Application Notes

©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 2 APPLICATION NOTE binding capacity, providing a depot and carrier for many endogenous and exogenous compounds 1,2 . It represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays pseudo-enzymatic properties 2 . HSA appears to be formed by three homologous domains and each domain is made up by two separate helical domains that are connected by random coil 3 with a variety of binding sites. The conformational adaptability of HSA involves more than the immediate vicinity of the binding site(s), this being the root for the observed ligand-dependent allosteric conformational transitions 3,4 . Notably, HSA undergoes pH- and allosteric effector-dependent reversible conformational isomerization 3 that affects its binding to drugs. For example, although Warfarin and Ibuprofen bind to sites distinct from the sites for fatty acid binding, they compete with fatty acid binding to HSA. Allosteric interactions have been reported to affect binding equilibria between HSA and Ibuprofen, Warfarin and Lorazepam 3 . Aside of mentioned natural and synthetic ligands, HSA also binds to phosphorothioates and oligonucleotides conjugated to ligands like Ibuprofen 5 , which is of importance when developing oligonucleotide-based drugs. The interaction of antisense oligonucleotides with HSA determines their pharmacokinetics (transport and distribution in target tissues) and pharmacodynamic (binding to the mRNA target) properties and hence their eventual pharmacology 5 . To develop safer and more effective oligonucleotide drug candidates, it would be valuable to enhance the interaction of these molecules with proteins involved in transport and absorption and to minimize the interaction with proteins responsible for undesirable side effects. Currently, the most commonly used method for the quantification of interaction between the oligomers and HSA is the electrophoretic mobility shi assay (EMSA) 6,7 . In this assay a solution of protein and nucleic acid are combined, and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. A er electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. EMSA is o en considered to bear number of limitations and disadvantages like the fact that dissociation can occur during the electrophoresis since samples are not at equilibrium during the run 7 , the use of radioactive material ( 32 P), problematic waste disposal and inconvenient detection and subsequent quantification. The MST assay on the other hand, offers an alternative approach with excellent sensitivity, the advantage of detecting the interaction in equilibrium

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