Double β-alanine Substitutions Incorporated in 12-ring Pyrrole-Imidazole Polyamides for Lengthened DNA Minor Groove Recognition

Abstract

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide (PI polyamide) are increasingly used in basic and applied biomedical research. Although β-alanine (β) substitutions in 8-ring PI polyamides have been well analyzed, efficacy of double β substitutions for lengthened DNA minor groove recognition has not been elucidated in vivo. Here, we show effectiveness of double β substitutions in PI polyamides to retain high affinity of specific lengthened DNA binding and suppress target gene expression in cells. Initially we synthesized four 12-ring PI polyamides targeting the AP-1 site within MMP-9 gene promoter, 1-4, including a β/β pair (3) and two adjacent Py/β and β/Im pairs (4) and investigated the binding kinetics with oligoDNA containing preferred sequence (5’-AGTCAGCA-3’) by surface plasmon resonance assays and MMP-9 mRNA expression in MDA-MB-231 cells. The PI polyamides 3 and 4 showed high affinities of target DNA binding (KD=4.33×10-8 ) and (KD=5.00×10-8 ), respectively and significant down regulation of MMP-9 expression. We then considered adjacent Im/β and β/Im pairs should solve the problem of repeating GC sequence. A PI polyamide 5 targeting the E2F site, 5’-TTGGCGC-3, within the MYCN promoter was synthesized and tested binding affinity and MYCN suppression in MYCN amplified CHP134 human neuroblastoma cells. The PI polyamide 5 showed high affinity (KD=3.07×10-8 ) binding of the target sequence and significantly suppressed MYCN mRNA expression. Those results demonstrated a possible use of the adjacent double β substitutions for 12-ring PI polyamides, particularly in G/C rich regions and suggested substitutions of β springs in PI polyamides may extend applications for in vivo biomedical research targeting lengthened genomic DNA.