Recognition of the DNA minor groove by pyrrole-imidazole polyamides

doi:10.1016/S0959-440X(03)00081-2

Current Opinion in Structural Biology

Volume 13, Issue 3, June 2003, Pages 284-299

https://doi.org/10.1016/S0959-440X(03)00081-2 Get rights and content

Abstract

Many diseases, such as cancer, are related to aberrant gene expression. Regulating transcription by chemical methods could be important in human medicine. Minor groove-binding polyamides offer one chemical approach to DNA recognition.

Introduction

Distamycin A is a crescent-shaped natural product that preferentially binds to A,T sequences in the minor groove of DNA as 1:1 and 2:1 ligand–DNA complexes 1., 2.. Analogs of the N-methylpyrrole (Py) rings of these polyamides afford a set of five-membered heterocycles that can be combined — as unsymmetrical ring pairs — in a modular fashion to recognize predetermined DNA sequences with affinity and specificity comparable to DNA-binding proteins (Figure 1, Figure 2) 3., 4.•. We describe here recent advances in the field of DNA-binding polyamides, including structural verification of binding models, new heterocycles for recognition, cellular and nuclear uptake properties, and recent biological applications.

Section snippets

Pairing rules

In a formal sense, the four Watson–Crick base pairs can be differentiated on the minor groove floor by the specific positions of hydrogen-bond donors and acceptors, as well as by subtle differences in molecular shape [5]. A key study in the early 1990s demonstrated that the N-methylimidazole (Im)-containing polyamide ImPyPy bound to the 5 bp sequence 5′-WGWCW-3′ (where W=A or T) instead of the 5′-WGWWW-3′ sequence, which would be expected for a 1:1 polyamide–DNA complex [6]. This surprising

Affinity and specificity

Covalently linking the two antiparallel polyamide strands results in molecules with increased affinity and specificity. Currently, the ‘standard’ motif is the eight-ring hairpin, in which a γ-aminobutyric acid (γ-turn) linker connects the carboxylic terminus of one polyamide to the amino terminus of another (Figure 1, Figure 2). Compared to the unlinked homodimers, hairpin polyamides display approximately 100-fold higher affinity, with the γ-turn demonstrating selectivity for A,T over G,C base

Binding site size

For biological applications, binding site size may be critical because longer sequences would be expected to occur less frequently in the genome. Yet, beyond five contiguous rings, the binding affinity of polyamides decreases [28]. Crystal structures of polyamide–DNA complexes have consistently shown that the polyamide rise per residue matches the pitch of the B-DNA helix — that is, the spacing of the polyamide rings matches the spacing of the DNA base pairs 8., 10., 11.. However, polyamides,

Exploration of new ring systems for minor groove recognition

To explore more broadly the structural landscape for minor groove recognition, a panel of five-membered aromatic heterocycles was synthesized and incorporated into DNA-binding polyamides (Figure 6) 34., 35.. Because we have found that the Hp residue can degrade over time in the presence of acid or free radicals, a more robust thymine-selective element will be needed for biological applications [35]. Remarkably, none of the heterocycles tested (in the context of an eight-ring hairpin) revealed

Synthetic methods

The investigation of minor groove-binding polyamides was greatly accelerated by the implementation of solid-phase synthesis [39]. Originally demonstrated on Boc-β-Ala-PAM resin with Boc-protected monomers, it was also shown that Fmoc chemistry could be employed with suitably protected monomers and Fmoc-β-Ala-Wang resin [40]. Recently, Pessi and co-workers [41] used a sulfonamide-based safety-catch resin to prepare derivatives of hairpin polyamides. Upon activation of the linker, resin-bound

Inhibition of gene expression

Polyamides can bind with high affinity to a wide range of DNA sites and can often competitively displace proteins from DNA (Figure 10, Figure 11). One approach to modifying gene expression involves inhibition of key transcription factor (TF)–DNA complexes in a designated promoter, thus interfering with the recruitment of RNA polymerases (Figure 12). Significantly, because there are considerably fewer oncogenic TFs than potentially oncogenic signaling proteins, TF inhibition represents a

