Regulation of the activity of an anti-thrombine aptamer and of the glmS-ribozyme with light

Im ersten Teil dieser Arbeit wurde eine Variante des Anti-Thrombin-Aptamers HD1 entwickelt, die vor Belichten aktiv war und sich durch Belichten deaktivieren ließ. Dazu wurde das Wildtyp-Aptamer am 5'-Ende um eine GAAA-Schleife und eine Gegenstrangregion, bestehend aus vier Nukleotiden, erweitert. Dies reichte für eine vollständige Inaktivierung des Aptamers aus. In die Gegenstrangregion wurde ein photolabil geschütztes Nukleotid eingebaut, das die Bildung einer Haarnadelstruktur vorübergehend verhindert. Dazu wurde ein Desoxycytidin-Derivat synthetisiert, das an seiner N4-Position mit einer 1-(2-Nitrophenyl)ethyl-Gruppe modifiziert war. Durch die Maskierung der Antisense-Region wies das Aptamer vor Belichtung blutgerinnungshemmende Aktivität auf, allerdings in geringerem Maße als das Wildtyp-Aptamer. Durch Belichten wurde die Gegenstrangregion freigesetzt und dadurch die aktive Konformation des Aptamers zerstört, sodass es keine blutgerinnungshemmende Wirkung mehr besaß. In einem daran anknüpfenden Projekt sollte eine mit Licht ausschaltbare HD1-Variante mit verbessertem Schaltverhalten entwickelt werden, deren Aktivität vor dem Belichten mit der des Wildtyp-Aptamers vergleichbar ist. Tests zeigten, dass eine 5'-Erweiterung des Aptamers stets einen Aktivitätsverlust zur Folge hatte. Getestet wurden verschiedene Linker-Sequenzen, D-Spacer (Abasic Sites) und nicht nukleotidische Linker wie Glykollinker oder alkylische Linker. Eine Erweiterung am 3'-Ende brachte dagegen fast immer Aptamervarianten hervor, deren Aktivität die des Wildtypaptamers überstiegen. Um diese verbesserten Aptamervarianten zu deaktivieren, war eine Antisense-Region bestehend aus bis zu neun Nukleotiden nötig. Für eine photolabil geschützte Variante wurde zusätzlich ein Desoxyadenosinderivat mit N6-1-(2-Nitrophenyl)ethylmodifikation synthetisiert. Es zeigte sich, dass eine photolabile Schutzgruppe nicht ausreichte um die Antisense- Region zu neutralisieren. Aptamervarianten mit vier oder fünf photolabilen Schutzgruppen in der Antisenseregion waren vor dem Belichten aktiver als das Wildtyp-Aptamer HD1 und konnten durch Belichten vollständig deaktiviert werden. In einem weiteren Projekt dieser Arbeit wurde eine photolabil geschützte Glukosamin-6- phosphat-Variante synthetisiert, um eine lichtabhängige Spaltung des glmS-Ribozyms aus Bacillus subtilis zu induzieren. Dazu wurde GlcN6P an der Aminofunktion über eine Carbonyllinker mit einer 2-(2-Nitrophenyl) propylgruppe modifiziert. In vitro konnte gezeigt werden, dass mit dieser Verbindung durch Belichten die Spaltung eines glmS-EGFP-mRNA-Konstrukts induziert werden konnte. In HeLa-Zellen wurde untersucht, ob sich dieses System zur Regulation der EGFP-Expression eignet. Da erste Versuche erfolglos blieben, wurde eine lipophile, zellgängige Variante des photolabil geschützten GlcN6Ps synthetisiert. Versuche, in denen dieses Derivat getestet wird, werden zur Zeit von unseren Kooperationspartnern durchgeführt. In einem weiteren Projekt wurden Desoxyguanosinderivate für die DNA-Festphasensynthese synthetisiert, die an ihrer O6-Position mit einer p-Hydroxyphenacylgruppe bzw. mit einer 1-(3-Nitrodibenzofuran-2-yl)ethylgruppe modifiziert wurden. Diese wurden in ein Desoxyoligonukleotid eingebaut und es konnte gezeigt werden, dass die photolabilen Schutzgruppen durch Belichten abgespalten werden. Beide photolabilen Modifikationen waren allerdings unter den basischen DNA-Abspaltbedingungen zu instabil, als dass sie sich für den routinemäßigen Einsatz zur Herstellung lichtaktivierbarer Nukleinsäuren eignen würden. Im letzten Teil der Arbeit wurde eine photolabile Schutzgruppe entwickelt, die über einen zusätzlichen Aminolinker verfügt [2-(4-(Aminomethyl)-2-nitrophenyl)-propanol]. Die Aminofunktionalität war für die Dauer der DNA/RNA-Festphasensynthese mit einer Trifluoracetylgruppe geschützt, die unter den basischen Abspaltbedingungen ebenfalls entfernt wird. Mit dieser photolabilen Schutzgruppe wurden ein Thymidinderivat an der O4-Position und ein Desoxyguanosinderivat an der O6-Position modifiziert. Das Desoxyguanosinderivat wurde erfolgreich in der Oligonukleotidfestphasensynthese eingesetzt. Die photolabile Schutzgruppe konnte durch Belichten vollständig von der synthetisierten Nukleinsäure abgespalten werden. Darüber hinaus gelang es, über die Aminofunktionalität die heterobifunktionalen Crosslinker SMCC und SMPB mit der Nukleinsäure zu verknüpfen. Auf diese Weise ist eine reversible Vernküpfung der Nukleinsäure mit einem nahezu beliebigen Bindungspartner möglich. Durch Belichten kann die Nukleinsäure in ihrer ursprünglichen Form wiederhergestellt werden.In the first part