Phototoxic aptamers selectively enter and kill epithelial cancer cells

Jen presented this paper on 4/23/09

Nucleic Acids Research, 2009, 37, 866

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.

A Bipedal DNA Brownian Motor with Coordinated Legs

Science, 2009, 324, 67

A substantial challenge in engineering molecular motors is designing mechanisms to coordinate the motion between multiple domains of the motor so as to bias random thermal motion. For bipedal motors, this challenge takes the form of coordinating the movement of the biped’s legs so that they can move in a synchronized fashion. To address this problem, we have constructed an autonomous DNA bipedal walker that coordinates the action of its two legs by cyclically catalyzing the hybridization of metastable DNA fuel strands. This process leads to a chemically ratcheted walk along a directionally polar DNA track. By covalently cross-linking aliquots of the walker to its track in successive walking states, we demonstrate that this Brownian motor can complete a full walking cycle on a track whose length could be extended for longer walks. We believe that this study helps to uncover principles behind the design of unidirectional devices that can function without intervention. This device should be able to fulfill roles that entail the performance of useful mechanical work on the nanometer scale.

Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties

Science, 2009, 324, 59

We report a strategy to create photodegradable poly(ethylene glycol)–based hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Postgelation control of the gel properties was demonstrated to introduce temporal changes, creation of arbitrarily shaped features, and on-demand pendant functionality release. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition was used to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments with the scope to answer fundamental questions about material regulation of live cell function and may affect an array of applications from design of drug delivery vehicles to tissue engineering systems.

Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

Science, 2009, 323, 1718

P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of 6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

A Role for RNAi in the Selective Correction of DNA Methylation Defects

Science, 2009, 323, 1600

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important role for RNAi in protecting genomes against long-term epigenetic defects.

Variants of the Antibody Herceptin That Interact with HER2 and VEGF at the Antigen Binding Site

Science, 2009, 323, 1610

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such “two-in-one” antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Polyfluorophores on a DNA Backbone: A Multicolor Set of Labels Excited at One Wavelength

J. Am. Chem. Soc., 2009, 131, 3923

We recently described the assembly of fluorescent deoxyriboside monomers (“fluorosides”) into DNA-like phosphodiester oligomers (oligodeoxyfluorosides or ODFs) in which hydrocarbon and heterocyclic aromatic fluorophores interact both physically and electronically. Here we report the identification of a multicolor set of water-soluble ODF dyes that display emission colors across the visible spectrum, and all of which can be simultaneously excited by long-wavelength UV light at 340−380 nm. Multispectral dye candidates were chosen from a library of 4096 tetramer ODFs constructed on PEG-polystyrene beads using a simple long-pass filter to observe all visible colors at the same time. We resynthesized and characterized a set of 23 ODFs containing one to four individual chromophores and included 2−3 spacer monomers to increase aqueous solubility and minimize aggregation. Emission maxima of this set range from 376 to 633 nm, yielding apparent colors from violet to red, all of which can be visualized directly. The spectra of virtually all ODFs in this set varied considerably from the simple combination of monomer components, revealing extensive electronic interactions between the presumably stacked monomers. In addition, comparisons of anagrams in the set (isomers having the same components in a different sequence) reveal the importance of nearest-neighbor interactions in the emissive behavior. Preliminary experiments with human tumor (HeLa) cells, observing two ODFs by laser confocal microscopy, showed that they can penetrate the outer cellular membrane, yielding cytoplasmic localization. In addition, a set of four distinctly colored ODFs was incubated with live zebrafish embryos, showing tissue penetration, apparent biostability, and no apparent toxicity. The results suggest that ODF dyes may be broadly useful as labels in biological systems, allowing the simultaneous tracking of multiple species by color, and allowing visualization in moving systems where classical fluorophores fail.

Green nanotechnology from tea: phytochemicals in tea as building blocks for production of biocompatible gold nanoparticles

J. Mater. Chem., 2009

Phytochemicals occluded in tea have been extensively used as dietary supplements and as natural pharmaceuticals in the treatment of various diseases including human cancer. Results on the reduction capabilities of phytochemicals present in tea to reduce gold salts to the corresponding gold nanoparticles are presented in this paper. The phytochemicals present in tea serve a dual role as effective reducing agents to reduce gold and also as stabilizers to provide a robust coating on the gold nanoparticles in a single step. The tea-generated gold nanoparticles (T-AuNPs), have demonstrated remarkable in vitro stability in various buffers including saline, histidine, HSA, and cysteine solutions. T-AuNPs with phytochemical coatings have shown significant affinity toward prostate (PC-3) and breast (MCF-7) cancer cells. Results on the cellular internalization of T-AuNPs through endocytosis into the PC-3 and MCF-7 cells are presented. The generation of T-AuNPs follows all principles of green chemistry and T-AuNPs have been found to be non toxic as assessed through MTT assays. No man made chemicals, other than gold salts, are used in this truly biogenic, green nanotechnological process thus paving the way for excellent opportunities for their application in molecular imaging and therapy.

Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor

J. Am. Chem. Soc., 2009, 131, 4262

The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

A small molecule that directs differentiation of human ESCs into the pancreatic lineage

Nature Chemical Biology, 2009, 5, 258

Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors—cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (-)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells.