Nucleic acid aptamers for target validation and therapeutic applications.

In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life (hours to days), are nontoxic and nonimmunogenic, and are easily produced using standard nucleic acid synthesis methods. These properties make aptamers ideal for target validation and as a new class of therapeutics. As a target validation tool, aptamers provide important information that complements that provided by other methods. For example, siRNA is widely used to demonstrate that protein knock-out in a cellular assay can lead to a biological effect. Aptamers extend that information by showing that the dose-dependent modulation of protein activity can be used to derive a therapeutic benefit. That is, aptamers can be used to demonstrate that the protein is a good target for drug development. As a new class of therapeutics, aptamers bridge the gap between small molecules and biologics. Like biologics, biologically active aptamers are rapidly discovered, have no class-specific toxicity, and are adept at disrupting protein-protein interaction. Like small molecules, aptamers can be rationally engineered and optimized, are nonimmunogenic, and are produced by scalable chemical procedures at moderate cost. As such, aptamers are emerging as an important source of new therapeutic molecules.

Inosine 5'-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.

Tomographic mammography using a limited number of low-dose cone-beam projection images.

A method is described for using a limited number (typically 10-50) of low-dose radiographs to reconstruct the three-dimensional (3D) distribution of x-ray attenuation in the breast. The method uses x-ray cone-beam imaging, an electronic digital detector, and constrained nonlinear iterative computational techniques. Images are reconstructed with high resolution in two dimensions and lower resolution in the third dimension. The 3D distribution of attenuation that is projected into one image in conventional mammography can be separated into many layers (typically 30-80 1-mm-thick layers, depending on breast thickness), increasing the conspicuity of features that are often obscured by overlapping structure in a single-projection view. Schemes that record breast images at nonuniform angular increments, nonuniform image exposure, and nonuniform detector resolution are investigated in order to reduce the total x-ray exposure necessary to obtain diagnostically useful 3D reconstructions, and to improve the quality of the reconstructed images for a given exposure. The total patient radiation dose can be comparable to that used for a standard two-view mammogram. The method is illustrated with images from mastectomy specimens, a phantom, and human volunteers. The results show how image quality is affected by various data-collection protocols.

Combined structural and functional imaging of the breast.

Scintimammography, or single gamma nuclear imaging of the breast, has shown promise as a way of characterizing certain biological properties of suspicious breast masses. Conventional scintimammography, performed using large clinical gamma cameras and prone patient positioning suffers from several drawbacks including poor sensitivity for small (> 1 cm) lesions and no reliable method for correlating scintigraphic findings with those of other imaging modalities. We are developing a system designed to overcome some of these problems. The system combines x-ray mammography with scintimammography on a common gantry. The x-ray and gamma ray images are obtained in quick succession, with the breast in a common configuration under mild compression. A digital x-ray detector is used, permitting rapid assessment of lesion location prior to gamma imaging, and enabling fusion of the x-ray transmission and gamma emission information in a single digital image. In a pilot clinical diagnostic study, the system has demonstrated high pathology-proven accuracy in differentiating benign and malignant masses.

Aptamer-based biosensor arrays for detection and quantification of biological macromolecules.

We have developed a chip-based biosensor for multiplex analysis of protein analytes. The biosensor utilizes immobilized DNA and RNA aptamers, selected against several different protein targets, to simultaneously detect and quantify levels of individual proteins in complex biological mixtures. Aptamers were each fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding. We show that solid-phase aptamer-protein interactions recapitulate binding interactions seen in solution. Furthermore, we demonstrate specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts. It is expected that this technology could speed diagnosis of cancer by enabling direct detection of the expression and modification of proteins closely correlated with disease.

High-sensitivity CCD-based X-ray detector.

The detector is designed for imaging measurements requiring relatively high sensitivity and high spatial resolution. The detector can discriminate single X-ray photons, yet has the wide dynamic range ( approximately 10000:1) associated with integrating detectors. A GdO2S2 phosphor screen converts the incoming X-ray image into an optical image. The optical image is coupled (without demagnification) to the CCD image sensor using a fiber optic faceplate. The CCD (Philips Semiconductors) has an area of 4.9 x 8.6 cm with 4000 x 7000 12 microm pixels. A single 12 keV X-ray photon produces a signal of 100 e-. With 2 x 2 pixel binning, the total noise per 24 microm pixel in a 100 s image is approximately 30 e- the detective quantum efficiency is >0.6 at 1 X-ray photon per pixel, and the full image can be read out in <4 s. The spatial resolution is 50 microm. The CCD readout system is fully computer-controlled, allowing flexible operation in time-resolved experiments. The detector has been characterized using visible-light images, X-ray images and time-resolved muscle diffraction measurements.