Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.

Long-term course of pulmonary arterial hypertension in adults with congenital heart disease under targeted therapy: a retrospective analysis of a single tertiary center.

Background: Pulmonary arterial hypertension (PAH), a common complication in adults with congenital heart disease (CHD), leads to significant morbidity and mortality. Targeted PAH medication is available, but PAH-CHD patient data are limited. Several questions regarding indication, treatment escalation, and combination therapy remain unanswered. The aim of this study was therefore to evaluate PAH-specific treatment in adults with PAH-CHD to better understand PAH-specific therapy management. Methods: In this cross-sectional study we retrospectively examined clinical, demographic, and cardiac-catheterization data and medical management for PAH-CHD, and analyzed clinical course and midterm outcome. Results: Over up to 14 years (median, 6.2 years), 103 PAH-CHD patients (66% female) receiving targeted PAH-therapy for pre-tricuspid-shunt (15.5%), post-tricuspid-shunt (32.0%), and complex CHD (52.4%) were followed. Based on modified clinical European Society of Cardiology (ESC) classification, patients were assigned to the following subgroups: Eisenmenger syndrome (ES) (45.6%), severe pulmonary vascular disease (PVD) in complex CHD (20.4%), post-repair patients (19.4%), prevalent systemic-to-pulmonary shunt (3.9%), coincidental/small defects (0%), and Fontan circulation (10.7%). Changes in targeted PAH therapy were observed 249 times, with up to 6 (median, 2) therapy changes over a median period of 1.3 years. Over the study course, the medical treatment strategy changed towards combination therapy (baseline, 13.6%; study-end, 41%), resulting mostly in stabilized functional class or even improvement in cases of prevalent systemic-to-pulmonary shunt, ES, and patients with repaired CHD. Functional class deterioration, however, was seen in patients with severe PVD due to complex CHD, and Fontan patients. Of the 103 patients in the study, 25 died (24.3%). Patients with repaired CHD and patients with systemic-to-pulmonary shunt or ES showed the best survival rates. Mortality was remarkably higher in patients with severe PVD in complex CHD and Fontan patients. Conclusions: Many patients with PAH-CHD benefited from targeted PAH therapy over a median period of 6.2 years. Treatment decisions after targeted PAH-medication initiation were based mainly on clinical assessment. To counteract disease progression, an escalation towards combination therapy was observed during the study course. We consider survival rates under targeted PAH medication to be favorable, particularly in the ES subgroup. Nevertheless, further research is needed to optimize the use of PAH medication, especially in patients with complex CHD.