Figure 2. Enriched ssODNs interact with the surface of exosomes.

(a) The enriched 5′-biotinylated ssODN library L2 (blue curves) reveals enhanced binding, measured by flow-cytometry, to UC isolated exosomes, compared to the 5′-biotinylated starting library L0 (cyan curves), or absence of ssODNs (w/o; black curves) using SA-PE as a staining agent, and gated only on double-positive CFSE+DiD+ events (Supplementary Fig. 2). The curves show the distribution of relative fluorescence intensities for observed events. Each curve represents an independent binding experiment (n = 3). (b) Total number of positive SA-PE staining events from (a) (number of events >RFU 400) of biotinylated L0 (cyan), and biotinylated L2 (blue), compared to absence of ssODNs (w/o; black), in exosome binding. In each binding reaction 12 nM of each library was used. (c) Post-ADAPT quantification of binding of individual aptamers as part of the library and individually as illustrated in Supplementary Fig. S3. The top panel shows 23 representative individual aptamers, selected either with high “H” or low “L” normalized counts from L3 NGS data after binding pooled plasma from breast biopsy negative donors. There is at least a 5-fold difference between counts of H and L ssODNs. These 23 sequences were re-synthesized and tested individually in the same binding assay, but in equal concentrations, unlike their original representation in L3. PEG-precipitated aptamer/plasma complexes were directly subjected to qPCR (bottom panel). Inlay: Magnification of qPCR results of ssODNs incubated with PBS instead of plasma (red). (d) Silver-stained reducing SDS-PA gel of pulled down proteins from PPT plasma with the indicated ssODNs (H1, H11, L4, L15), immobilized on streptavidin magnetic beads, and the control without ssODN (NO). Dashed arrows indicate pulled down proteins C1QA, C1QB, and C1QC. The dotted arrow indicates the heavy chain of IgM (IgM_HC). SA: streptavidin. RC: reverse complement sequence.