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The Many Uses for in vitro Transcribed RNA

Leah Cronan

Promega Corporation
Publication Date 05/2019; tpub_211

Abstract

This article describes recent advancements in RNA research made possible through use of the T7 RiboMAX™ Express Large-Scale RNA Production System for in vitro transcription.

Introduction

Our Riboprobe® Systems are great for preparation of microgram amounts of single-stranded RNA transcripts with radiolabeled ribonucleotides, which are useful as RNA probes. But what if you need large amounts of non-radiolabeled RNA that is biologically active? Our RiboMAX™ Systems will do the trick. But, what if you need a large amount of non-radiolabeled RNA that is biological active, and you need it done quickly? We have a tool for that, too.

The T7 RiboMAX™ Express Large-Scale RNA Production System (Cat.# P1320) is an in vitro transcription system designed for the consistent production of large amounts of RNA in a short amount of time. Milligram amounts of high-quality RNA transcripts up to 14kb have been generated using this kit.

RNA transcription is typically not the end of the story: it can be the setup for a great beginning. Transcribed RNA has many uses, a few of which are described below.

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Figure 1. Schematic illustrating how the T7 RiboMax™ Express Large-Scale RNA Production System functions.

RNA Structure and Function

Four of the six known coronaviruses are sources of common cold infections around the world. The severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) viruses have been known to cause severe, sometimes deadly illness. Between November 2002 and July 2003, an outbreak of SARS with a case-fatality rate of 9.6% was first reported in Asia and spread quickly throughout the world (1). MERS was first reported in 2012 in Saudi Arabia and has caused the deaths of 806 people out of 2,279 reported infected people in 27 countries (2).

With the help of the T7 RiboMAX™ Express kit, Madhugiri et al were able to transcribe a large amount of high-quality human corona virus RNA for use in RNA structure probing with primer extension (3). This allowed the researchers 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. Read the full article here.

RNA:Protein Interactions

Grafting is a horticultural technique commonly used for propagation of woody plants such as fruit trees or roses. Grafting is the combining of the scion, or upper part of a plant, with the rootstock of another plant. The rootstock and scion can be of the same or different species, and will influence certain traits of each other. This allows for particular phenotypic traits, like taste or appearance, to be combined with disease-resistant rootstock to produce a hardier plant.

On a molecular level, mRNAs bind to RNA binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. This protects the mRNAs as they move over the long distances through graft unions to regulate plant growth and development. To study these RNP complexes in more detail, Wang et al used the T7 RiboMAX™ Express kit to produce biotin-labeled mRNA, which they used to perform electrophoretic mobility-shift assays (EMSAs) with RBPs of interest (4). The researchers concluded that the stability of the RNP complex increases when mRNA binds more than one RNP, and found different combinations of RNPs lead to differences in RNP complex stability. Read the full article here.

RNAi

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 the potential for a 100% mortality rate in the penaeid shrimp family, which includes tiger prawn and Atlantic white shrimp. Currently, there is no treatment for this virus.

Alenton et al used RNAi to study host-pathogen molecular interactions in shrimp infected with WSSV (5). RNAi is the use of double-stranded RNA (dsRNA) to silence expression of the corresponding gene. 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. The T7 RiboMAX™ Express kit was used to create dsRNA for VP9 and control target genes. The researchers found that silencing of VP9 increased the viral clearance in the shrimp, as well as overall survival rates. Read the full article here.

Viral Inoculation

According to the CDC, hepatitis C virus (HCV) infection was a contributing factor in 50% of cases of liver cancer in the United States (6). HCV is an oncogenic RNA virus that can be screened for and is treatable. The p53 protein is a tumor suppressor protein that assists in DNA damage-control through initiation of senescence, cell cycle arrest, or apoptosis. p53 is inactivated by many oncogenic DNA viruses. Mutation-induced changes in function or regulation of p53 have also been found in the majority of human cancers.

Mitchell et al used the T7 RiboMAX™ Express kit to transcribe genome-length HCV RNA from replication-incompetent control plasmids (7). 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 and enzymes, in HCV-infected cells. The researchers 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. Read the full article here.

Real-Time RT-PCR

Chronic myeloid leukemia (CML) is a disease of hematopoietic stem cells that leads to uncontrolled proliferation of abnormal blood cells. One treatment option is targeted therapy in the form of a tyrosine kinase inhibitor. While using this treatment, patients must be monitored to watch for signs of minimal residual disease (MRD) in order to verify progress and avoid relapse.

The most sensitive method of monitoring MRD is through molecular testing using real-time qPCR. During development of their own highly sensitive real-time qPCR test for MRD, Kitamura et al made use of the T7 RiboMAX™ Express kit to create the RNA standards used in the assay (8). The assay the researchers developed is fast and easy to perform, making it useful in hospital laboratories and other low-throughput situations. Read the full article here.

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References

  1. SARS Investigative Team, U.S. Centers for Disease Control and Prevention (2003) Revised U.S. Surveillance Case Definition for Severe Acute Respiratory Syndrome (SARS) and Update on SARS Cases — United States and Worldwide, December 2003. MWWR Morb. Mortal. Wkly. Rep. 52, 1202–6.
  2. World Health Organization (28 March 2019) "Middle East Respiratory Syndrome Coronavirus (MERS-CoV)." World Health Organization, www.who.int/emergencies/mers-cov/en/
  3. Madhugiri, R. et al. (2018) Structural and function conservation of cis-acting RNA elements in 5´-terminal genome regions. Virology 517, 44–55.
  4. 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.
  5. Alenton, R.R. (2016) Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii. Virus Res. 214, 65–70.
  6. 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
  7. 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.
  8. 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.