Artificial reporter gene providing MRI contrast based on proton exchange

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.

Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins

Nature Biotechnology 27, 378 – 386 (2009)

Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow
cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single
measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically
from cell surface–exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori
knowledge. We apply our cell surface–capturing (CSC) technology, which covalently labels extracellular glycan moieties on
live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as
well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled
differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable
detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means.

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy

Nature Biotechnology 27, 77 – 83 (2008)

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase–activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome–wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

Direct multiplexed measurement of gene expression with color-coded probe pairs

Nature Biotechnology, 26, 2008, 317

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

Rapid genome sequencing with short universal tiling probes

Nature Biotechnology, 26, 2008, 676

The increasing availability of high-quality reference genomic sequences has created a demand for ways to survey the sequence differences present in individual genomes. Here we describe a DNA sequencing method based on hybridization of a universal panel of tiling probes. Millions of shotgun fragments are amplified in situ and subjected to sequential hybridization with short fluorescent probes. Long fragments of 200 bp facilitate unique placement even in large genomes. The sequencing chemistry is simple, enzyme-free and consumes only dilute solutions of the probes, resulting in reduced sequencing cost and substantially increased speed. A prototype instrument based on commonly available equipment was used to resequence the Bacteriophage and Escherichia coli genomes to better than 99.93% accuracy with a raw throughput of 320 Mbp/day, albeit with a significant number of small gaps attributed to losses in sample preparation.

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.

Protein microarrays with carbon nanotubes as multicolor Raman labels

Nature Biotechnology, 26, 2008, 1285

The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM—a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegener’s granulomatosis, diluted up to 107-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure 12C and 13C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.