of this thesis a variant of the anti thrombin aptamer HD1 was developed, that was active before irradiation and could be deactivated with light. For this purpose the wildtype aptamer was extended at the 5'-end with a GAAA-loop and an antisense region consisting of four nucleotides. This resulted in a complete depletion of the aptamer activity. A photoactivatable nucleotide was then introduced in the antisense region to prevent the formation of a hairpin structure. Therefore a deoxycytidine derivative caged at the N4-position with a 1-(2-nitrophenyl)ethyl group was synthesized. Due to the caged antisense region the aptamer led to a prolonged blood clotting time before irradiation. However, the activity of the prolonged and caged aptamer variant did not reach the activity of the wildtype aptamer. Upon irradiation the antisense region was released and this resulted in a destruction of the active conformation of the aptamer and the loss of its anticoagulative effect. A following project was dedicated to the development of an HD1 variant that could be deactivated with light with a better switching behaviour. The activity of the caged aptamer before irradiation should match the activity of the wildtype aptamer. Preliminary studies showed that a 5'-elongation of the aptamer led always to a significant loss of activity. Different linker sequences were tested, including D-Spacer (abasic sites) and nonnucleotidic linkers like glycol derived linkers or alkylic linkers. In contrast, an elongation at the 3'-end always led to aptamer variants that were even more active than the wildtype aptamer. To deactivate these improved aptamers it was necessary to apply an antisense region consisting of up to nine nucleotides. For the preparation of a photolabile protected aptamer variant a deoxyadenosine derivative was synthesized in addition to the caged deoxycytidine derivative, caged at the N6-position with a 1-(2-nitrophenyl)ethyl group. One caged nucleotide proved to be ineffective in masking the 9mer antisense region. Aptamer variants with four or five photolabile protected nucleotides in the antisense region finally showed a better activity than the wildtype aptamer HD1 before irradiation and could be completely deactivated with light. In an additional project of this thesis a photolabile protected glucosamin-6-phosphat derivative was synthesized in order to get an instrument for the light-dependent induction of the cleavage reaction of the glmS-ribozyme from bacillus subtilis. A 2-(2-nitrophenyl) propyl group was attached to the amino function of the GlcN6P via a carbonyl linker. In vitro in could be shown that this compound was able to induce the cleavage of a glmSEGFP-mRNA construct upon irradiation. In HeLa cells it was investigated if this system would be suitable for the regulation of EGFPexpression. Since the first experiments led to negative results, a lipophilic, cell penetrating variant of caged GlcN6P was synthesized. Experiments with this derivative are currently performed by our collaboration partners. In an additional project two deoxyguanosine derivatives for DNA solid phase synthesis were synthesized, modified at the O6-position roup and a 1-(3-nitrodibenzofuran-2-yl)ethyl group respectively. These derivatives were applied for solid phase synthesis and it could be shown that the photolabile protecting groups were cleaved from the oligonucleotide upon irradiation. However, both photolabile protecting groups were not very stable on the O6-position of deoxyguanosin during the basic DNA cleavage step, what leads to the conclusion that these photolabile protecting groups may be not suitable for the routine synthesis of light activatable oligonucleotides. In the last part a photolabile protecting group with an additional amino linker was developed [2-(4-(aminomethyl)-2-nitrophenyl)propanol]. During the DNA/RNA solid phase synthesis the amino function was protected with a trifluoroacetyl group that also could be removed after synthesis during the basic cleavage conditions. A thymidine derivative and a deoxyguanosine derivative were synthesized that were modified with this photolabile protecting group at the O4-position (T) and the O6-position (dG) respectively. The deoxyguanosine derivative was successfully applied for oligonucleotide synthesis. Upon irradiation the photolabile protecting group could be entirely removed from the oligonucleotide. Furthermore it was possible to connect the amino function of the photolabile protected oligonucleotide to the bifunctional crosslinkers SMCC or SMPB. In this way it is possible to create a reversible linkeage between the nucleic acid and almost any kind of binding partner. By irradiation the nucleic acid can be restored in its native form

