Improved method for capturing proteins holds promise for biomedical research

Antibodies are the backbone of the immune system—capable of targeting proteins associated with infection and disease. They are also vital tools for biomedical research, the development of diagnostic tests and for new therapeutic remedies.

Producing antibodies suitable for research however, has often been a difficult, costly and laborious undertaking.

Now, John Chaput and his colleagues at the Biodesign Institute at Arizona State University have developed a new way of producing antibody-like binding agents and rapidly optimizing their affinity for their target proteins. Such capture reagents are vital for revealing the subtleties of protein function, and may pave the way for improved methods of detecting and treating a broad range of diseases.

The team's results appear in today's issue of the journal ChemBioChem.

Antibodies are Y-shaped structures, capable of binding in two or more places with specific target proteins. Synthetic antibodies are much simpler forms that attempt to mimic this behavior. As Chaput explains, creating affinity reagents with strong binding properties can be accomplished by combining two weak affinity segments on a synthetic scaffold. The resulting affinity reagent, if properly constructed, can amplify the binding properties of the individual segments by two or three orders of magnitude.

"This dramatic change in affinity has the ability to transform ordinary molecules into a high affinity synthetic antibody," Chaput says. "Unfortunately, the chemistry used to make these reagents can be quite challenging and often requires a lot of trial-and-error. With NIH funding, my group has reduced the complexity of this problem to simple chemistry that is user friendly and easily amenable to high throughput automation. Such technology is absolutely necessary if we want to compete with traditional monoclonal antibody technology. "

Traditionally, antibodies for research have been extracted from animals induced to produce them in response to various protein antigens. While the technique has been invaluable to medical science, obtaining antibodies in this way is a cumbersome and costly endeavor. Instead, Chaput and his team produce synthetic antibodies that do not require cell culture, in vitro selection or the application of complex chemistry. They call their reagents DNA synbodies.

The new strategy—referred to as LINC (for Ligand Interaction by Nucleotide Conjugates) uses DNA as a programmable scaffold to determine the optimal distance needed to transform two weak affinity binding segments or ligands into a single high affinity protein capture reagent. The result is an artificial antibody, capable of binding to its antigen target with both high affinity and high specificity. The process is rapid and inexpensive. It also offers considerable flexibility, as the distance between the two ligand components bonded to the short, double-stranded DNA scaffold can be fine-tuned for optimum affinity.

In earlier work, the group identified ligand candidates by producing thousands of random sequence peptide chains—strings of amino acids, connected like pearls on a necklace. The peptide sequences were affixed to a glass microarray slide and screened against a target protein to pinpoint those that were capable of recognizing distinct protein binding sites. Two promising ligand candidates could then be combined to form a DNA synbody.

In the current study, the group instead makes use of pre-existing ligands with documented affinity for various disease-related proteins. The method involves the use of well-characterized ligands as building components for high quality DNA synbodies, eliminating the initial screening procedure and expanding the potential to tinker with the two-piece synbody in order to optimize affinity.

The peptides of choice for the study were those with high affinity for something called growth factor receptor bound protein 2 (Grb2). Grb2 has many cell-signaling functions and is an important focus of research due to its association with cellular pathways involved in tumor growth and metastasis.

By scouring the scientific literature, the group identified two peptides that recognize distinct sites on the surface of Grb2. Chaput points out, "this is a nice example where a few hours in the library can save you weeks in the lab."

The next step was to create an assortment of synbody constructs based on these peptides. To do this, one peptide was attached to the end of a short DNA strand, while the other peptide was attached to the complementary DNA strand further along its length (see figure 1).

The two peptide strands could be attached to the scaffold in either a forward or reverse direction and could be interchanged, with either occupying the terminal end of the first DNA strand. Further, the distance between peptide segments along the DNA strands could be adjusted to yield the best target affinity.

Experiments examined binding affinity for peptide chains separated by 3, 6, 9, 12, 15 and 18 base pairs along the DNA strand, (a distance range of 1.0-6.1 nm). Inspection revealed the best results for a synbody constructed of peptides separated by 12 base pairs at a distance of 4.1 nm, compared with the other 5 constructs.

The results for the best synbody in the study were impressive, demonstrating a binding affinity five- to ten-fold stronger than commercially available antibodies for Grb2, despite the synbody's comparatively primitive architecture. In further tests, the synbody was shown to exhibit high specificity—isolating Grb2 from other proteins in a complex biological mixture and selectively binding with its target.

The technique offers a new approach to producing high qualityaffinity reagents for disease research, diagnostic testing and the development of effective therapeutics.

