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Two of the most important features in the field of nanotechnology are self-assembly with nanometre-scale precision, and the self-alignment of functionalised nanomaterials. Here, we discuss the binding feasibility of single-strand spacer-added DNA building blocks to biotin–streptavidin (SA) complexes. We use atomic force microscopy, photoluminescence (PL) spectroscopy, and dynamic simulation to study the topological, optical, and vibrational characteristics of DNA lattices. To construct the DNA lattices, we use two distinct DNA building blocks, i.e. a double-crossover tile with a biotin (DXB), and a double-crossover tile with a flexible single-strand spacer containing a biotin (DXSB). Biotinylated DXB and DXSB lattices grown on the substrate eventually attracted streptavidins (SA, a tetramer protein) and formed DXB + SA, and DXSB + SA lattices, respectively. Furthermore, we examine the feasibility of alignments of an individual DXB (DXSB) tile on SA-bound DXB (DXSB) lattices, and a SA-conjugated Au nanoparticle (NP) on DXB (DXSB) lattices. To use more than two binding sites of biotins on SA (to serve as a connector between biotinylated tiles), the introduction of flexible single-strand spacers in DX tiles helped to overcome geometrical hindrance. In addition, the PL spectra of DXB and DXSB lattices with SA–Au conjugates are analysed to understand the periodic bindings of Au NPs on DXB (DXSB) lattices. We also conduct dynamic simulations of modal analysis and molecular dynamics simulation, which provide the vibrational characteristics and evidence of the importance of single-strand spacer-added DNA samples. Patterning of nanomaterials with specific functionalities with high precision using a simple method would be useful for the manufacture of high-density nanoelectronic devices and extreme-sensitivity biosensors.