TU Darmstadt / ULB / TUbiblio

Development of RNA aptamers binding environmental and food contaminants

Kramat, Janice (2023)
Development of RNA aptamers binding environmental and food contaminants.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00024484
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

The pollution of the environment by various substances is a central issue of our time. Humans introduce a wide variety of pollutants into nature through industrial processes, the use of certain substances in agriculture and the application of pharmaceuticals in factory farming. These contaminants can exert negative effects on the environment and living organisms due to their presence or specific effect and return to humans through various pathways. The detection of such substances in environmental samples or food is therefore essential for uncovering their entry routes and distribution. Analytical methods such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS) are already used for this purpose. Both methods offer precise analysis and can detect even the smallest traces of various substances. However, this requires specially qualified staff, extensive laboratory equipment and very expensive instruments. In addition, the analyses take a certain amount of time and cannot be used flexibly or portably. Detection methods that do not have these disadvantages are based on sensor technology using biological elements. They represent rapid, simple, comparatively inexpensive analysis platforms that can be used on site. These are so-called biosensors and are based on a harmonised interplay of different components. The basis for the recognition of an analyte is provided by the bioreceptors. These can consist of whole microorganisms, isolated proteins such as enzymes or antibodies, or even short DNA or RNA molecules. They are responsible for the sensing of the analyte and enable the generation of a signal. This is subsequently converted into a visible or measurable signal by a transducer. Finally, a third component can be used to amplify and process this signal. Thus, biosensors provide not only a qualitative but also a quantitative evaluation of a wide variety of analytes. Based on the problems caused by environmental as well as food contamination, the aim of this study has been the development of short, single-stranded RNA sequences as suitable bioreceptors. These are also called aptamers and their application as recognition elements in biosensors make them aptasensors. Aptamers are able to bind a variety of different ligands. These include whole cells, proteins, but also ions or small compounds. In this work, the latter were selected as target molecules for RNA aptamers. The substance classes of artificial sweeteners as sugar substitutes, bisphenols, which are necessary to produce synthetic materials, as well as antibiotics used in human medicine and factory farming were chosen in particular. The individual representatives of these classes were acesulfame K, cyclamate, saccharin and sucralose as sweeteners, the bisphenols A, F, S and 4-hydroxyacetophenone, a degradation product of bisphenol A, as well as the antibiotics kanamycin A and levofloxacin. Some of these substances can already be detected in the environment and partially cause damage to nature and living organisms or are suspected of doing so. Therefore, they represent interesting analytes for aptamer-based biosensors. The origin of the development of aptamers involved different in vitro selections of ligand-binding RNA molecules from an extensive sequence library. The method used for this was derived from SELEX (systematic evolution of ligands by exponential enrichment), which was developed in 1990. It is called Capture-SELEX and is based on an iterative process. This always includes the immobilisation of RNA molecules, elution of these through interaction with the chosen ligand, amplification of the selected sequences and their preparation to be used as a new RNA pool for the next selection round. This led to an enrichment of potential aptamers for the target molecules bisphenol A, kanamycin A as well as levofloxacin. The selection against bisphenol A revealed that binding did not occur to the ligand itself but to ethanol, which was required for the solubility of the bisphenol in aqueous solution. Based on this discovery, further steps in the development of an RNA aptamer were focused on monohydric alcohols as ligands. In