Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides

Abstract

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.

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

“…The conjugated groups exhibited distinct distribution profiles, notably increasing SSO delivery to liver, kidney, bone marrow and lung. A variety of sensitive techniques have emerged recently for the detection and quantification of oligonucleotides in biological samples (28,65,66). We employed CL-qPCR to quantify the SSOs in tissues and recorded similar values to those reported using other methods (10's-100's ng/mg tissue).…”

Investigation of the Activity of Antisense Oligonucleotides Targeting Multiple Genes by RNA-Sequencing

“…The numbers of LNA-modified ASO molecules that enter the cells and the nucleus by gymnosis/unassisted delivery and the corresponding target knockdown have been measured before [32].…”

A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics

“…Cytosolic/nuclear penetration refers to the fraction of RNA therapeutic that has successfully accessed the cytosolic/nuclear compartment. While RNA therapeutics can be active in the cytosol or in the nucleus ( 49 ), and some studies have shown selective localization in one compartment over the other ( 50–52 ), for the sake of this discussion we will group these compartments together as ‘cytosolic/nuclear’ to distinguish them from compartments that prevent activity, such as endosomes and lysosomes. Ultimately, cytosolic/nuclear material is what leads to the observation of productive uptake, while the difference between total cellular uptake and cytosolic/nuclear penetration constitutes non-productive uptake (Figure 1 ).…”

“…Other label-free assays take advantage of the selective nature of hybridization by using a labeled complementary strand for isolation and quantification of the RNA therapeutic. One example is the adaptation of the enzyme-linked immunosorbent assay (ELISA) to measure total cellular uptake and tissue distribution of antisense oligonucleotides (Figure 4C ) ( 50 , 120–127 ). In most of these examples, a cell lysate is incubated with a biotinylated oligonucleotide complementary to the RNA therapeutic.…”

“…In this section, we discuss methods that can selectively quantitate cytosolic and nuclear material. Many of the assays described in the previous section on total cellular uptake can offer information about subcellular localization through the use of subcellular fractionation ( 50 , 72 , 75 , 107 , 136 ). Ultracentrifugation of cell lysates allows for separation of cellular structures, which in principle allows for the separation of membrane-bound, endosomal, cytosolic and nuclear-localized material ( 137 ).…”