Gene activation

Polyamides can upregulate transcription in two main ways: derepression or recruitment of transcriptional machinery. For example, a hairpin polyamide was shown to block binding of the repressor IE86 to DNA, thereby upregulating transcription of the human cytomegalovirus MIEP [60]. A more complex case involves derepression of the integrated HIV-1 long terminal repeat (LTR). The human protein LSF binds in the promoter region at the LTR and recruits YY1, which then recruits histone deacetylases

Nucleosomes

In eukaryotic cells, DNA is tightly packaged by compaction into chromatin and changes in chromatin structure can alter the accessibility of specific sequences and actively affect components of the molecular machinery in the nucleus. The fundamental repeating unit of chromatin is the nucleosome, comprising a 20–80 bp DNA linker region and the nucleosome core particle — roughly two tight superhelical turns of DNA (147 bp in length) wrapped around a disk of eight histone proteins. The ability of

Nuclear uptake

DNA-binding polyamides can inhibit and influence a wide variety of protein–DNA interactions in solution, yet effectiveness in cell culture has proved to be dependent on cell type. A series of fluorescently labeled polyamides was prepared to analyze the intracellular distribution of these molecules in a panel of cell lines [69^•]. In cell types that had shown robust responses to polyamides, such as primary human T cells [47], polyamide–dye conjugates were observed to enter the nuclei of live

Double-stranded DNA detection

We are hopeful that solutions to several key problems connected with molecular recognition and biological trafficking are in sight. Although the scope and limitations of these advances must be examined, we are now keenly interested in using polyamides as multipurpose tools for DNA detection. Fluorescent derivatives may find broad application. Laemmli and co-workers [33^•] developed the telomere-specific dye conjugates mentioned previously, and Trask and co-workers [71] have painted human

Outlook and future directions

Our long-term aspiration has always been the control of gene expression in living systems. This goal can now be pursued with renewed vigor. Polyamides and the pairing rules allow for the ‘digital read-out’ of predetermined DNA sequences and, now that it appears that polyamides can be modified with improved cell uptake and nuclear localization properties in mammalian cells, the question of specificity on a genomic scale will be critical. What will be the minimum requirements for selectively

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

•
of special interest
••
of outstanding interest

Acknowledgements

The work described in this review was supported by the National Institutes of Health. BSE is supported by a predoctoral fellowship from the Howard Hughes Medical Institute.

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Recognition of the DNA minor groove by pyrrole-imidazole polyamides

Abstract

Introduction

Section snippets

Pairing rules

Affinity and specificity

Binding site size

Exploration of new ring systems for minor groove recognition

Synthetic methods

Inhibition of gene expression

Gene activation

Nucleosomes

Nuclear uptake

Double-stranded DNA detection

Outlook and future directions

References and recommended reading

Acknowledgements

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Chem. Biol.

Nature

Trends Biochem. Sci.

Structure and synthesis of distamycin A

Nature

Structural characterization of a 2:1 distamycin A·D(CGCAAATTGGC) complex by two-dimensional NMR

Proc. Natl. Acad. Sci. U.S.A.

Chemical approaches to control gene expression

Gene Expr.

Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands

Nature

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J. Am. Chem. Soc.

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Proc. Natl. Acad. Sci. U.S.A.

Structural basis for G⋅C recognition in the DNA minor groove

Nat. Struct. Biol.

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Biochemistry

A structural basis for recognition of A·T and T·A base pairs in the minor groove of B-DNA

Science

Structural effects of DNA sequence on T⋅A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

J. Mol. Biol.

Hairpin peptide motif. A new class of oligopeptides for sequence-specific recognition in the minor-groove of double-helical DNA

J. Am. Chem. Soc.

Recognition of DNA by designed ligands at subnanomolar concentrations

Nature

NMR characterization of hairpin polyamide complexes with the minor groove of DNA

J. Am. Chem. Soc.

Orientation preferences of pyrrole-imidazole polyamides in the minor groove of DNA

J. Am. Chem. Soc.

Stereochemical control of the DNA binding affinity, sequence specificity, and orientation preference of chiral hairpin polyamides in the minor groove

J. Am. Chem. Soc.