addition to ethanol, isopropanol and methanol were also examined as target molecules in Capture-SELEX experiments. No binding between RNA and the ligand could be detected for methanol. Six different RNA sequences could be identified for ethanol and isopropanol using cloning and sequencing techniques. These sequences were evaluated for their ability to distinguish between the two alcohols, which led to the ‘aptamer I’ that preferentially binds isopropanol. Based on the prediction of a secondary structure, various truncations of aptamer I were made subsequently. These were again assayed for their ability to distinguish ethanol and isopropanol as ligands. Certain truncations led to a loss of the binding ability of the RNA to the applied ligands. Others still exhibited binding but did not show any improvement in specificity. Attention has to be paid to the possibility that the monohydric alcohols ethanol and isopropanol are ligands that are capable of forming non-specific interactions with RNA. Therefore, further research is needed to determine whether it is a 'classical' aptamer-ligand binding. The in vitro selection against levofloxacin resulted in an enrichment of target-binding aptamers. Subsequently, the entire selection was analysed using Next Generation Sequencing. The resulting data were bioinformatically evaluated in cooperation with the research group ‘Self-Organising Systems’ of the Department of Electrical Engineering and Information Technology. In addition to assessing the enrichment process of levofloxacin-binding RNA molecules, eight different sequences of potential aptamers were identified. These were tested for their ability to distinguish between two closely related ligands. In addition to the intended target molecule, the structurally very similar ciprofloxacin was used. This resulted in the aptamer LXC, which had the best distinguishing ability and a dissociation constant in the low micromolar range for binding to levofloxacin. Subsequently, based on the predicted secondary structure of LXC, truncations as well as mutations of the sequence were carried out. These were evaluated by isothermal titration calorimetry (ITC). It resulted in the levofloxacin-binding aptamer trLXC. Based on the ITC mutation studies and subsequent in-line probing experiments, the predicted secondary structure could be confirmed. Furthermore, it allowed the identification of regions involved in ligand binding. Thus, a levofloxacin-binding aptamer ready to be used as a receptor in a biosensor was successfully developed and characterised.

Typ des Eintrags: Dissertation
Erschienen: 2023
Autor(en): Kramat, Janice
Art des Eintrags: Erstveröffentlichung
Titel: Development of RNA aptamers binding environmental and food contaminants
Sprache: Englisch
Referenten: Süß, Prof. Dr. Beatrix ; Niopek, Prof. Dr. Dominik
Publikationsjahr: 2023
Ort: Darmstadt
Kollation: 123 Seiten
Datum der mündlichen Prüfung: 30 August 2023
DOI: 10.26083/tuprints-00024484
URL / URN: https://tuprints.ulb.tu-darmstadt.de/24484
Kurzbeschreibung (Abstract):

The pollution of the environment by various substances is a central issue of our time. Humans introduce a wide variety of pollutants into nature through industrial processes, the use of certain substances in agriculture and the application of pharmaceuticals in factory farming. These contaminants can exert negative effects on the environment and living organisms due to their presence or specific effect and return to humans through various pathways. The detection of such substances in environmental samples or food is therefore essential for uncovering their entry routes and distribution. Analytical methods such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS) are already used for this purpose. Both methods offer precise analysis and can detect even the smallest traces of various substances. However, this requires specially qualified staff, extensive laboratory equipment and very expensive instruments. In addition, the analyses take a certain amount of time and cannot be used flexibly or portably. Detection methods that do not have these disadvantages are based on sensor technology using biological elements. They represent rapid, simple, comparatively inexpensive analysis platforms that can be used on site. These are so-called biosensors and are based on a harmonised interplay of different components. The basis for the recognition of an analyte is provided by the