Background: Nucleosome positioning plays an important role in regulation of the gene activity in eukaryotic cell.DNA sequence is believed to be one of the major factors affecting the nucleosome positioning and sequence-dependent nucleosome positioning is largely dependent on DNA deformability properties.One of basic binary periodical patterns was well established: in purine/pyrimidine alphabet YRRRRRYYYYYR by theoretical calculation model, but its validity need to be further verified.Methods: One of chemical synthetic R5Y5 sequences version was cloned into multiple cloning sites of pUC19.And then the sequence was labeled by biotin and purified after PCR.A salt dialysis method in vitro was employed to assembly nucleosome and in which 601 sequences was used as control.Results: The sequence has strong nucleosome formation ability in the 1.2 ratio of octamer to DNA.But in the 0.8 ratio it has low affinity to histones.More, its stability to assembly nucleosome is not as high as 601.Conclusions: The R5Y5 sequences ability to assembly nucleosome was perhaps strong in vitro and the result was the basic to further investigate the sequence-dependent of nucleosome positioning .