The linker 10-hydroxydecanoic acid was then attached to the amines of the TG-beads to provide an alcohol terminus for coupling of phosphoramidites. The unmodified MinE07 aptamer was synthesised on the beads to check the linker attachment and optimise the synthesis method. To assure the compatibility of the TG-beads with the flow cytometer and to analyse the sensitivity of its fluorescent detector, a range of fluorescently labelled TG-beads were prepared. TG-beads were labelled with rhodamine B at 100% (TGRhodB100), 73% TGRhodB73), 36% (TGRhodB36), 17% (TGRhodB17) and 1.7% (TGRhodB1.7) loading. The eight fluorescently labelled TG-bead samples were passed through the flow cytometer to detect the level of fluorescence compared with the unlabelled TG-beads. The histograms show that the flow cytometer can distinguish between different intensities of fluorescence, that the TG-beads could be detected and counted by the flow cytometer as beads, and that in this setup bead autofluorescence is negligible compared with genuine dye emission (Fig. ). In summary, we have shown that TG-beads that are suitable for aptamer synthesis are also compatible for fluorescence-based TG-bead sorting by flow cytometry. Step 2: Synthesis of modified monomers. We chose to modify the uridine (U) monomers at the C5 position since this is synthetically accessible, and gives modifications of medical relevance which would sufficiently validate our method. The modifications were designed to cover aryl (-Ph) and aliphatic hydrophobic (-Vi), halogenated (-I), and natural uridine (i.e. H) substituents, potentially enhancing or disrupting interactions steric hinderance, hydrophobic effects, π-stacking, or halogen bonding. MinE07 uses nucleosides in which the 2' hydroxyl has been replaced by fluorine (f) on the pyrimidine nucleotides; this modification was retained in our monomers. All modified monomers (fU-Ph, fU-Vi, and fU-I) were synthesised through iodination and carbon-carbon cross coupling reactions as needed, with appropriate protecting groups to give dimethoxytrityl-protected activated phosphoramidites suitable for automated oligonucleotide synthesis (S1, Step 3: Aptamer library synthesis. Automated synthesis of MinE07Library was conducted by combining our monomers (fU-Ph, fU-Vi, and fU-I) with commercially made phosphoramidites (rA, rG, fC, fU) and using the hydroxy-modified TG-beads (Fig. ). After swelling in dichloromethane, a photocleavable linker was added to all beads so that after selection the aptamers can be liberated for analysis using UV light. Following this step, cycles of automated phosphoramidite synthesis were performed, with split-and-mix procedures at each instance of U. The final library contained on average 67 beads each displaying 10 17 copies of each sequence of modifications. The trityl monitor was used as a semiquantitively check synthetic efficiency (S2, Figs. 46). Figure . Synthesis of the MinE07Library on TentaGel (TG) beads, starting with conversion of the amine to alcohol functionality, appendage of the photocleavable linker (PC), followed by oligonucleotide synthesis with split-and-mix steps at each uridine using the fU, fU-Ph, fU-Vi, and fU-I phosphoramidite monomers as shown.