Besides the acquisition of pharmacokinetic parameters of antisense oligonucleotide microRNA (miRNA) inhibitors, such as measuring in vivo concentration, their pharmacodynamic characteristics are also of interest. An emerging and straightforward method for studying molecular interactions is microscale thermophoresis (MST). This technique makes it possible to study interactions between miRNAs and various oligonucleotide inhibitors, independent of the chemical modifications of the inhibitors or their respective target structure, with very little sample volume required compared to competitive techniques, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Interaction studies between these inhibitors and their respective target structures were performed, and they allowed the assessment of binding characteristics and parameters, such as EC50 for a number of these inhibitors, with little effort. Furthermore, MST could be utilized for obtaining kinetic binding data of the Argonaute-2 protein with a miRNA, which showed a possible RNA-induced silencing complex (RISC)-mediated turnover of inhibited miRNAs.
Cardiac diseases are the most frequent causes of death in industrialized countries. Pathological remodeling of the heart muscle is caused by several etiologies such as prolonged hypertension or injuries that can lead to myocardial infarction and in serious cases also the death of the patient. The micro-RNA miR-132 has been identified as a master-switch in the development of cardiac hypertrophy and adverse remodeling. In this study, MALDI-TOF mass spectrometry (MS) was utilized to establish a robust and fast method to sensitively detect and accurately quantify anti-microRNA (antimiR) oligonucleotides in blood plasma. An antimiR oligonucleotide isolation protocol containing an ethanol precipitation step with glycogen as oligonucleotide carrier as well as a robust and reproducible MS-analysis procedure has been established. Proteinase K treatment was crucial for releasing antimiR oligonucleotides from plasma-as well as cellular proteins and reducing background derived from biological matrices. AntimiR oligonucleotide detection was achieved from samples of studies in different animal models such as mouse and pig where locked nucleic acids-(LNA)-modified antimiR oligonucleotides have been used to generate pharmacokinetic data.
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