In order to investigate the inhibition of gene expression by a new type of hairpin polyamide-CPI conjugate 1, its ability to inhibit transcription in cell free system was investigated. Sequence-selective alkylation of double-stranded DNA by 1 was investigated by denaturing gel electrophoresis using 1000 bp DNA fragment which codes for green fluorescence protein (GFP) under the control of T7 promoter. Analysis of DNA sequence indicated that 1 alkylated predominantly at the site of 5'-AGTCA-3' in coding region of GFP. The transcript by T7 RNA polymerase using the alkylated DNA as a template was analyzed by PAGE. The results clearly indicate that 1 inhibits transcription by alkylation of coding region at a nanomolar concentration.