A wide range of multivalent scaffolds was assembled by using only five different PNA oligomers and various DNA templates. The flexibility of the PNA–DNA duplexes could be increased by introducing nick-sites and partially unpaired regions, as confirmed by MD simulations. The self-organized glyco-assemblies were used in a spatial screening of accessible carbohydrate binding sites in the Erythrina cristagalli lectin (ECL). This systematic investigation revealed a distance dependence which is in agreement with the crystal structure analysis.