RNA and siRNA user applications

Phosphorothioate protected ssRNA for toll-like receptor research

Toll-like receptors (TLRs) are fundamental for the innate immune response. Two of these orphan receptors, TLR7 and TLR8, have been identified to act as sensors for single-stranded RNA (ssRNA). Whereas double stranded ribonucleic acid (dsRNA) was already known as a danger signal associated with viral infection and the stimulation of innate immune cells, the immunostimulatory potential of ssRNA is poorly understood and respective receptors were unknown. Only recently, members of the TLR family were found to play an important role in the sensing of ssRNAs. Activation of TLRs leads to the generation of an adaptive immune response, resulting in the eradication of pathogens. Guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DCs) and macrophages to secrete interferon-α and proinflammatory, as well as regulatory, cytokines.
Beside a research group at the TU Munich two other groups at the Imperial Cancer Research (ICRF) in London and the other at the Howard Hughes Medical Institute at Yale University in New Haven also reported on this phenomenon. Researchers in the group of Stefan Bauer at the TU Munich stated that this discovery has potential for the development of novel methods of immunization for vaccine design, using RNA as adjuvant.
Mediators of the innate immune system for the sensing of bacterial pathogens such as CpG-rich motifs have already entered clinical trials.

Related IBA products & quality

Products of our GeneTAGnology division have been used, amongst others, by a research group at the TU Munich to identify members of the Toll-like receptor (TLR) family as sensors for single stranded RNA.

IBA ssRNA as well as our other RNA products are manufactured in state-of-the art facilities. Due to the phosphorothioate modifications, the stability of this kind of ssRNA-oligonucleotides is increased significantly. Quality is checked routinely according to our stringent quality standards. Special care is taken to use only highest quality raw materials. IBAs ssRNA products are especially recognized for their superior performance in cell culture assays looking for an immune response, avoiding any kind of endotoxin-related unspecific effects.

For references click here.

Investigation of the RNAi enzyme complex RISC

Cy5-labeled and unlabeled siRNAs, custom synthesized by IBA GmbH, were microinjected into human cells and show the Exportin-5 mediated cytoplasmic localization (Figure 1; Ohrt and Schwille, 2008).

Cy-5-labeled and unlabaled siRNAs microinjected into human cells
Figure 1: Injected single labeled siRNAs in ER293 cells. Scale bars are 10μm. (Kindly provided by Dr. Thomas Ohrt)

By using Fluorescence Cross-Correlation Spectroscopy (FCCS), incorporation of a labeled guide strand into the RNA-induced silencing complex (RISC), which is the key enzyme of the RNAi mechanism, can be measured in living cells.
Figure 2 shows an outline of another experiment, which is useful for target-RNA-RISC interaction: The EGFP-labeled Ago protein in RISC contains an unlabeled siRNA and its interaction with the Cy5-labeled complementary target-RNA (50 nt long labeled RNA from IBA) can be visualized by FCCS measurements. A non-perfect match, as it is shown in the figure, leads to a reduction of the RNAi silencing efficiency which leads to increased interaction levels of RISC with its target-RNA.
Ohrt et al. (2008) showed that RISC is loaded in the cytoplasm and can then be imported into the nucleus to access targets in both cellular compartments.

Cy-5 labeled RNA

Figure 2: An outline of the experiment is illustrated. EGFP-Ago2 serves as a fluorescent label for RISC that contains unlabeled RNA and can interact with the Cy5-labeled target-RNA. The interaction with the target-RNA is determined by FCCS in vivo. The phosphate group of the unlabeled RNA is shown; Cy5 is shown as a red circle. (Kindly provided by Dr. Thomas Ohrt)

Literature:

Thomas Ohrt and Petra Schwille (2008)
siRNA modifications and sub-cellular localization: a question of intracellular transport?
Current Pharmaceutical Design 14:3674-3685
Thomas Ohrt, Jörg Mütze, Wolfgang Staroske, Lasse Weinmann, Julia Höck, Karin Crell, Gunter Meister and Petra Schwille (2008) 
Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.
Nucleic Acids Research 36(20):6439–6449

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Appl Note_RISC.pdf Appl Note_RISC.pdf (550.0 kB)
RNA hairpin folding

RNA sequences were manually designed by Nagel et al. and custom synthesized by IBA. These sequences can fold into either of two mutually exclusive hairpins (Figure) and thus folding of the two competing hairpins from the single-stranded RNA was detected by secondary structure probing. To trap the RNA in the kinetically favored conformation, it was denatured either by heat or by acid, followed by rapid cooling or a pH-jump to neutral pH, respectively. Both methods gave identical results, indicating that the trapping procedure does not influence the kinetic competition between the two mutually exclusive hairpins.
Taken together Nagel et al. could determine some parameters of the RNA hairpin folding rate and verify that the folding program "Barriers" is capable of predicting the folding ratios of competing structures in an RNA chain.

Figure 1: Schematic representation of the folding, in which the A nucleotides can base pair with B, resulting in the yellow loop, or B with C, resulting in the dark blue loop. In the kinetic experiments there should not be an interchange between the two mutually exclusive structures, while in thermodynamic experiments they should be in equilibrium.
(Kindly provided by Dr. Maarten de Smit)

Literature:

J. H. A. Nagel, C. Flamm, I. L. Hofacker, K. Franke, M. H. de Smit, P. Schuster and C. W. A. Pleij (2006) 
Structural parameters affecting the kinetics of RNA hairpin formation. 
Nucleic Acids Research34(12): 3568–3576.

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Appl Note_RNA hairpin.pdf Appl Note_RNA hairpin.pdf (470.4 kB)
RNase assembly

The endoribonuclease "RNase mitochondrial RNA processing" ("RNase MRP") is composed of an RNA molecule and several protein subunits. For their investigation a part of the RNA in RNase MRP (the P3 arm) was custom synthesized by IBA (Fig.). It was used for binding experiments by isothermal titration calorimetry. Hands-Taylor et al. could show that the P3 RNA interacts with the heterodimer of two RNase subunits (Rpp20 and Rpp25) in a stoichiometry of 1:1:1, thus mediating assembly of the RNase holoenzyme. Importantly, the formation of a 1:1 heterodimer is an obligate prerequisite for RNA binding. The single subunits cannot (Rpp20) or only weakly (Rpp25) interact with P3 RNA.

Figure: Expected secondary structure of the P3 arm of the "RNase MRP" RNA, custom synthesized by IBA.
(Kindly provided by
Dr. Maria R. Conte)

Literature:

Katherine L. D. Hands-Taylor, Luigi Martino, Renée Tata, Jeffrey J. Babon, Tam T. Bui, Alex F. Drake, Rebecca L. Beavil, Ger J. M. Pruijn, Paul R. Brown and Maria R. Conte (2010)
Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA. 
Nucleic Acids Research 38(12):4052-66

"In our experience, the RNA oligos purchased from IBA are of a very high quality and purity, ideal for quantitative biophysical analysis. In our samples we found no contamination, i.e. from prematurely terminated oligos, which could be a problem in quantitative characterisations."
Dr. Maria R. Conte, Structural Biology, King's College London, UK

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Appl Note_RNase.pdf Appl Note_RNase.pdf (322.3 kB)