Tanlab's Weblog

Purification of Proteins Associated with Specific Genomic Loci

tanlab — Mon, 30 Nov 2009 20:02:27 +0000

Eukaryotic DNA is bound and interpreted by numerous protein complexes in the context of chromatin. A description of the full set of proteins that regulate specific loci is critical to understanding regulation. Here, we describe a protocol called proteomics of isolated chromatin segments (PICh) that addresses this issue. PICh uses a specific nucleic acid probe to isolate genomic DNA with its associated proteins in sufficient quantity and purity to allow identification of the bound proteins. Purification of human telomeric chromatin using PICh identified the majority of known telomeric factors and uncovered a large number of novel associations. We compared proteins found at telomeres maintained by the alternative lengthening of telomeres (ALT) pathway to proteins bound at telomeres maintained by telomerase. We identified and validated several proteins, including orphan nuclear receptors, that specifically bind to ALT telomeres, establishing PICh as a useful tool for characterizing chromatin composition.

Chemical Approaches to DNA Nanotechnology

tanlab — Sun, 13 Sep 2009 22:16:14 +0000

http://www3.interscience.wiley.com/cgi-bin/fulltext/122579503/PDFSTART

Diagnosing lung cancer in exhaled breath using gold nanoparticles

tanlab — Wed, 09 Sep 2009 01:36:43 +0000

Conventional diagnostic methods for lung cancer1,2 are unsuitable
for widespread screening2,3 because they are expensive and
occasionally miss tumours. Gas chromatography/mass spectrometry
studies have shown that several volatile organic compounds,
which normally appear at levels of 1–20 ppb in healthy
human breath, are elevated to levels between 10 and 100 ppb in
lung cancer patients4–6. Here we show that an array of sensors
based on gold nanoparticles can rapidly distinguish the breath
of lung cancer patients from the breath of healthy individuals in
an atmosphere of high humidity. In combination with solidphase
microextraction7, gas chromatography/mass spectrometry
was used to identify 42 volatile organic compounds
that represent lung cancer biomarkers. Four of these were
used to train and optimize the sensors, demonstrating good
agreement between patient and simulated breath samples.
Our results show that sensors based on gold nanoparticles
could form the basis of an inexpensive and non-invasive diagnostic
tool for lung cancer.

Artificial reporter gene providing MRI contrast based on proton exchange

tanlab — Fri, 04 Sep 2009 14:04:25 +0000

Existing magnetic resonance reporter genes all rely on the
presence of (super)paramagnetic substances and employ water
relaxation to gain contrast. We designed a nonmetallic,
biodegradable, lysine rich–protein (LRP) reporter, the prototype
of a potential family of genetically engineered reporters
expressing artificial proteins with frequency-selective contrast.
This endogenous contrast, based on transfer of radiofrequency
labeling from the reporter’s amide protons to water protons, can
be switched on and off.

Imaging the Beta-Cell Mass: Why and How

tanlab — Fri, 04 Sep 2009 13:56:46 +0000

Diabetes is a disorder characterized by beta-cell loss or exhaustion
and insulin deficiency. At present, knowledge is
lacking on the underlying causes and for the therapeutic recovery
of the beta-cell mass. A better understanding of diabetes
pathogenesis could be obtained through exact monitoring
of the fate of beta-cells under disease and therapy
conditions. This could pave the way for a new era of intervention
by islet replacement and regeneration regimens.
Monitoring the beta-cell mass requires a reliable method for
noninvasive in vivo imaging. Such a method is not available at
present due to the lack of a beta-cell-specific contrast agent.
The only existing method to monitor islet cells in vivo consists
of labeling islet transplants with iron nanoparticles prior
to transplantation and visualization of the transplanted islets
by magnetic resonance imaging (MRI). Therefore, accurate
assessment of the native beta-cell mass is still limited to autopsy
studies. Endeavors to find a biological structure specific
for beta-cells led to the discovery of potential candidates
that have been tested for noninvasive imaging. Among
them are the ligand to the vesicular monoamine transporter
type 2 (VMAT-2), which is called dihydrotetrabenazine
(DTBZ), antibodies to zinc transporter (ZnT-8) and the
monoclonal antibody IC2. While DTBZ and antibodies to
ZnT-8 showed binding activities to more than beta-cells, the
anti-IC2 monoclonal antibody showed binding properties
exclusively to insulin-producing beta-cells. This effect was
demonstrated in many previous investigations, and has been
further substantiated more recently. Thus, at present, IC2
seems to be the only useful marker for noninvasive functional
imaging of native beta-cells. Experiments with a radioisotope-
chelated IC2 structure on pancreas ex vivo
showed that the tracer specifically bound to the beta-cell
surface and could be detected by nuclear imaging. In the
near future, these promising findings may offer a new way
to monitor the beta-cell mass in vivo under disease and therapy
conditions so that we can learn more about diabetes
pathogenesis and options for disease prevention.
Keywords: diabetes · beta-cell · noninvasive imaging ·
IC2 · islet transplantation · MRI · PET · photoacoustic

Inhibiting Plasmodium falciparum growth and heme detoxification pathway using heme-binding DNA aptamers

tanlab — Tue, 28 Jul 2009 19:31:21 +0000

The human parasite Plasmodium falciparum enzymatically digests hemoglobin during its intra-erythrocytic developmental stages in acidic food vacuole compartments. The released heme is rapidly detoxified by polymerization into the chemically inert pigment, hemozoin. Several heme-binding anti-malarial compounds, such as chloroquine, efficiently inhibit this process, and this is believed to be the predominant mechanism by which these drugs induce parasite toxicity. In an effort to expand the biochemical tools available for exploration of this pathogen’s basic biology, we chose this heme-detoxification pathway as a model system for exploring the suitability of DNA aptamers for modulating this essential parasite biochemical pathway. In this report, we demonstrate that heme-binding DNA aptamers efficiently inhibit in vitro hemozoin formation catalyzed by either a model lipid system or parasite-derived extracts just as or more potently than chloroquine. Furthermore, when parasites are grown in red cells loaded with heme-binding aptamers, their growth is significantly inhibited relative to parasites exposed to non-heme-binding DNA oligonucleotides. Both the timing of parasite-induced toxicity and the concentration of heme-binding aptamer required for inducing toxicity correlate well with the uptake of red cell cytosolic components by the parasite, and the requirement for compounds with similar in vitro hemozoin inhibitory potency to preconcentrate within the parasite before observing toxicity. Thus, these heme-binding aptamers recapitulate the in vitro hemozoin inhibition activity and induce parasite toxicity in a manner consistent with inhibition of this pathway. Altogether, these data demonstrate that aptamers can be versatile tools with applicability in functionally dissecting important P. falciparum-specific pathways both in vitro and in vivo.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus.

tanlab — Fri, 26 Jun 2009 14:45:12 +0000

Very simple idea. They just used a pool of five binding aptamers selected against bacteria and showed that the pool had better enrichment then any single apatamer. Their method for ssDNA is also interesting–the forward and reverse primers have 30nt difference. Therefore when run on a gel they separate and the forward strand can be cut out and purified.

Deconvolution of a Complex Target Using DNA Aptamers

tanlab — Fri, 01 May 2009 17:31:31 +0000

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 34193–34201

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets.Wepropose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected
populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to
the analysis of other complex biological targets.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

tanlab — Fri, 01 May 2009 17:25:22 +0000

Gene Therapy (2007) 14, 682–689

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirusmediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

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

tanlab — Fri, 01 May 2009 17:19:45 +0000

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.