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.
Highlights
Denmark, Campusvej 55, 5230 Odense M, Denmark e Department of Oncology, Odense University Hospital, Sdr
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
Aptamers containing LNA nucleotides were selected against recombinant human CD73 using Systematic Evolution of Ligands by EXponential enrichment (SELEX) and next-generation sequencing (NGS) with custom NGS bioinformatics software
Summary
Campusvej 55, 5230 Odense M, Denmark e Department of Oncology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark † Electronic supplementary information (ESI) available. LNA (Locked Nucleic Acid) is an RNA-mimic containing a 20-O,40-C-methylene bridge, which locks the furanose ring in a C30-endo conformation.[13,14,15] LNA nucleotides have been proven to improve thermal stability of nucleic acid duplexes and confer nuclease-resistance when incorporated into nucleic acid oligomers.[16,17] Aptamers containing LNA nucleotides by post-SELEX modification have been reported (reviewed in[16] and18), but the effects of these modifications on aptamer function can be difficult to predict. In investigating the properties of LNA nucleotides in aptamers, we and others, have focused on selecting aptamers using SELEX methods, front-loaded with LNA and other modified nucleotides.[19,20,21,22,23]
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