Characterizing Antibodies Targeting Antisense Oligonucleotide Phosphorothioate and 2'-

The efficacy of nucleic acid therapeutics (NATs) such as antisense oligonucleotides (ASOs) and small interfering RNAs relies on multiple stages of extra- and intracellular trafficking. Assessing uptake and efficacy often relies on fluorescent tagging of the NAT for imaging, although the exogenous tag undoubtedly influences the kinetics of intracellular transport and does not represent the compound used clinically. Therefore, better methods to assess the cellular and tissue distribution of NATs are needed. Here, we have validated new panels of antibody reagents that target clinically relevant nucleic acid modifications for visualizing ASOs both in vitro and in vivo. Using the ModDetect™ library of antibodies, we have tested ASOs in vitro for intracellular localization by immunocytochemistry and for biodistribution in mouse tissues by immunohistochemistry. Antibodies specific for the commonly used phosphorothioate (PS) or 2'-O-methoxyethyl (2'-MOE) modifications successfully detected gapmer ASOs, facilitating colocalization studies with endosomal markers in 2D and 3D cell models. In addition, we assessed colocalization of anti-PS signals with fluorescently tagged ASOs. Our data demonstrate the utility of these reagents for the NAT field, where modified nucleic acids can be detected irrespective of the nucleotide sequence, rendering the system amenable for multiple clinical and preclinical workflows and quantitative immunoassays.