Provided by Arizona State University (news : web)

Capturing the fugitive... in art

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Winslow Homer (1836-1910) For to Be a Farmer's Boy 1887 (Gift of Mrs George T. Langhorne in memory of Edward Carson Waller, AIC 1963.760). This image had long puzzled scholars due to the seemingly unfinished and flat sky in a highly finished work. Credit: ? The Art Institute of Chicago
What do Winslow Homer's For to Be a Farmer's Boy (1887) and Vincent van Gogh's The Bedroom (1889) have in common?
First, they are both displayed at the Art Institute of Chicago (AIC). Homer's painting represents a high point in the career of America's premiere watercolorist, while Van Gogh's painting is perhaps one of most recognizable paintings in the world. However, they also share a key physical trait.
"These breathtaking artworks are both painted with colorants that are sensitive to light, or, as we say in museums, they are 'fugitive,' meaning they quickly vanish if exposed to too much light," says Francesca Casadio, A.W. Mellon senior conservation scientist at the AIC. "Fading can dramatically change the color balance of fragile works of art and go so far as to obfuscate, in part, the artist's intended effect."
In For to Be a Farmer's Boy, the sky is starkly blank--gone are the vibrant colors that Homer is known to have used for his evocative renditions of skies and seas. Yet, through research funded by the National Science Foundation's (NSF) Chemistry and Materials Research in Cultural Heritage Science (CHS) program, a new story is being revealed about Homer's painting.


Laboratory replicas of watercolor brushstrokes containing cochineal (Carmine Naccarat) and madder lakes before (below) and after (above) fading for 20 months in a sun-exposed window. Credit: ? Kristi Dahm, The Art Institute of Chicago
After painstakingly peering through binocular microscopes, art conservators working behind the scenes at AIC discovered some microscopic particles of colored pigments trapped in the artwork's paper fibers. Precise identification of such pigments was key to "recognizing the implicit emotional, narrative and symbolic content" of the artist's work, according to AIC curator Martha Tedeschi.
Effectively identifying the red "lake" pigments that Homer used is difficult using conventional analytical techniques. For example, Raman spectroscopy, normally used to fingerprint artists' palettes, is strongly affected by the overwhelming fluorescence of natural colorants.
Nanotechnology comes to the rescue
Now, art historians and art conservators have one more tool in their arsenal to preserve our cultural treasures: Surface Enhanced Raman Spectroscopy (SERS). Although this technique has been around for almost 30 years, only recently has SERS fully realized its potential, thanks to the nanotechnology boom.

SERS is an ideal technique for art analysis--it is highly sensitive and can detect vanishingly small amounts of organic pigments that have long eluded identification by other approaches. Yet, only a handful of research groups are working on this application.
"Imagine a child in a sandbox with toys. The toys are the molecules--we want to study them, but they are hidden under the sand and you cannot see them," explains Richard P. Van Duyne, the Charles E. and Emma H. Morrison Professor of Chemistry at Northwestern University, who is best known for the discovery of SERS. "Our technique gets rid of the sand so that you can see the toys and identify what they are."

A digital recreation of Homer's For to Be a Farmer's Boy, simulating the appearance of the sky at sunset before fading, matched to the original, faded image. Credit: ? Kristi Dahm, Loren McDonald, The Art Institute of Chicago
By using a colloidal suspension of silver nanoparticles as a "performance enhancing drug," researchers, for the first time, can identify natural organic colorants on a single grain of pigment otherwise invisible to the naked eye.
SERS analysis
Indeed, only a handful of pigment particles were available from the Homer watercolor. Compared to reference 19th century watercolor pigments available at AIC, these colorants were identified as Indian purple (cochineal precipitated with copper sulfate) and madder purple, two natural dyestuffs derived from an insect and vegetable-root sources, respectively.
The results indicate that in Homer's For to Be a Farmer's Boy, the "empty" sky once depicted a vibrant autumn sunset, with organic purples and reds, in addition to inorganic reds and yellows. Although these results are promising, there is still a need to improve the identification of pigments from severely faded paintings. This is because pigment particles are normally applied in a complex medium, which increases the fluorescence background of the spectra, much like adding more sand to the sandbox covers up the toys (molecules) below.
In order to reduce the fluorescence, research professor Nilam Shah, also of Northwestern, will be developing ad hoc-tailored nanoparticles optimized to resonate with infrared lasers, which are less damaging to the artworks, and more universal. These next-generation nanoparticles hold promise as tools to unlock information on dyes, pigments and binding media, as present in Van Gogh's The Bedroom.
Capturing the fugitive
Typically, researchers who use SERS for materials identification search an unknown compound against a database of references, much like matching fingerprints of known criminals to forensic evidence collected on the crime scene.
Thanks to the theoretical expertise of George Schatz, Morrison Professor of Chemistry at Northwestern, this painstaking database search will no longer be a limiting factor. In fact, researchers will be able to calculate from theory not only the expected SERS spectrum of unknown fugitive dyes, but also the tell-tale signs of dyes disappearing after prolonged exposure to light.
Taking into account the pigment identification by SERS and nuances of shade and tone that are typical of Homer's paint handling, art conservators proposed a digital re-creation of the Homer watercolor. By shining laser light on particles buried in the artwork, SERS investigators have now unearthed the materials evidence that allows viewers to truly experience the hues of Homer's faded sunsets for the first time in modern times.

Provided by National Science Foundation (news : web)