Application Notes

Quantifying Oligonucleotides Binding to Human Serum Albumin with MST: A Faster and Precise Approach for Drug Candidates ADME Profiling

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©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 5 APPLICATION NOTE Conclusions Antisense oligonucleotides are an emerging therapeutic option to treat diseases with known genetic origin. Their pharmacokinetics and pharmacodynamics are dependent on their interaction with plasma proteins like HSA. We have shown that MST offers unprecedented advantages over classical assay like EMSA for the quantification of oligonucleotide binding properties to HSA. It delivers fast and precise K d values. The high information content of MST data enables straightforward assay development including the selection of the optimal assay buffer. For this particular interaction we recommend the use of Cy5-labeled oligonucleotide as a tracer. This tracer can be used for the screenings of a variety of different oligonucleotide constructs providing flexibility and most convenient assay setup. Materials and Methods Oligonucleotide Synthesis All nucleotides were provided by AxoLabs GmbH (Kulmbach, Germany) and were produced by automated chemical solid-phase synthesis using commercially available building blocks. A er cleavage from the solid support, the synthesized oligos were purified by high performance liquid chromatography (HPLC). MST experiments For the MST experiments the following oligonucleotides were tested: cholesterol- conjugated oligonucleotide, fully phosphorothioate modified oligonucleotide, Cy5 coupled to a fully phosphorothioate modified oligonucleotide and Cy5 coupled to a cholesterol-conjugated oligonucleotide. HSA (Cat# A9731, Lot# SLBC7527V, Sigma Aldrich) was used as a binding partner. Buffer phosphate buffered saline (PBS, pH 7.4) with or without 0.05% Tween-20 was used as assay buffer. The Cy5-labeled oligomer was prepared at 20 nM and working concentration solutions and dilution series were prepared either in Protein LoBind Eppendorf Tubes (Cat# 003 108.094, Eppendorf) or non-binding surface 384-well assay plate (Cat# 4513, Corning). For the competition assays 100 µM of HSA was preincubated for 15 min with 50 nM of Cy5-labeled oligomer in PBS. The probes were filled in Monolith NT.115 Premium Capillaries (Cat# MO-K025, NanoTemper Technologies). Instrumentation and data analysis Measurements were performed on a NanoTemper Monolith NT.115 instrument. Final Cy5-oligonucleotide concentration of 20 nM yielded fluorescence intensities of about ~400 counts at an LED power of 20%. The samples were measured at high MST power using MO.Control so ware to acquire the data. The parameters were deduced a er 1.5 s MST on-time and the resulting dose-response curves fitted with MO.Affinity Analysis either to a one-site binding model to extract K d values or, in the case of the competition assays, fitted by Hill model to determine the EC50 values.

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