bioreceptors. These can consist of whole microorganisms, isolated proteins such as enzymes or antibodies, or even short DNA or RNA molecules. They are responsible for the sensing of the analyte and enable the generation of a signal. This is subsequently converted into a visible or measurable signal by a transducer. Finally, a third component can be used to amplify and process this signal. Thus, biosensors provide not only a qualitative but also a quantitative evaluation of a wide variety of analytes. Based on the problems caused by environmental as well as food contamination, the aim of this study has been the development of short, single-stranded RNA sequences as suitable bioreceptors. These are also called aptamers and their application as recognition elements in biosensors make them aptasensors. Aptamers are able to bind a variety of different ligands. These include whole cells, proteins, but also ions or small compounds. In this work, the latter were selected as target molecules for RNA aptamers. The substance classes of artificial sweeteners as sugar substitutes, bisphenols, which are necessary to produce synthetic materials, as well as antibiotics used in human medicine and factory farming were chosen in particular. The individual representatives of these classes were acesulfame K, cyclamate, saccharin and sucralose as sweeteners, the bisphenols A, F, S and 4-hydroxyacetophenone, a degradation product of bisphenol A, as well as the antibiotics kanamycin A and levofloxacin. Some of these substances can already be detected in the environment and partially cause damage to nature and living organisms or are suspected of doing so. Therefore, they represent interesting analytes for aptamer-based biosensors. The origin of the development of aptamers involved different in vitro selections of ligand-binding RNA molecules from an extensive sequence library. The method used for this was derived from SELEX (systematic evolution of ligands by exponential enrichment), which was developed in 1990. It is called Capture-SELEX and is based on an iterative process. This always includes the immobilisation of RNA molecules, elution of these through interaction with the chosen ligand, amplification of the selected sequences and their preparation to be used as a new RNA pool for the next selection round. This led to an enrichment of potential aptamers for the target molecules bisphenol A, kanamycin A as well as levofloxacin. The selection against bisphenol A revealed that binding did not occur to the ligand itself but to ethanol, which was required for the solubility of the bisphenol in aqueous solution. Based on this discovery, further steps in the development of an RNA aptamer were focused on monohydric alcohols as ligands. In addition to ethanol, isopropanol and methanol were also examined as target molecules in Capture-SELEX experiments. No binding between RNA and the ligand could be detected for methanol. Six different RNA sequences could be identified for ethanol and isopropanol using cloning and sequencing techniques. These sequences were evaluated for their ability to distinguish between the two alcohols, which led to the ‘aptamer I’ that preferentially binds isopropanol. Based on the prediction of a secondary structure, various truncations of aptamer I were made subsequently. These were again assayed for their ability to distinguish ethanol and isopropanol as ligands. Certain truncations led to a loss of the binding ability of the RNA to the applied ligands. Others still exhibited binding but did not show any improvement in specificity. Attention has to be paid to the possibility that the monohydric alcohols ethanol and isopropanol are ligands that are capable of forming non-specific interactions with RNA. Therefore, further research is needed to determine whether it is a 'classical' aptamer-ligand binding. The in vitro selection against levofloxacin resulted in an enrichment of target-binding aptamers. Subsequently, the entire selection was analysed using Next Generation Sequencing. The resulting data were bioinformatically evaluated in cooperation with the research group ‘Self-Organising Systems’ of the Department of Electrical Engineering and Information Technology. In addition to assessing the enrichment process of levofloxacin-binding RNA molecules, eight different sequences of potential aptamers were identified. These were tested for their ability to distinguish between two closely