Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide.

Intracellular delivery of oligonucleotides is important for their use as therapeutic drugs. The conjugation of molecules interacting with cell membrane proteins to enhance their internalization into cells is an effective strategy for delivering oligonucleotides. In the present study, we focused on creating aptamers, which are single-stranded oligonucleotides that bind target molecules with high affinity and specificity, as membrane protein-binding molecules. With an evolutionary selection approach using a random DNA library containing a uracil derivative with a hydrophobic functional group at the 5 position, we successfully obtained aptamers that are efficiently internalized into A549 cells. The efficacies of the aptamers were tested by further conjugation with -targeting antisense oligonucleotides (ASOs), and the expression levels of RNA were examined. The aptamer-ASO conjugates were taken up by A549 cells, although there was no observable reduction in RNA levels. In contrast, the activity of the aptamer-ASO conjugate was potentiated when endosomal/lysosomal escape was enhanced by the addition of chloroquine. Thus, we showed that the hydrophobic modification of the nucleobase moiety is useful for developing highly internalizing aptamers and that endosomal/lysosomal escape is important for the intracellular delivery of ASOs by aptamers.