RNA Binding Properties of Novel Gene Silencing Pyrrole-Imidazole Polyamides

Abstract

Pyrrole-imidazole (PI) polyamides are a novel group of gene-silencing compounds, which bind to a minor groove of double stranded (ds)DNA in a sequence-specific manner. To explore the RNA binding properties of PI polyamides targeting rat transforming growth factor-β1 (TGF-β1 Polyamide) and influenza A virus (PA polyamide), we designed dsRNAs with an identical sequence to the target DNA and analyzed RNA binding properties of the polyamide. Biacore assay showed fast binding of TGF-β1 Polyamide to the dsRNA, whereas mismatch polyamide did not bind to the dsRNA. Dissociation equilibrium constant (KD) value was 6.7×10 −7 of the target dsRNA. These results indicate that PI polyamide could bind to RNA with a 2 log lower binding affinity than its DNA-binding affinity. We designed a PI polyamide targeting the panhandle stem region of influenza A virus. KD value of the PI polyamide to dsRNA targeting influenza A virus was 4.6×10 −7. Gel-shift assay showed that TGF-β1 and PA polyamides bound to the appropriate dsDNA, whereas these PI polyamides did not show obvious gel-shift with the appropriate dsRNA. Structural modeling suggests that PI polyamide binds to the appropriate B-form dsDNA in the minor groove, whereas it does not fit in the minor groove to dsRNA. Thus PI polyamides have a lower binding affinity with target dsRNA than they do with dsDNA. The distinct binding properties of PI polyamides to dsRNA and dsDNA may be associated with differences of secondary structure and chemical binding properties between target RNA and DNA.Key words pyrrole-imidazole polyamide; double-stranded RNA; affinity; Biacore; gel-shift assay; influenza virus Engineered inactivation of gene function is important for elucidating the function of particular genes and may be used in gene therapy, treatment of viral infection, cancer and other diseases caused by aberrant gene expression. Gene function can be inactivated at the DNA level by nucleic acid medicines such as anti-gene 1) or antisense peptide nucleic acid, or at the RNA level by antisense oligodeoxynucleotides, 2) ribozymes, 3) small interfering RNA (siRNA), 4) and aptamers. 5)Since these nucleic acid agents are easily degraded by nucleases, suitable chemical modifications or drug-delivery systems, including vectors, are required for their therapeutic applications. Transcriptional regulation is essential for gene expression. Initiation of transcription requires binding of transcription factors to the cognate DNA response elements in the gene promoter. Thus, we developed pyrrole-imidazole (PI) polyamides as a novel gene-suppressing agent. PI polyamides were first identified from duocarmycin A and distamycin A and are small synthetic molecules that are composed of the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids. 6) Synthetic PI polyamides can bind to specific nucleotide sequences in the minor groove of double-stranded (ds) DNA with high affinity and specificity. PI polyamides are completely resistant to nucleases and could be delivered into tissues without any drug...

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