related ligands. In addition to the intended target molecule, the structurally very similar ciprofloxacin was used. This resulted in the aptamer LXC, which had the best distinguishing ability and a dissociation constant in the low micromolar range for binding to levofloxacin. Subsequently, based on the predicted secondary structure of LXC, truncations as well as mutations of the sequence were carried out. These were evaluated by isothermal titration calorimetry (ITC). It resulted in the levofloxacin-binding aptamer trLXC. Based on the ITC mutation studies and subsequent in-line probing experiments, the predicted secondary structure could be confirmed. Furthermore, it allowed the identification of regions involved in ligand binding. Thus, a levofloxacin-binding aptamer ready to be used as a receptor in a biosensor was successfully developed and characterised.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Die Verschmutzung der Umwelt durch verschiedenste Substanzen ist ein zentrales Thema der heutigen Zeit. Der Mensch bringt durch industrielle Prozesse, der Nutzung bestimmter Substanzen in der Landwirtschaft sowie der Anwendung von Medikamenten in der Massentierhaltung unterschiedlichste Schadstoffe in die Natur ein. Diese Verunreinigungen können durch ihre Anwesenheit oder spezifische Wirkung negative Effekte auf die Umwelt sowie Lebewesen ausüben und gelangen durch verschiedene Kreisläufe wieder zurück zum Menschen. Die Detektion solcher Stoffe in Umweltproben oder Lebensmitteln ist somit für die Aufdeckung ihrer Eintragungswege sowie Verbreitung essentiell. Dafür nutzt man bereits Analysemethoden wie die Hochleistungsflüssigkeitschromatographie (HPLC) oder Massenspektrometrie (MS). Beide Verfahren bieten eine präzise Untersuchung und können sogar geringste Spuren verschiedener Substanzen ermitteln. Dafür werden jedoch geschultes Personal, umfangreiches Laborequipment sowie sehr teure Messinstrumente benötigt. Zusätzlich beanspruchen die Analysen eine gewisse Zeit und können nicht flexibel oder mobil eingesetzt werden. Untersuchungsmethoden, welche diese Nachteile nicht aufweisen, stützen sich auf die Sensorik mittels biologischer Elemente. Sie stellen schnelle, einfache, vergleichsweise preisgünstige sowie vor Ort einsetzbare Analyseplattformen dar. Diese werden auch als Biosensoren bezeichnet und basieren auf einem aufeinander abgestimmten Zusammenspiel aus verschiedenen Komponenten. Die Grundlage des Erkennens eines Analyten bieten dabei die Biorezeptoren. Diese können aus ganzen Mikroorganismen, isolierten Proteinen wie Enzymen oder Antikörpern oder auch kurzen DNA- oder RNA-Molekülen bestehen. Sie übernehmen das ‚sensing‘ und ermöglichen die Erzeugung eines Signals. Dieses wird anschließend von einem weiteren Sensor, dem Transducer, in ein sicht- oder messbares Signal umgewandelt. Final kann es noch mittels einer dritten Komponente zur Signalverstärkung und -verarbeitung kommen. Biosensoren gewährleisten somit neben einer qualitativen auch eine quantitative Auswertung von verschiedensten Analyten. Auf Grundlage der Probleme durch Verunreinigungen von Umwelt sowie Lebensmitteln war das Ziel der hier vorgelegten Arbeit geeignete Biorezeptoren in Form von kurzen einzelsträngigen RNA-Sequenzen zu entwickeln. Diese werden auch als Aptamere bezeichnet und ihre Nutzung als Erkennungselemente in Biosensoren machen diese zu Aptasensoren. Aptamere sind in der Lage eine Vielzahl von verschiedenen Liganden zu binden. Dazu zählen ganze Zellen, Proteine, aber auch Ionen oder kleine molekulare Verbindungen. Im Rahmen dieser Arbeit wurden letztere als Zielmoleküle für RNA-Aptamere ausgewählt. Dabei wurde sich genauer auf die Substanzklassen der synthetischen Süßungsmittel als Zuckerersatzstoffe, die Bisphenole, welche zur Herstellung von Kunstoffen benötigt werden sowie die in Human- und Veterinärmedizin genutzten Antibiotika festgelegt. Die einzelnen Vertreter dieser Klassen waren Acesulfam K, Cyclamat, Saccharin und Sucralose als Süßstoffe, die Bisphenole A, F, S und 4-Hydroxyaceto-phenon, ein Abbauprodukt von Bisphenol A, sowie die Antibiotika Kanamycin A und Levofloxacin. Einige dieser Stoffe können bereits in der Umwelt detektiert werden und verursachen teilweise Schäden in der Natur sowie Lebewesen oder werden diesbezüglich verdächtigt. Sie stellen somit interessante Analyten für Aptamer-basierte Biosensoren dar. Ausgangspunkt für die Entwicklung entsprechender Aptamere bildeten