Strong and Specific Recognition of CAG/CTG Repeat DNA (5’‐dWGCWGCW‐3’) by a Cyclic Pyrrole‐Imidazole Polyamide

Abstract

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease.Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (T m ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.Although DNA repeat sequences are normally present in the human genome, abnormally elongated repeats can lead to a variety of diseases. [1,2] The abnormal elongation of CAG/CTG repeat sequences causes Huntington's disease, spinocerebellar ataxia, and myotonic dystrophy. Compounds that bind to the CAG/CTG repeat sequences have been studied to develop therapeutic agents for these neurological disorders. [3][4][5] Notably, Pearson et al. recently achieved a reduction in the number of repeats in vivo using a compound that binds to the hairpin structure formed by the CAG repeats. [6] These studies targeted the r(CUG) repeats [3,4] or d(CAG/CAG) hairpin structures in the CAG repeat regions, [5,6] whereas our group previously developed pyrrole-imidazole polyamides (PIPs) that bind to the d(CAG/ CTG) sequences in a sequence-specific manner. [7,8] PIPs are one of the well-studied DNA-binding compounds, which were developed by Dervan et al. [9,10] They bind to the minor groove of B-DNA, recognizing Watson-Crick base pairs by antiparallel pairings of their N-methylpyrrole (Py) and N-meth-[a] Y.

Pyrrole–Imidazole Polyamides – A Frontrunner in Nucleic Acid‐Based Small Molecule Drugs

“…Adaptedunder the terms of the Creative Commons CC BY license. [ 102 ] Copyright 2021 The Authors, published by Wiley‐VCH.…”

“…designed and synthesized cyclic (cPIP 1) and hairpin (hPIP 2) versions of PIPs targeting the 5’‐WGCWGCW‐3’ sequence (Figure 5B). [ 102 ]…”

“…[100,101] Recently, Hirose et al designed and synthesized cyclic (cPIP 1) and hairpin (hPIP 2) versions of PIPs targeting the 5'-WGCWGCW-3' sequence (Figure 5B). [102] The binding affinity of these cPIP 1 and hPIP 2 with the sequences 5'-CGAGCAGCACG-3'/ 3'-GCTCGTCGTGC-5' as their target were studied using the double-stranded melting temperature (T m ) assay and it was found that cPIP 1 has relatively higher binding affinity than hairpin PIPs (ΔΔT m = 12.4 °C). The binding affinity study of these PIPs has also been stretched out to the extended sequences 5'-(CAG) 10 -3' (5'-CAGCAGCAGCAGCAGCAGCAGCAGCAGCAG-3') and 5'-(CTG) 10 -3' (5'-CTGCTGCTGCTGCTGCTGCTGCTGCTGCTG-3'), which are known to form a self-complementary hairpin structure.…”

“…B) Structures of cyclic (cPIP 1) and hairpin (h PIP) pyrrole imidazole polyamides. Adaptedunder the terms of the Creative Commons CC BY license [102]. Copyright 2021 The Authors, published by Wiley-VCH.…”

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A–A-Mismatch-Containing DNA

“…Moreover, β-alanine increases the flexibility of PIP and is sometimes used in place of Py; Py−β and β–β pairs recognize A–T or T–A pairs . Using this sequence specificity, many studies have been conducted using various PIPs as drug candidates, , to visualize telomere, , and as epigenetic regulators. − A cyclic-PIP (cPIP) that strongly binds to a sequence containing CAG/CTG repeats (5′-WGCWGCW-3′, W: A or T) has been developed . Furthermore, biological experiments using this cPIP revealed that it specifically binds to the hairpin structure that contains the mismatched base pairs formed thereby suppressing the genes that cause the expanded-repeat-associated diseases .…”