1 NAC, Institut for Fysik og Kemi, Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU2 Ditzel group, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU3 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU4 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU5 Oncology, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU6 Danish Genome Institut7 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU8 Ditzel group, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU9 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.
Molecular Biosystems, 2015, Vol 11, Issue 5, p. 1260-1270