A Structural Basis for Recognition of A·T and T·A Base Pairs in the Minor Groove of B-DNA

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Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.chemical genomics ͉ ligand-DNA recognition

Section: Resultsmentioning

confidence: 99%

Defining the sequence-recognition profile of DNA-binding molecules

Warren

Kratochvil

Hauschild

et al. 2006

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238

“…The unanticipated result of this study is the observation that an Hp residue in this polyamide sequence context can distinguish one of the four Watson⅐Crick base pairs, an A,T. Whether the hydroxyl moiety lies asymmetrically in the cleft between A and T and makes a specific hydrogen bond to the O2 of T, as observed for Hp͞Py recognition of T⅐A (24), awaits structural studies.…”

Section: Discussionmentioning

confidence: 99%

Toward rules for 1:1 polyamide:DNA recognition

Urbach

Dervan

2001

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Polyamides composed of four amino acids, imidazole (Im), pyrrole (Py), hydroxypyrrole (Hp), and ␤-alanine (␤), are synthetic ligands that form highly stable complexes in the minor groove of DNA. Although specific pairing rules within the 2:1 motif can be used to distinguish the four Watson⅐Crick base pairs, a comparable recognition code for 1:1 polyamide:DNA complexes had not been described. To set a quantitative baseline for the field, the sequence specificities of Im, Py, Hp, and ␤ for the four Watson⅐Crick base pairs were determined for two polyamides, Im-␤-ImPy-␤-Im-␤-ImPy-␤-Dp (1, for Im, Py, and ␤) and Im-␤-ImHp-␤-Im-␤-ImPy-␤-Dp (2, for Hp), in a 1:1 complex within the DNA sequence context 5 -AAA-GAGAAGAG-3 . Im residues do not distinguish G,C from A,T but bind all four base pairs with high affinity. Py and ␤ residues exhibit >10-fold preference for A,T over G,C base pairs. The Hp residue displays a unique preference for a single A⅐T base pair with an energetic penalty.

“…Our previous investigations have established a framework for molecular recognition of the minor groove of DNA by polyamides that can target predetermined DNA binding sites (3)(4)(5). Cell culture experiments have shown that cellular uptake of Py-Im polyamides targeting six-base pair sequences can be observed (6).…”

mentioning

confidence: 99%

Gene expression changes in a tumor xenograft by a pyrrole-imidazole polyamide

Raskatov

Nickols

Hargrove

et al. 2012

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Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5′-WGGWWW-3′ with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. An FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which was interrogated by quantitative reverse transcription-PCR and correlated with cell culture expression levels.tumor RNA-sequencing | eXpress | in vivo circulation | efficacy