcribed viral RNA was electroporated into HCV-permissive HepG2 cells to initiate viral infection. This allowed the team to study the effects on the p53 protein and other related proteins in HCV-infected cells. The results suggest that inhibition of p53 is actually caused by a host response to viral RNA replication—the activation of protein kinase R (PKR). This activation of PKR decreases the synthesis of all proteins in general, including p53, which dampens DNA damage response.

RNA Standards for Real-Time qPCR Assays

Chronic myeloid leukemia (CML) is a disease affecting hematopoietic stem cells that causes uncontrolled proliferation of abnormal blood cells. One treatment option is targeted therapy using tyrosine kinase inhibitors. During treatment, patients must be monitored for signs of minimal residual disease (MRD) to verify progress and avoid relapse. The most sensitive method for monitoring MRD is through molecular testing using real-time qPCR.

Kitamura et al. developed their own highly sensitive real-time qPCR assay for MRD, using the T7 RiboMAX™ Express Large Scale RNA Production System to create the RNA standards for the assay (11). The new assay is fast and easy to perform, making it useful in hospital laboratories and other low-throughput applications.

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References

  1. Tang, S.J. et al. (2020) Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development. Nat Commun 11, 799.
  2. Wang, S. et al. (2019) PbTTG1 forms a ribonucleoprotein complex with polypyrimidine tract-binding protein PbPTB3 to facilitate the long-distance trafficking of PbWoxT1 mRNA. Plant Sci. 280, 424–32.
  3. Yamada, Y. et al. (2020) Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene. Sci Rep 10, 7511.
  4. Bai, H. et al. (2020) CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. BMC Genomics 21, 67.
  5. Fujimori, S. et al. (2012) Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data. Sci Rep 2, 691.
  6. World Health Organization (28 March 2019) "Middle East Respiratory Syndrome Coronavirus (MERS-CoV)." World Health Organization, www.who.int/emergencies/mers-cov/en/
  7. Madhugiri, R. et al. (2018) Structural and function conservation of cis-acting RNA elements in 5´-terminal genome regions. Virology 517, 44–55.
  8. Alenton, R.R. (2016) Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii. Virus Res. 214, 65–70.
  9. U.S. Centers for Disease Control and Prevention (2016) Viral Hepatitis and Liver Cancer [Factsheet]. Retrieved from www.cdc.gov/nchhstp/newsroom/docs/factsheets/viral-hep-liver-cancer.pdf
  10. Mitchell, J.K. et al. (2017) Hepatitis C virus indirectly disrupts DNA damage-induced p53 responses by activating protein kinase R. mBio 8, e00121-17.
  11. Kitamura, H. et al. (2019) A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib. PLoS ONE 14, e0207170.