RESUMO
Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040-0.042 µM for variants as compared to 0.15 µM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies.
RESUMO
Cold-stress syndrome (CSS) is a leading natural cause of mortality in free-ranging Florida manatees Trichechus manatus latirostris, but comprehensive investigations into blood analyte derangements and prognostic indicators in CSS are lacking. The objectives of this study were to (1) compare admission blood analyte data of manatees pre and post rehabilitation for CSS to identify clinicopathological derangements, (2) identify blood analyte prognostic indicators for survival, and (3) correlate post-mortem anatomic pathological changes with clinicopathological findings to improve the understanding of CS pathophysiology. CSS manatees admitted to a rehabilitation facility between 2007 and 2017 were included: 59 manatees with data for clinicopathological analysis (7 non-survivors and 49 survivors) and 14 manatees with necropsy data (7 with and 7 without blood analyte data). Main interpretive clinicopathological findings indicated systemic inflammation, bone marrow damage, diuresis, malnutrition, tissue necrosis, fat mobilization, hepatic impairment, acid-base imbalances, and gastrointestinal ulceration. The best diagnostically performing prognostic indicators for survival included platelet concentration, aspartate aminotransferase, calcium, and blood urea nitrogen. The main anatomic pathological findings were cutaneous lesions (n = 14), lipid depletion (n = 12), upper gastrointestinal ulceration and/or hemorrhage (n = 9), and pneumonia (n = 5). Based on the identified blood prognostic indicators interpreted in the context of anatomic pathological findings, multi-organ tissue injury, gastrointestinal ulceration and/or hemorrhage, and hemodynamic and platelet derangements are the presumptive major factors of CSS manatee mortality. These results contribute to the understanding of the complex CSS pathophysiology and offer the use of blood analyte prognostic indicators as a clinically applicable tool for the medical care of manatees during rehabilitation, thereby contributing to increased rehabilitation success and conservation of the Florida manatee.
