Initial observations of 99mTc labelled locked nucleic acids for antisense targeting

The most recent DNA analogues to become commercially available are known as locked nucleic acids (LNAs). The aim of this study was to evaluate the properties of LNAs for antisense targeting.

A 15 mer LNA antisense to RIalpha mRNA was studied in cell culture. The antisense LNA (5'-amine linker-TGCCTCCTCACTGGC) was purchased along with its sense control LNA. Surface plasmon resonance was used to compare affinity constants with uniform 18 mer phosphorothioate (PS) DNA and uniform 18 mer phosphodiamidate morpholinos (MORFs, another DNA analogue). After radiolabelling with 99mTc via MAG3, the antisense and sense LNAs were added at 5 nM to wells containing ACHN cells in culture and accumulations measured over 24 h. Subcellular partition was determined after 16 h of incubation by separating membrane bound, cytoplasmic and nuclear fractions. The cell studies were conducted both with naked LNAs and with liposomes (oligofectamine) as carrier.

Radiochemical purity was about 95% after purification on a P4 column and each LNA was radiolabelled at about 20 GBq.micromol(-1) (100.microCi.microg(-1)). The surface plasmon resonance results showed a more favourable dissociation constant for the duplex with DNA of the 15 mer LNA (0.55 x 10(-10).M(-1)) compared to the duplex with 18 mer DNA and 18 mer MORFS (2.05 and 1.06 x 10(-10).M(-1), respectively). Because of lower dissociation constants, the hybridization affinities of LNAs are therefore higher than those of uniform and identical PS DNAs or MORFs. The cellular accumulations suggested an antisense effect in that the antisense LNA accumulation was higher than sense both when added naked (1.8% vs. 0.4% at 24 h) and with liposome carrier (3.8% vs. 1.0% at 24 h). Thus while absolute cellular uptake was lower than that observed by this laboratory with other oligomers, the antisense/sense differential was higher. The number of antisense LNAs accumulating per cell specifically (i.e., antisense minus sense) was about 45,000 naked and about 100,000 with carrier. Subcellular partition showed that both LNAs were partitioned to each fraction with antisense accumulations greater than sense and carrier accumulations greater than naked as before. That as much as 2.9% of the antisense LNA (with carrier) was in the cytoplasmic or nuclear factions demonstrates that the LNA was internalized.

LNAs appear to be attractive oligomers for antisense targeting and other radiopharmaceutical applications.