title: Selection of LNA-containing DNA aptamers against recombinant human CD73.
abstract: 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.
authors.affiliation: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark. jwe@sdu.dk.
authors.forename: Ida C
authors.initials: IC
authors.lastname: Elle
authors.forename: Kasper K
authors.initials: KK
authors.lastname: Karlsen
authors.forename: Mikkel G
authors.initials: MG
authors.lastname: Terp
authors.forename: Niels
authors.initials: N
authors.lastname: Larsen
authors.forename: Ronni
authors.initials: R
authors.lastname: Nielsen
authors.forename: Nicola
authors.initials: N
authors.lastname: Derbyshire
authors.forename: Susanne
authors.initials: S
authors.lastname: Mandrup
authors.forename: Henrik J
authors.initials: HJ
authors.lastname: Ditzel
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D019295:Computational Biology / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins; D020349:Surface Plasmon Resonance
chemical_list: D052157:Aptamers, Nucleotide; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C118485:latent nuclear antigen (LNA); D015720:5'-Nucleotidase