RNA aptamers specific for transmembrane p24 trafficking protein 6 and Clusterin for the targeted delivery of imaging reagents and RNA therapeutic to human β cells

The ability to detect and target β cells in vivo can drastically refine the way diabetes is studied and treated. By an unsupervised Systematic evolution of ligands by exponential enrichment (SELEX) we identified two RNA aptamers that specifically recognize mouse and human β cells in vitro and in vivo. Here we took advantage of commercially available high density protein arrays to identify putative target of the two islet specific aptamers. Briefly, 5' biotynilated RNA aptamer 1-717 and m12-3773 were chemically produced , complexed with Alexafluor 647-streptavidin and used as probe on the HuProt™ v2.0 19K protein array. Putative binders were further confirmed by cold target inhibition assays, silencing experiments, and surface plasmon resonance. Commercially available human protein arrays were blocked with yeast RNA-enriched blocking buffer and stained with astreptavidin Alexafluor 647 conjugated with 5' biotinylated aptamers. After several washes, images were acquired with a microarray scanner and the fluorescence signal was quantified. Data from 2 different replica (for a total of 4 independent spots per each protein) were background (average fluorescence around the protein spots) corrected, quantile normalized and the differential binding of the aptamer 1-717 and m12-3773 to the spotted protein evaluated.