Co-reporter:Graham S. Erwin;Devesh Bhimsaria;Asuka Eguchi ; Aseem Z. Ansari
Angewandte Chemie International Edition 2014 Volume 53( Issue 38) pp:10124-10128
Publication Date(Web):
DOI:10.1002/anie.201405497
Abstract
Targeting the genome with sequence-specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole-based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high-affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.
Co-reporter:Graham S. Erwin;Devesh Bhimsaria;Asuka Eguchi ; Aseem Z. Ansari
Angewandte Chemie 2014 Volume 126( Issue 38) pp:10288-10292
Publication Date(Web):
DOI:10.1002/ange.201405497
Abstract
Targeting the genome with sequence-specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole-based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high-affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.
Co-reporter:Dr. Kimberly J. Peterson-Kaufman;Dr. Clayton D. Carlson;Dr. José A. Rodríguez-Martínez ; Aseem Z. Ansari
ChemBioChem 2010 Volume 11( Issue 14) pp:1955-1962
Publication Date(Web):
DOI:10.1002/cbic.201000255
Abstract
Nature constructs intricate complexes containing numerous binding partners in order to direct a variety of cellular processes. Researchers have taken a cue from these events to develop synthetic molecules that can nucleate natural and unnatural interactions for a diverse set of applications. These molecules can be designed to drive protein dimerization or to modulate the interactions between proteins, lipids, DNA, or RNA and thereby alter cellular pathways. A variety of components within the cellular machinery can be recruited with or replaced by synthetic compounds. Directing the formation of multicomponent complexes with new synthetic molecules can allow unprecedented control over the cellular machinery.
Co-reporter:Clayton D. Carlson;Naveeda Qadir;Mary S. Ozers;Devesh Bhimsaria;Aseem Z. Ansari;Franco Cerrina;Karl E. Hauschild;Christopher L. Warren;Youngsook Lee
PNAS 2010 Volume 107 (Issue 10 ) pp:4544-4549
Publication Date(Web):2010-03-09
DOI:10.1073/pnas.0914023107
Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher
gene regulatory networks or engineer molecules that act on genomes. Here we compare the DNA sequence specificities for different
classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized
and interpreted using an interactive “specificity landscape” which simultaneously displays the affinity and specificity of
a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and
often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity
constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of
a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater
accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every
DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic
DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe
will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity
of DNA binding molecules.
Co-reporter:Karl E. Hauschild, James S. Stover, Dale L. Boger, Aseem Z. Ansari
Bioorganic & Medicinal Chemistry Letters 2009 Volume 19(Issue 14) pp:3779-3782
Publication Date(Web):15 July 2009
DOI:10.1016/j.bmcl.2009.04.097
Determining the sequence specifity of DNA binding molecules is a non-trivial task. Here we describe the development of a platform for assaying the sequence specificity of DNA ligands using label free detection on high density DNA microarrays. This is achieved by combining Cognate Site Identification (CSI) with Fluorescence Intercalation Displacement (FID) to create CSI–FID. We use the well-studied small molecule DNA ligand netropsin to develop this high throughput platform. Analysis of the DNA binding properties of protein- and small molecule-based libraries with CSI–FID will advance the development of genome-anchored molecules for therapeutic purposes.
Co-reporter:Rocco Moretti, Leslie J. Donato, Mary L. Brezinski, Ryan L. Stafford, Helena Hoff, Jon S. Thorson, Peter B. Dervan and Aseem Z. Ansari
ACS Chemical Biology 2008 Volume 3(Issue 4) pp:220
Publication Date(Web):April 18, 2008
DOI:10.1021/cb700258r
The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Although direct interactions are important, the contribution of indirect interactions toward cooperative assembly of Hox and Exd remains unresolved. Here we use minor groove binding polyamides as structural wedges to induce perturbations at specific base steps within the Exd binding site. We find that allosteric modulation of DNA structure contributes nearly 1.5 kcal/mol to the binding of Exd to DNA, even in the absence of direct Hox contacts. In contrast to previous studies, the sequence-targeted chemical wedges reveal the role of DNA geometry in cooperative assembly of Hox−Exd complexes. Programmable polyamides may well serve as general probes to investigate the role of DNA modulation in the cooperative and highly specific assembly of other protein−DNA complexes.