Light-Activatable Nucleic Acids 'Caged' at the Nucleobases

The attachment of photolabile groups to biologically active molecules offers a very versatile way to put a biological effect under the control of an external trigger signal and thus confers spatiotemporal and dose control to this effect. Over the last years we have prepared a number of oligonucleotide derivatives that are modified in such a way. These derivatives are usually referred to as 'caged compounds' and in particular our photolabile 'caging groups' are located at the nucleobases of oligonucleotides so that the Watson–Crick interaction is temporarily impossible. Thus several nucleic acid-based applications have now become controllable with light

Dependence of aptamer activity on opposed terminal extensions : improvement of light-regulation efficiency

Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable "caged" intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5'- to 3'-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation

Dependence of aptamer activity on opposed terminal extensions: improvement of light-regulation efficiency

Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable ‘caged’ intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5′- to 3′-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation

Dependence of aptamer activity on opposed terminal extensions: improvement of light-regulation efficiency

Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable ‘caged’ intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5′- to 3′-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation

Activity alterations in the bed nucleus of the stria terminalis and amygdala during threat anticipation in generalized anxiety disorder

Sustained anticipatory anxiety is central to Generalized Anxiety Disorder (GAD). During anticipatory anxiety, phasic threat responding appears to be mediated by the amygdala, while sustained threat responding seems related to the bed nucleus of the stria terminalis (BNST). Although sustained anticipatory anxiety in GAD patients was proposed to be associated with BNST activity alterations, firm evidence is lacking. We aimed to explore temporal characteristics of BNST and amygdala activity during threat anticipation in GAD patients. Nineteen GAD patients and nineteen healthy controls (HC) underwent functional magnetic resonance imaging (fMRI) during a temporally unpredictable threat anticipation paradigm. We defined phasic and a systematic variation of sustained response models for blood oxygen level-dependent responses during threat anticipation, to disentangle temporally dissociable involvement of the BNST and the amygdala. GAD patients relative to HC responded with increased phasic amygdala activity to onset of threat anticipation and with elevated sustained BNST activity that was delayed relative to the onset of threat anticipation. Both the amygdala and the BNST displayed altered responses during threat anticipation in GAD patients, albeit with different time courses. The results for the BNST activation hint towards its role in sustained threat responding, and contribute to a deeper understanding of pathological sustained anticipatory anxiety in GAD

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics

Activity alterations in the bed nucleus of the stria terminalis and amygdala during threat anticipation in generalized anxiety disorder

Sustained anticipatory anxiety is central to Generalized Anxiety Disorder (GAD). During anticipatory anxiety, phasic threat responding appears to be mediated by the amygdala, while sustained threat responding seems related to the bed nucleus of the stria terminalis (BNST). Although sustained anticipatory anxiety in GAD patients was proposed to be associated with BNST activity alterations, firm evidence is lacking. We aimed to explore temporal characteristics of BNST and amygdala activity during threat anticipation in GAD patients. Nineteen GAD patients and nineteen healthy controls (HC) underwent functional magnetic resonance imaging (fMRI) during a temporally unpredictable threat anticipation paradigm. We defined phasic and a systematic variation of sustained response models for blood oxygen level-dependent responses during threat anticipation, to disentangle temporally dissociable involvement of the BNST and the amygdala. GAD patients relative to HC responded with increased phasic amygdala activity to onset of threat anticipation and with elevated sustained BNST activity that was delayed relative to the onset of threat anticipation. Both the amygdala and the BNST displayed altered responses during threat anticipation in GAD patients, albeit with different time courses. The results for the BNST activation hint towards its role in sustained threat responding, and contribute to a deeper understanding of pathological sustained anticipatory anxiety in GAD