Rational design of specific binding hairpin Py–Im polyamides targeting human telomere sequences

Abstract

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are organic molecules that can recognize predetermined DNA sequences in a sequence-specific manner. Human telomeres contain regions of (TTAGGG)n repetitive nucleotide sequences at each end of chromosomes, and these regions protect the chromosome from deterioration or from fusion with neighboring chromosomes. The telomeres are disposable buffers at the ends of chromosomes that are truncated during cell division. Tandem hairpin Py-Im polyamide TH59, which recognizes human telomere sequences, was reported by Laemmli's group in 2001. Here, we synthesized three types of Py-Im polyamides 1-3 based on TH59 for specific recognition of human telomere repeat sequences. Thermal melting temperature (Tm) measurements and surface plasmon resonance analysis were used to evaluate the abilities of the three types of Py-Im polyamides to discriminate between three kinds of DNA sequences. Significantly, the results showed that polyamides 1 and 2 have better affinities to TTAAGG than to TTAGGG. In contrast, polyamide 3 displayed good specificity to human telomere sequence, TTAGGG, as expected on the basis of Py-Im binding rules.

An overview of recent advances in duplex DNA recognition by small molecules

“…Sugiyama et al designed and synthesized a series of telomere-targeting synthetically challenging tandem hairpin Py/Im polyamides which could recognize >10 base pairs with flexible linker conjugated with a fluorescent dye (either Texas Red (TR) or Cyanine 3 (Cy3)) using a Fmoc-based solid phase synthetic approach; two of the representative conjugates 23 and 24 are shown in the Fig. 8 [ 86 – 87 ]. The authors investigated the binding affinity and sequence specificity of these conjugates for the human telomeric repeat TTAGGG in mouse MC12 and human HeLa cells.…”

Comparative Analysis of DNA‐Binding Selectivity of Hairpin and Cyclic Pyrrole‐Imidazole Polyamides Based on Next‐Generation Sequencing

“…For several decades, PAGE has been one of the most useful methods of defining PIP‐binding sites, by using a technique called DNAse I footprinting, or o identify alkylated sites by PIP conjugates . Melting temperature ( T m ) measurement and surface plasmon resonance (SPR) analysis have also provided data on the interaction between DNA sequences and PIP compounds, and the results have aided investigations into affinity and specificity.…”

Pip-HoGu: An Artificial Assembly with Cooperative DNA Recognition Capable of Mimicking Transcription Factor Pairs

“…A thermal stabilization assay (T m assay) was performed to evaluate the cooperative binding potency and how it was influenced by the gap distance. 29 In the positive-binding mode, the overall thermal stability of Ada1−Cyd1 had a ΔT mP value of 9−15 °C (ΔT mP = T mP − T m ) in a gap-distance-dependent manner (Figure 2B, Table S1). In negative-binding mode, however, there were no gap-distance-dependent effects on the thermal stability of Ada1−Cyd1 with ΔT mN values around 9− 10 °C (Table S1).…”

“…In contrast, in the negative-binding mode, Ada must bridge two PIP-binding sites plus the gap distance, making it impossible for Ada to interact with Cyd. A thermal stabilization assay (Tm assay) was performed to evaluate the cooperative binding potency and how it was influenced by the gap distance 29 . In the positive-binding mode, the overall thermal stability of Ada1-Cyd1 had a ∆TmP value of 9-15 °C (∆TmP = TmP -Tm) in a gap-distance-dependent manner ( Figure 2B, Table S1).…”

“…A thermal stabilization assay ( T m assay) was performed to evaluate the cooperative binding potency and how it was influenced by the gap distance . In the positive-binding mode, the overall thermal stability of Ada1 – Cyd1 had a Δ T mP value of 9–15 °C (Δ T mP = T mP – T m ) in a gap-distance-dependent manner (Figure B, Table S1).…”