Objective To determine enhancer activity of a short sequence DNA (AA, 5-GTGAAATAAATGCAAATAAAGT) and its derived sequences.To determine whether the synthetic single stranded AA sequence and its derived sequences form a secondary structure via intrachain base complementation.Method AA sequence and its de rive sequences were inserted between the GFP gene and the Alu repeats in the C1-Alu14 vector (14 head-to-tail tandem Alu elements were inserted downstream of the GFP gene in the pEGFP-C1 vector) to construct expression vectors that were transfected into HeLa cells.Fluorescence microscopy, flow cytometry and RT-PCR were used to de tect the GFP protein and mRNA expression in different expression vectors.PAGE method was used to detect whether synthetic single stranded DNA sequences form a secondary structure.Results AA sequence induced strong GFP gene expression and that its derived sequence 7pieA (5-GTGAAAAAAATGCAAAAAAAGT) did not.The five T bases of the AA sequence were mutated to A, C or G.The sequences retaining the 7th and/or 17th T possessed strong enhan cer activity.RT-PCR and RNA synthesis inhibition analysis using actinomycin D revealed that the enhanced GFP gene expression induced by the AA sequence occurred at the transcriptional level.Our previous study reported that common characteristics of short sequence enhancers include an incomplete palindrome and symmetric structure.The PAGE result showed that the AA sequence formed a secondary structure.Conclusion DNA secondary structure of AA sequence is related to its enhancer activity.