Entwicklung neuer Diagnostikmethoden für genetische Erkrankungen basierend auf der Click-Chemie

Jede Krankheit kann man vermutlich auf eine genetische Störung zurückführen. Die Methoden u. a. zur Detektion von Chromosomenaberrationen sind daher von großer Bedeutung für Diagnostik und Therapiezwecke. Heutzutage beruht die Diagnostik von genetischen Krankheiten auf der Detektion des entsprechenden defekten Gens oder auf der Messung entweder des jeweiligen Transkripts durch die quantitative „real-time-Polymerase Chain Reaction“ (RT-qPCR)oder des jeweiligen Proteins via z.B. Massenspektrometrie. Auch die Durchflusszytometrie wird als Standardmethode verwendet, um für Krebszellen spezifische Oberflächenproteine zu detektieren und zu quantifizieren. Vor allem wird sie für die Analyse des Zellzyklus bei der Diagnose benutzt. Ein Vorteil der Methode ist die gleichzeitige Analyse einer großen Anzahl von Zellen (z.B. Blutzellen). Eine Abweichung in der Zellproliferationsrate oder eine Anomalität in der Zellvermehrung deuten auf eine genetische Mutation hin. Obwohl diese Methoden gut etabliert sind und in fast jedem Diagnostiklabor verwendet werden, bleiben sie indirekte Nachweismethoden. Direkte Informationen bekommt man mit der Fluoreszenz-in-situ-Hybridisierung FISH, die es ermöglicht, die betreffenden Gene in situ sichtbar zu machen. Hiermit wird ein Ziel-Gen mit einer fluoreszierenden Sonde markiert und in seinem ursprünglichen Platz in der Zelle mit Hilfe der Fluoreszenzmikroskopie detektiert. Wichtige Informationen, die durch das Aufschließen der Zellen und das Isolieren der DNA oder RNA verloren gehen können, bleiben mit der FISH erhalten. Sie ermöglicht es, Informationen auf der Ebene einzelner Zellen zu gewinnen. Außerdem kann die Expressionsaktivität von einer Zelle zur andern unterschieden werden. Expressionsaktive Zellen werden von den expressionsstillen differenziert. Die Detektionsgrenze erkrankter Zellen ist ein ernstes Problem, denn Krebspatienten müssen trotz erster Erfolgszeichen nach der Therapie wiederholt und regelmäßig medizinisch kontrolliert werden. Erst nach fünf Jahren ohne Rückfall der Krankheit (Remission) können die behandelnden Ärzte und die betroffenen Patienten von Therapieerfolg sprechen. Trotz der Entwicklung sowohl neuer Farbstoffe als auch unzähliger neuer Methoden für die Fluoreszenzmikroskopie, die ein verbessertes Signal-zu-Hintergrund-Verhältnis anbieten, ist die Detektionsgrenze weiterhin der limitierende Faktor. Diese Methoden beruhen auf der Herstellung von Sonden, die willkürlich mit Farbstoffen versehen werden. Die ungenaue limitierte Markierung in einer uneinheitlichen Sondenherstellung führt zu unterschiedlichen Ergebnissen. In dieser Arbeit wurde die in der Gruppe von Thomas Carell für Nukleinsäuren entwickelte Cu(I)-katalysierte Alkin-Azid-Cykloaddition (CuAAC) benutzt, um neue mit fluoreszenten Farbstoffen markierten Sonden zu entwickeln, die eine bessere Sensitivität und ein niedrigeres Signal-zu-Hintergrund-Verhältnis bieten.Any disease can be traced back to a genetic disorder. The methods for the detection of chromosome aberrations are therefore of great importance for diagnostics and therapy purposes. Nowadays, the diagnosis of genetic diseases is based on the detection of the corresponding defective gene or on the measurement of either the corresponding transcript by quantitative real-time PCR (RT-qPCR) or the corresponding protein via mass spectrometry. Flow cytometry is also used as standard method to detect, measure and quantify surface proteins specific for cancer cells. Flow cytometry is mainly used in diagnosis for analysis of the cell cycle. One advantage of this is the simultaneous analysis of a large number of cells (e.g. blood cells). A deviation in the cell proliferation rate or an anomaly in cell proliferation indicates a genetic disorder of the cells. Although these methods are well established and used in almost every diagnostic laboratory, they remain indirect gene detection methods. Direct information is obtained using fluorescence in situ hybridisation (FISH), which makes it possible to visualise the relevant genes in situ. Thus, a target gene (sample) is marked with a fluorescent probe and detected in its original position whithin the cell. Important information that can be lost by breaking down the cells and isolating the DNA or RNA is retained with FISH. It makes it possible to obtain information at the level of individual cells. In addition, expression activity can be differentiated from one cell to another. Expression pattern of active cells are differentiated from those that are silent. Fluorescence microscopy is used to visualize these probes. This detection limit of mutated cells is a serious problem, because cancer patients must be medically checked regularly despite the first signs of success after therapy. Only after five years without relapse of the disease (remission) can the physicians and the concerned patients speak about therapeutic success. Despite the development of new dyes and numerous methods for fluorimetric microscopy, which offer an improved signal-to-background ratio, detection is still limited. These methods are based on the production of samples, which are randomly and unquantitatively labelled. The inaccurate limited labelling gives inconsistent probe preparations which provides differences in results. Another problem is the cell itself, which could contain a genetic mutation but do not express it. In this work the Cu(I)-catalysed alkyne-azide cycloaddition (CuAAC) on nucleic acids and on DNA, which was developed in the group of Thomas Carell, was used to develop new fluorescence labelled probes, that offer better sensitivity and a lower signal-to-background rati

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

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

Synthesis of (<i>R</i>)‑Configured 2′-Fluorinated mC, hmC, fC, and caC Phosphoramidites and Oligonucleotides

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′-(<i>R</i>)-fluorinated versions of the phosphoramidites of 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). For oligonucleotides containing 2′-(<i>R</i>)-F-fdC, we show that these compounds cannot be cleaved by the main BER enzyme thymine-DNA glycosylase (TDG)

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using Cu-I-catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy

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

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