Publications by authors named "Nada Raddaoui"

5 Publications

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Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry.

Chembiochem 2020 08 20;21(15):2214-2218. Epub 2020 Apr 20.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany.

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.
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http://dx.doi.org/10.1002/cbic.202000081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496099PMC
August 2020

Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ.

Chembiochem 2017 09 24;18(17):1716-1720. Epub 2017 Jul 24.

Center for Integrated Protein Science (CiPSM), Department of Chemistry, LMU München, Butenandtstrasse 5-13, 81377, München, Germany.

The in vivo incorporation of alkyne-modified bases into the genome of cells is today the basis for the efficient detection of cell proliferation. Cells are grown in the presence of ethinyl-dU (EdU), fixed and permeabilised. The incorporated alkynes are then efficiently detected by using azide-containing fluorophores and the Cu -catalysed alkyne-azide click reaction. For a world in which constant improvement in the sensitivity of a given method is driving diagnostic advancement, we developed azide- and alkyne-modified dendrimers that allow the establishment of sandwich-type detection assays that show significantly improved signal intensities and signal-to-noise ratios far beyond that which is currently possible.
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http://dx.doi.org/10.1002/cbic.201700209DOI Listing
September 2017

Synthesis of (R)-Configured 2'-Fluorinated mC, hmC, fC, and caC Phosphoramidites and Oligonucleotides.

Org Lett 2016 09 19;18(17):4368-71. Epub 2016 Aug 19.

Center for Integrated Protein Science, Department of Chemistry, Ludwig-Maximilians-Universität München , Butenandtstraße 5-13, 81377 Munich, Germany.

Investigation of the function of the new epigenetic bases requires the development of stabilized analogues that are stable during base excision repair (BER). Here we report the synthesis of 2'-(R)-fluorinated versions of the phosphoramidites of 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). For oligonucleotides containing 2'-(R)-F-fdC, we show that these compounds cannot be cleaved by the main BER enzyme thymine-DNA glycosylase (TDG).
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http://dx.doi.org/10.1021/acs.orglett.6b02110DOI Listing
September 2016

Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction.

Chromosome Res 2016 09 19;24(3):299-307. Epub 2016 Apr 19.

Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany.

In situ hybridisation is a powerful tool to investigate the genome and chromosome architecture. Nick translation (NT) is widely used to label DNA probes for fluorescence in situ hybridisation (FISH). However, NT is limited to the use of long double-stranded DNA and does not allow the labelling of single-stranded and short DNA, e.g. oligonucleotides. An alternative technique is the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), at which azide and alkyne functional groups react in a multistep process catalysed by copper(I) ions to give 1,4-distributed 1,2,3-triazoles at a high yield (also called 'click reaction'). We successfully applied this technique to label short single-stranded DNA probes as well as long PCR-derived double-stranded probes and tested them by FISH on plant chromosomes and nuclei. The hybridisation efficiency of differently labelled probes was compared to those obtained by conventional labelling techniques. We show that copper(I)-catalysed azide-alkyne cycloaddition-labelled probes are reliable tools to detect different types of repetitive sequences on chromosomes opening new promising routes for the detection of single copy gene. Moreover, a combination of FISH using such probes with other techniques, e.g. immunohistochemistry (IHC) and cell proliferation assays using 5-ethynyl-deoxyuridine, is herein shown to be easily feasible.
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http://dx.doi.org/10.1007/s10577-016-9522-zDOI Listing
September 2016

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Nat Chem Biol 2014 Jul 18;10(7):574-81. Epub 2014 May 18.

Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
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http://dx.doi.org/10.1038/nchembio.1532DOI Listing
July 2014