David M. Chenoweth

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 Å widening of the minor groove and compression of the major groove along with a >18 ° 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.

Localized light-induced protein dimerization in living cells using a photocaged dimerizer

Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein-protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.

DNA-CNT Nanowire Networks for DNA Detection

The ability to detect biological analytes in a rapid, sensitive, operationally simple, and cost-effective manner will impact human health and safety. Hybrid biocatalyzed-carbon nanotube (CNT) nanowire-based detection methods offer a highly sensitive and specific platform for the fabrication of simple and effective conductometric devices. Here, we report a conductivity-based DNA detection method utilizing carbon nanotube-DNA nanowire devices and oligonucleotide-functionalized enzyme probes. Key to our sensor design is a DNA-linked-CNT wire motif, which forms a network of interrupted carbon nanotube wires connecting two electrodes. Sensing occurs at the DNA junctions linking CNTs, followed by amplification using enzymatic metalization leading to a conductimetric response. The DNA analyte detection limit is 10 fM with the ability to discriminate single, double, and triple base pair mismatches. DNA-CNT nanowires and device sensing gaps were characterized by scanning electron microscopy (SEM) and confocal Raman microscopy, supporting the enhanced conductometric response resulting from nanowire metallization.

Structural Basis for Cyclic Py-Im Polyamide Allosteric Inhibition of Nuclear Receptor Binding

Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor−DNA interfaces, and modulate gene expression pathways in cell culture experiments. In this paper we describe a high-resolution X-ray crystal structure of a β-amino turn-linked eight-ring cyclic Py-Im polyamide bound to the central six base pairs of the sequence d(5′-CCAGTACTGG-3′)2, revealing significant modulation of DNA shape. We compare the DNA structural perturbations induced by DNA-binding transcripton factors, androgen receptor and glucocorticoid receptor, in the major groove to those induced by cyclic polyamide binding in the minor groove. The cyclic polyamide is an allosteric modulator that perturbs the DNA structure in such a way that nuclear receptor protein binding is no longer compatible. 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.

Separation using planar chromatography with electroosmotic flow

Planar chromatography with electroosmotic flow is used to separate either a mixture of dyes using 80% aqueous ethanol as the mobile phase or a mixture of miscellaneous compounds using 45% aqueous acetonitrile as the mobile phase. Both mobile phases are 1.0 mM in N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) buffer. Separations using this technique are faster and more efficient than the same separations by conventional TLC. The respective relationships between migration velocity and applied potential, and between analysis time and distance migrated, are presented.

Next Generation Hairpin Polyamides with (R)-3,4-Diaminobutyric Acid Turn Unit

The characterization of a new class of pyrrole-imidazole hairpin polyamides with beta-amino-gamma-turn units for recognition of the DNA minor groove is reported. A library of eight hairpins containing ( R)- and ( S)-3,4-diaminobutyric acid (beta-amino-gamma-turn) has been synthesized, and the impact of the molecules on DNA-duplex stabilization was studied for comparison with the parent gamma-aminobutyric acid (gamma-turn) and standard ( R)-2,4-diaminobutyric acid (alpha-amino-gamma-turn)-linked eight-ring polyamides. For some, but not all, sequence compositions, melting temperature analyses have revealed that both enantiomeric forms of the beta-amino-gamma-turn increase the DNA-binding affinity of polyamides relative to the ( R)-alpha-amino-gamma-turn. The ( R)-beta-amine residue may be an attractive alternative for constructing hairpin polyamide conjugates. Biological assays have shown that ( R)-beta-amino-gamma-turn hairpins are able to inhibit androgen receptor-mediated gene expression in cell culture similar to hairpins bearing the standard ( R)-alpha-amino-gamma-turn, from which we infer they are cell-permeable.

Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization

Summary Cell polarity, growth and signaling require organelle transport by cytoskeletal motor proteins that are precisely regulated in time and space. Probing these complex, dynamic processes requires experimental techniques with comparable temporal and spatial precision. Inducible dimerization offers the ability to recruit motor proteins to organelles in living cells. Approaches include rapamycin-induced dimerization of motors and cargo-bound binding partners [1] or the recent application of the TULIP light-inducible dimerization system [2,3]. In the latter system, motor recruitment is activated by blue light, and relaxes to an OFF state in the dark within seconds. While rapid relaxation is desirable for some applications, many experiments require sustained motor recruitment. Here, we use a photocaged chemical dimerizer to achieve sustained, spatially-defined motor recruitment to individual organelles with a single pulse of light. We demonstrate the general applicability of the system by recruiting microtubule plus end-directed kinesin-1 and minus end-directed dynein motors to peroxisomes and mitochondria in HeLa cells and primary neurons, leading to alterations in organelle transport on timescales from 10 minutes after photoactivation.

Spindle asymmetry drives non-Mendelian chromosome segregation

How selfish genes get their way At the core of Mendelian genetics is the concept that gametes are equally likely to carry either of the two parental copies of a gene. Selfish genetic elements can cheat, however, by subverting Mendelian segregation to increase their representation in the gametes. Akera et al. show how the inherent asymmetry of female meiosis is translated to an asymmetry within the spindle machinery that segregates the chromosomes (see the Perspective by McNally). Experiments in mouse eggs revealed how asymmetry is exploited by selfish genetic elements to increase their transmission to the egg. Science, this issue p. 668; see also p. 594 Selfish genetic elements exploit the asymmetry inherent in female meiosis to bias their transmission to eggs. Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through a process known as meiotic drive. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle. This implies some asymmetry between the two sides of the spindle, but the molecular mechanisms underlying spindle asymmetry are unknown. Here we found that CDC42 signaling from the cell cortex regulated microtubule tyrosination to induce spindle asymmetry and that non-Mendelian segregation depended on this asymmetry. Cortical CDC42 depends on polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.

Addressable Terminally-Linked DNA-CNT Nanowires

Despite many advances in carbon nanotube (CNT) research, several issues continue to plague the field with regard to the construction of well-defined hybrid CNT materials. Regiospecific covalent functionalization, nonspecific surface absorption, and carbon nanotube aggregation/bundling present major difficulties when working with these materials. In this communication, we circumvent these problems and report a new addressable hybrid material composed of single-walled carbon nanotubes terminally linked by oligonucleotides into a nanowire motif. We show that the oligonucleotide junctions are addressable and can be targeted by gold nanoparticles.

Pyrrole‐Imidazole Polyamides Distinguish Between Double‐Helical DNA and RNA

Groove specificity: pyrrole-imidazole polyamides are well-known for their specific interactions with the minor groove of DNA. However, polyamides do not show similar binding to duplex RNA, and a structural rationale for the molecular-level discrimination of nucleic acid duplexes by minor-groove-binding ligands is presented.