Co-reporter:Anna K. Mapp and Aseem Z. Ansari
ACS Chemical Biology 2007 Volume 2(Issue 1) pp:62
Publication Date(Web):January 19, 2007
DOI:10.1021/cb600463w
Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation. Keywords: Activator ATF: An activator ATF up-regulates specific genes or sets of genes by binding to a particular sequence of DNA and interacting with one or more components of the transcriptional machinery. Molecules that indirectly affect gene activation, for example, by stimulating signal transduction cascades or altering DNA structure, are thus not activator ATFs.; Artificial transcription factor (ATF): An ATF is a designer molecule that seeks out specific genes or groups of genes and directly regulates them either positively or negatively. An ATF typically contains at least two functional domains, a DNA binding domain and a regulatory domain.; Coactivator: A protein that interacts with the transcriptional activation domain of a DNA-bound transcriptional activator and participates in the gene-activation process.; DNA binding domain (DBD): One of the two key domains of an activator ATF, the DBD provides the gene-targeting specificity of the molecule.; Transcriptional activation domain (TAD): One of the two key domains of an activator ATF, the TAD dictates the timing and extent of transcriptional up-regulation through binding interactions with one or more components of the transcriptional machinery.; Transcriptional activator: These natural transcription factors are key players in the cascade of events that lead to gene activation. Minimally composed of a DNA binding domain and a transcriptional activation domain, activators function in a signal-responsive fashion to regulate the timing and extent of gene-specific activation.
Co-reporter:Elenita I. Kanin;Ryan T. Kipp;Charles Kung;Matthew Slattery;Agnes Viale;Steven Hahn;Kevan M. Shokat;Aseem Z. Ansari;
Proceedings of the National Academy of Sciences 2007 104(14) pp:5812-5817
Publication Date(Web):March 28, 2007
DOI:10.1073/pnas.0611505104
The process of gene transcription requires the recruitment of a hypophosphorylated form of RNA polymerase II (Pol II) to a
gene promoter. The TFIIH-associated kinase Cdk7/Kin28 hyperphosphorylates the promoter-bound polymerase; this event is thought
to play a crucial role in transcription initiation and promoter clearance. Studies using temperature-sensitive mutants of
Kin28 have provided the most compelling evidence for an essential role of its kinase activity in global mRNA synthesis. In
contrast, using a small molecule inhibitor that specifically inhibits Kin28 in vivo, we find that the kinase activity is not essential for global transcription. Unlike the temperature-sensitive alleles, the
small-molecule inhibitor does not perturb protein–protein interactions nor does it provoke the disassociation of TFIIH from
gene promoters. These results lead us to conclude that other functions of TFIIH, rather than the kinase activity, are critical
for global gene transcription.
Co-reporter:Shane Foister;Mary L. Brezinski;Christopher L. Warren;Peter B. Dervan;Aseem Z. Ansari;George N. Phillips, Jr.;Karl E. Hauschild;Natasha C. S. Kratochvil
PNAS 2006 Volume 103 (Issue 4 ) pp:867-872
Publication Date(Web):2006-01-24
DOI:10.1073/pnas.0509843102
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.
Co-reporter:Karl E. Hauschild;Renee E. Metzler;Hans-Dieter Arndt;Rocco Moretti;Marni Raffaelle;Peter B. Dervan;Aseem Z. Ansari;
Proceedings of the National Academy of Sciences 2005 102(14) pp:5008-5013
Publication Date(Web):March 21, 2005
DOI:10.1073/pnas.0501289102
Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription
factors. A hairpin polyamide–YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to
an adjacent DNA site. Such small molecules function as protein–DNA dimerizers that stabilize complexes at composite DNA binding
sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree
of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker
tested confers a “switch”-like property on the protein–DNA dimerizer, in that it abolishes the ability of the YPWM moiety
to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription
factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.
Co-reporter:Aseem Z. Ansari, Marsha Rich Rosner, Julius Adler
Molecular Cell (23 December 2011) Volume 44(Issue 6) pp:841-843
Publication Date(Web):23 December 2011
DOI:10.1016/j.molcel.2011.12.009
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