verschiedene in vitro Selektionen von Liganden-bindenden RNA-Molekülen, welche aus einer umfangreichen Sequenz-Bibliothek stammten. Die dafür verwendete Methode leitete sich von der 1990 entwickelten SELEX (systematic evolution of ligands by exponential enrichment) ab. Sie wird als Capture-SELEX bezeichnet und basiert auf einem iterativen Prozess. Dieser umfasst die Immobilisierung von RNA-Molekülen, Elution dieser durch Interaktion mit dem gewählten Liganden, Amplifizierung der selektierten Sequenzen sowie ihrer Verwendung als neuer RNA-Pool für die nächste Selektionsrunde. Dies führte für die Zielmoleküle Bisphenol A, Kanamycin A sowie Levofloxacin zu einer Anreicherung von potentiellen Aptameren. Im Falle der Selektion gegen Bisphenol A kam es jedoch nicht zur Bindung an den Liganden selbst sondern Ethanol, welches für die Löslichkeit des Bisphenols in wässriger Lösung benötigt wurde. Aufgrund dieser Erkenntnis wurden weitere Schritte der Entwicklung eines RNA-Aptamers auf einwertige Alkohole als Liganden ausgerichtet. Neben Ethanol wurden so auch Isopropanol und Methanol als Zielmoleküle in Capture-SELEX-Experimenten untersucht. Für Letzteres konnte keine Bindung zwischen RNA und dem Liganden festgestellt werden. Somit konnten mittels Klonierung und Sequenzierung für Ethanol sowie Isopropanol sechs verschiedene RNA-Sequenzen identifiziert werden. Sie wurden nach ihrer Fähigkeit zwischen beiden Alkoholen zu unterscheiden bewertet. Dies führte zu dem bevorzugt Isopropanol-bindenden ‚Aptamer I‘. Anhand der Vorhersage einer Sekundärstruktur wurden anschließend verschiedene Verkürzungen des Aptamers I vorgenommen. Diese wurden erneut auf ihre Fähigkeit untersucht, Ethanol und Isopropanol als Liganden zu unterscheiden. Bestimmte Verkürzungen führten dabei zum Verlust der Bindungsfähigkeit der RNA zu den verwendeten Liganden. Andere zeigten zwar noch eine Bindung, aber keine Verbesserung der Spezifität. Die Möglichkeit, dass es sich bei den einwertigen Alkoholen Ethanol und Isopropanol um Liganden handelt, die in der Lage sind, unspezifische Wechselwirkungen mit der RNA zu bilden, sollte in Betracht gezogen werden. Daher sind weitere Untersuchungen erforderlich, um festzustellen, ob es sich um eine "klassische" Aptamer-Liganden-Bindung handelt. Die in vitro Selektion gegen Levofloxacin führte zu einer Anreicherung von Zielmolekül-bindenden Aptameren. Anschließend wurde die gesamte Selektion mittels Next Generation Sequencing analysiert. Die daraus resultierenden Daten wurden anschließend in der Kooperation mit der Arbeitsgruppe ‚Selbstorganisierende Systeme‘ des Fachbereichs Elektrotechnik und Informationstechnik bioinformatisch ausgewertet. Zusätzlich zu der Evaluation des Anreicherungsprozesses Levofloxacin-bindender RNA-Moleküle wurden acht verschiedene Sequenzen potentieller Aptamere identifiziert. Diese wurden auf ihre Fähigkeit getestet, zwischen zwei sehr ähnlichen Liganden zu unterscheiden. Dabei wurde neben dem eigentlichen Zielmolekül das strukturell sehr ähnliche Ciprofloxacin verwendet. Daraus ergab sich das Aptamer LXC, welches die beste Unterscheidungsfähigkeit und für die Bindung zu Levofloxacin eine Dissoziationskonstante im niedrigen mikromolaren Bereich aufwies. Anhand der vorhergesagten Sekundärstruktur von LXC wurden anschließend Verkürzungen sowie Mutationen der Sequenz durchgeführt. Diese wurden mittels isothermaler Titrationskalorimetrie (ITC) untersucht. Daraus resultierte final das Levofloxacin-bindende Aptamer trLXC. Anhand der ITC-Mutationsstudien sowie den darauffolgenden In-line Probing-Experimenten konnte die vorhergesagte Sekundärstruktur bestätigt werden. Weiterhin war eine Identifizierung von Regionen, welche an der Bindung des Liganden beteiligt sind, möglich. Somit konnte erfolgreich ein Levofloxacin-bindendes Aptamer, welches für den Einsatz als Rezeptor in einem Biosensor bereit ist, entwickelt und charakterisiert werden.

Deutsch
Status: Verlagsversion
URN: urn:nbn:de:tuda-tuprints-244847
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Synthetic RNA biology
Hinterlegungsdatum: 14 Sep 2023 12:42
Letzte Änderung: 15 Sep 2023 06:36
PPN:
Referenten: Süß, Prof. Dr. Beatrix ; Niopek, Prof. Dr. Dominik
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 30 August 2023
Export:
Suche nach Titel in: TUfind oder in Google
Frage zum Eintrag Frage zum Eintrag

Optionen (nur für Redakteure)
Redaktionelle Details anzeigen Redaktionelle Details anzeigen