Open AccessNuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides
Author(s) -
Hannah M. Pendergraff,
Steffen Schmidt,
Jonas Vikeså,
Christian Weile,
Charlotte Øverup,
Marie Lindholm,
Troels Koch
Publication year - 2020
Publication title -
nucleic acid therapeutics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.255
H-Index - 67
eISSN - 2159-3345
pISSN - 2159-3337
DOI - 10.1089/nat.2019.0810
Subject(s) - oligonucleotide , nucleic acid , locked nucleic acid , chemistry , biodistribution , computational biology , gene knockdown , biochemistry , biophysics , gene , biology , in vitro
Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.