A DNA nanomachine that maps spatial and temporal pH changes inside living cells

Mingxu presented this paper on 4/30/09

Nature Nanotechnology 2009

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by
external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the
construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based
on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8,
with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the
I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA
nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in
sensing, diagnostics and targeted therapies in living systems.

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.

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

Meghan presented this paper on 3/26/09

PNAS, 2009, 106, 3970

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.

RNA Aptamer Blockade of Osteopontin Inhibits Growth and Metastasis of MDA-MB231 Breast Cancer Cells

Dalia presented this paper on 3/6/09

Molecular Therapy, 2009, 17, 153

Osteopontin (OPN) is a secreted phosphoprotein which mediates tumorigenesis, local growth, and metastasis in a variety of cancers. It is a potential therapeutic target for the regulation of cancer metastasis. RNA aptamer technology targeting OPN may represent a clinically viable therapy. In this study, we characterize the critical sequence of an RNA aptamer, termed OPN-R3, directed against human OPN. It has a Kd of 18 nmol/l and binds specifically to human OPN as determined by RNA electrophoretic mobility assays. In MDA-MB231 human breast cancer cells examined under fluorescence microscopy, OPN-R3 ablates cell surface binding of OPN to its cell surface CD44 and v3 integrin receptors. Critical enzymatic components of the OPN signal transduction pathways, PI3K, JNK1/2, Src and Akt, and mediators of extracellular matrix degradation, matrix metalloproteinase 2 (MMP2) and uroplasminogen activator (uPA), are significantly decreased following exposure to OPN-R3. OPN-R3 inhibits MDA-MB231 in vitro adhesion, migration, and invasion characteristics by 60, 50, and 65%, respectively. In an in vivo xenograft model of breast cancer, OPN-R3 significantly decreases local progression and distant metastases. On the basis of this “proof-of-concept” study, we conclude that RNA aptamer targeting of OPN has biologically relevance for modifying tumor growth and metastasis.

One-step DNA-programmed growth of luminescent and biofunctionalized nanocrystals

Nature Nanotechnology, 4, 2009, 121-125

Kang presented this article on 02/26/09

Colloidal semiconductor nanocrystals are widely used as lumiphores in biological imaging because their luminescence is both strong and stable, and because they can be biofunctionalized. During synthesis, nanocrystals are typically passivated with hydrophobic organic ligands1, so it is then necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water. Finally, biological labels must be added to allow the detection of nucleic acids, proteins and specific cell types2, 3, 4, 5, 6, 7, 8. This multistep process is time- and labour-intensive and thus out of reach of many researchers who want to use luminescent nanocrystals as customized lumiphores. Here, we show that a single designer ligand—a chimeric DNA molecule—can controllably program both the growth and the biofunctionalization of the nanocrystals. One part of the DNA sequence controls the nanocrystal passivation and serves as a ligand, while another part controls the biorecognition. The synthetic protocol reported here is straightforward and produces a homogeneous dispersion of nanocrystal lumiphores functionalized with a single biomolecular receptor. The nanocrystals exhibit strong optical emission in the visible region, minimal toxicity and have hydrodynamic diameters of 6 nm, which makes them suitable for bioimaging4. We show that the nanocrystals can specifically bind DNA, proteins or cells that have unique surface recognition markers.

Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression

Basri presented this on 4/9/09

Nature, 457, 2009, 910-914

Multiple, complex molecular events characterize cancer development and progression1, 2. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.

Micromagnetic selection of aptamers in microfluidic channels

Hui presented this paper on 2/19/09.

PNAS, 2009

Aptamers are nucleic acid molecules that have been selected in vitro to bind to their molecular targets with high affinity and specificity. Typically, the systematic evolution of ligands by exponential enrichment (SELEX) process is used for the isolation of specific, high-affinity aptamers. SELEX, however, is an iterative process requiring multiple rounds of selection and amplification that demand significant time and labor. Here, we describe an aptamer discovery system that is rapid, highly efficient, automatable, and applicable to a wide range of targets, based on the integration of magnetic bead-based SELEX process with microfluidics technology. Our microfluidic SELEX (M-SELEX) method exploits a number of unique phenomena that occur at the microscale and implements a design that enables it to manipulate small numbers of beads precisely and isolate high-affinity aptamers rapidly. As a model to demonstrate the efficiency of the M-SELEX process, we describe here the isolation of DNA aptamers that tightly bind to the light chain of recombinant Botulinum neurotoxin type A (with low-nanomolar dissociation constant) after a single round of selection.

Noninvasive in vivo imaging of pancreatic islet cell biology

Dimitri presented this paper on 1/22/09

Nature Medicine, 14, 2008, 574

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes

Elizabeth presented this paper on 1/29/09

Nature Medicine, 15, 2008, 104

A long-term goal of cancer diagnosis is to develop tumor imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted ‘activatable’ fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH–activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2–positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface–targeting molecules that result in cellular internalization.

Electrostatic readout of DNA microarrays with charged microspheres

Noon presented this paper on 2/12/09

Nature Biotechnology, 26, 2008, 825

DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis1, 2, 3. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care4, 5. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-m lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.