Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands.
The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach for the regulation of gene expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogues have been shown to interfere with gene expression, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G x C from C x G and both of these from A x T/T x A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish AT from T x A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A x T from T x A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.
A structural basis for recognition of A.T and T.A base pairs in the minor groove of B-DNA.
Polyamide dimers containing three types of aromatic rings-pyrrole, imidazole, and hydroxypyrrole-afford a small-molecule recognition code that discriminates among all four Watson-Crick base pairs in the minor groove. The crystal structure of a specific polyamide dimer-DNA complex establishes the structural basis for distinguishing T.A from A.T base pairs. Specificity for the T.A base pair is achieved by means of distinct hydrogen bonds between pairs of substituted pyrroles on the ligand and the O2 of thymine and N3 of adenine. In addition, shape-selective recognition of an asymmetric cleft between the thymine-O2 and the adenine-C2 was observed. Although hitherto similarities among the base pairs in the minor groove have been emphasized, the structure illustrates differences that allow specific minor groove recognition.
Molecular recognition of the nucleosomal ‘‘supergroove''
Chromatin is the physiological substrate in all processes involving eukaryotic DNA. By organizing 147 base pairs of DNA into two tight superhelical coils, the nucleosome generates an architecture where DNA regions that are 80 base pairs apart on linear DNA are brought into close proximity, resulting in the formation of DNA ‘‘supergrooves.'' Here, we report the design of a hairpin polyamide dimer that targets one such supergroove. The 2-Å crystal structure of the nucleosome–polyamide complex shows that the bivalent ‘‘clamp'' effectively crosslinks the two gyres of the DNA superhelix, improves positioning of the DNA on the histone octamer, and stabilizes the nucleosome against dissociation. Our ﬁndings identify nucleosomal supergrooves as platforms for molecular recognition of condensed eukaryotic DNA. In vivo, supergrooves may foster synergistic protein–protein interactions by bringing two regulatory elements into juxtaposition. Because supergroove formation is independent of the translational position of the DNA on the histone octamer, accurate nucleosome positioning over regulatory elements is not required for supergroove participation in eukaryotic gene regulation.
Allosteric modulation of DNA by small molecules.
Many human diseases are caused by dysregulated gene expression. The oversupply of transcription factors may be required for the growth and metastatic behavior of human cancers. Cell permeable small molecules that can be programmed to disrupt transcription factor-DNA interfaces could silence aberrant gene expression pathways. Pyrrole-imidazole polyamides are DNA minor-groove binding molecules that are programmable for a large repertoire of DNA motifs. A high resolution X-ray crystal structure of an 8-ring cyclic Py/Im polyamide bound to the central 6 bp of the sequence d(5'-CCAGGCCTGG-3')2 reveals a 4 A widening of the minor groove and compression of the major groove along with a >18 degrees bend in the helix axis toward the major groove. This allosteric perturbation of the DNA helix provides a molecular basis for disruption of transcription factor-DNA interfaces by small molecules, a minimum step in chemical control of gene networks.
Design of sequence-specific DNA-binding molecules.
Base sequence information can be stored in the local structure of right-handed double-helical DNA (B-DNA). The question arises as to whether a set of rules for the three-dimensional readout of the B-DNA helix can be developed. This would allow the design of synthetic molecules that bind DNA of any specific sequence and site size. There are four stages of development for each new synthetic sequence-specific DNA-binding molecule: design, synthesis, testing for sequence specificity, and reevaluation of the design. This approach has produced bis(distamycin)fumaramide, a synthetic, crescent-shaped oligopeptide that binds nine contiguous adenine-thymine base pairs in the minor groove of double-helical DNA.
Affinity cleavage method
Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes.
Quantitative footprinting titration analyses.(left)Cleavage pattern generated by quantitative DNase I footprinting titration on a 3'-labeled DNA fragment in the presence of increasing ligand concentration (right). Langmuir binding titration isotherm obtained from DNase I data.
Quantitative Microarray Profiling of DNA-Binding Molecules
A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.
Guiding the Design of Synthetic DNA-Binding Molecules with Massively Parallel Sequencing.
Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.
Sequence-specific cleavage of double helical DNA by triple helix formation.
Homopyrimidine oligodeoxyribonucleotides with EDTA-Fe attached at a single position bind the corresponding homopyrimidine-homopurine tracts within large double-stranded DNA by triple helix formation and cleave at that site. Oligonucleotides with EDTA.Fe at the 5' end cause a sequence specific double strand break. The location and asymmetry of the cleavage pattern reveal that the homopyrimidine-EDTA probes bind in the major groove parallel to the homopurine strand of Watson-Crick double helical DNA. The sequence-specific recognition of double helical DNA by homopyrimidine probes is sensitive to single base mismatches. Homopyrimidine probes equipped with DNA cleaving moieties could be useful tools for mapping chromosome.
Sequence-Specific Alkylation of Double-Helical DNA by Oligonucleotide-Directed Triple-Helix Formation.
Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.
Site-Specific Cleavage of Human Chromosome 4 Mediated by Triple-Helix Formation.
Sequence-Specific Double-Strand Alkylation and Cleavage of DNA Mediated by Triple-Helix Formation.
Ribbon model of A'-bromoacetyloligonucleotide-directed double-strand cleavage of DNA by triple-helix formation at inverted adjacent binding sites. Cleavage occurs at a single nucleotide position on each Watson-Crick strand within each triple-helix complex. (B) Left: Autoradiogram of a high-resolution denaturing polyacrylamide gel revealing cleavage products from reaction of A'-bromoacetyloligonucleotide 5 with a 0.9-kbp restriction fragment from plasmid pUCLEU2C labeled at the 3'- and 5'-end with 32P. Lanes 1-4 are 5'-end-labeled DNA. Lanes 5-8 contain 3'-end-labeled DNA. Lanes 1 and 8 are A-specific chemical sequencing reactions for the 5'- and 3'-end-labeled fragments.2' Lanes 2 and 7 are G-specific chemical sequencing reactions for the 5'- and 3'-end-labe!ed fragments.30 Lanes 3 and 6 are controls that contain DNA incubated for 36 h in the absence of A'-bromoacetyloligonucleotide 5 followed by treatment with piperidine. Lancs 4 and 5 contain DNA incubated for 36 h with A-bromoacetyloligonucleotide 5 followed by treatment with piperidine. Right: Sequence of the oligonucleotide and DNA target site. The major sites of modification are indicated by arrows.
Cleavage of yeast chromosome III by /V-bromoacetyloligo- nucleotide 5. Left: Diagram of yeast chromosome III indicating location and sequence of oligonucleotide binding sites and the HIS4 locus used for cleavage analysis. The binding site sequence and sites of alkylation for subsequent site-specific DNA cleavage are indicated. Right: DNA from yeast strains SEY6210 (no target site) and SEY6210C (+ target site) allowed to react with /V-bromoacetyloligonucleotide 5. Lane I: Control with DNA from yeast strain SEY6210 lacking triple-helical target site incubated with /V-bromoacetyloligonucleotide 5 followed by treatment with piperidine. Lane 2: Control with DNA from yeast strain SEY62I0C incubated in the absence of /V-bromoacetyloligonucleotide 5 followed by treatment with piperidine. Lane 3: DNA from yeast strain SEY6210C incubated with /V-bromoacetyloligonucleotide 5, followed by treatment with piperidine. (A) Separation of yeast chromosomes by pulsed field gel electrophoresis in agarose and stained with ethidium bromide. (B) Autoradiogram of a DNA blot hybridization of gel in part A with a 250-bp HIS 4 fragment radiolabeled with '2P by random-primer extension. Locations of the intact chromosome II (340 kbp) and the 110-kbp cleavage fragment are indicated. Yield of site-specific cleavage is 90%.