DNA strand length has been found to be an important factor in many DNA-based nanoscale systems. Here, we apply molecular dynamics simulations in a synergistic effort with layer-by-layer experimental data to understand the effect of DNA strand length on the assembly of DNA films. The results indicate that short (less than 10 bases) and long (more than 30 bases) single-stranded DNAs do not exhibit optimal film growth, and this can be associated with the limited accessibility of the bases on the surface due toformation of self-protected interactions that prevent efficient hybridization. Interestingly, the presence of a duplex attached to a single strand significantly alters the persistence length of the polyT strands. Our study suggests that restrained polyT, compared to labile suspensions of free polyT, are more capable of hybridization and hence DNA-based assembly.