A polyamide containing N-methylpyrrole (Py) and N-methylimidazole (Im), designated PIPA, binds with high affinity and specificity to specific nucleotide sequences in the minor groove of double-helical DNA. Based on a recent report of the synthesis of PIPA for telomere visualization, the present paper focused on the size of the connecting part (hinge region) of two PIPA segments of the tandem hairpin PIPA, Dab(Im-Im-Py)-Py-Py-Py-Im-[Hinge]-Dab(Im-Im-Py)-Py-Py-Py-Im-beta Ala-NH(CH2)(3)N(CH3)-(CH2)(3)NH-[Dye]. The present paper also describes the characterization of binding by measuring the thermal melting temperature and surface plasmon resonance and by specific staining of telomeres (TTAGGG)n in human cells. Micro-heterogeneity was also investigated by high-resolution mass spectrometry. We found that the optimal compound as the hinge segment for telomere staining was [-NH(C2H4O)(2)(C2H4)CO-] with tetramethylrhodamine as the fluorescent dye.