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Giving PCR Some Sugar | PCR Feature

12/05/2012
Janelle Weaver, Ph.D.

A new method could make it possible to detect single molecules of glycans in individual cells, allowing scientists to explore new terrain in the field of glycobiology. Janelle Weaver reports.


Carbohydrates play key roles in a variety of biological processes, from providing energy to activating immune responses against microbes. But a complete understanding of carbohydrate function has been limited by the difficulty of detecting and quantifying low amounts of these molecules. There are few carbohydrate-specific antibodies, and unlike DNA and RNA, carbohydrates cannot be detected by amplification methods.

The Linhardt and his collaborators developed a novel approach called glyco-quantitative polymerase chain reaction (Glyco-qPCR) for the ultrasensitive detection and quantification of glycans in biological samples. Source: Rensselaer Polytechnic Institute





"When you look at how widely polymerase chain reaction is used worldwide, it's interesting to me to see how little it has been exploited to detect molecules other than DNA or RNA," says Robert Linhardt, a glycobiologist at Rensselaer Polytechnic Institute. Source: Rensselaer Polytechnic Institute

“The protein people worked this out a long time ago,” says Pamela Stanley, a glycobiologist at Albert Einstein College of Medicine. “So, in terms of novelty, Dr. Linhardt has conjugated a piece of DNA to a sugar or glycan.” Source: Griffith University

"When you look at how widely polymerase chain reaction is used worldwide, it's interesting to me to see how little it has been exploited to detect molecules other than DNA or RNA," says Robert Linhardt, a glycobiologist at Rensselaer Polytechnic Institute who has been studying carbohydrates for decades. "A single gene encodes a single protein, so you can detect proteins by virtue of the genes that encode them, and there's the ability to amplify detection with PCR. But carbohydrates are not like that."

Frustrated by the lack of sensitive methods for carbohydrate detection, Linhardt and his collaborators developed a novel approach called glyco-quantitative polymerase chain reaction (Glyco-qPCR) for the ultrasensitive detection and quantification of glycans in biological samples. This method, reported October 16 in Angewandte Chemie International Edition, could be used to study low-abundance glycans on cell surfaces and quantitatively analyze interactions between carbohydrates and proteins that are important for development and disease processes (1).

Glycoconjugates are found in low concentrations in complex biological mixtures, and glycan chains are chemically complex. “Almost always, you have very little material, usually in the range of micrograms, and it's very difficult to synthesize a large repertoire of glycans that are representative of any kind of glycome of any cell,” says Richard Cummings, who studies the role of glycoconjugates in cell adhesion and cell signaling at Emory University School of Medicine. “Ultimately, there are a lot of ideas circulating in the field of glycomics about enhancing sensitivity and detectability, so methods like this are going to be very important.”

Stronger Sugar Sensitivity

Glyco-qPCR uses high-affinity biotin–streptavidin interaction combined with qPCR amplification. In the proof-of-concept study, Linhardt and his team demonstrated the approach with glycans that contained a free reducing end, which the researchers labeled with a short DNA sequence, and a carboxy group, which they labeled with biotin. The attached DNA allowed the group to perform qPCR amplification to quantify the amount of the carbohydrate-DNA conjugates, while the biotin moiety enabled binding to streptavidin-coated magnetic beads for a thorough clean-up of the DNA-labeled carbohydrates from unreacted DNA prior to qPCR.

Because Glyco-qPCR can easily detect residual unreacted DNA—which would produce a false positive signal—the researchers introduced an enhanced clean-up procedure. That process consisted of four more rounds of washing and gel electrophoresis to completely remove unreacted DNA.

“The biggest challenge was actually the clean-up step,” says Linhardt. “When you do the chemistry to attach a nucleic acid onto a carbohydrate, even if your chemistry is pretty good, it's 99%. The amount of residual tag left is going to be such a problem and is going to give so much background when you try to detect one molecule, that you're never going to see what you want to detect, so you need a really good clean-up step.”

Using the carbohydrate-DNA conjugates as templates for qPCR amplification, the researchers detected as little as 1 zmol of disaccharide and less than 1 amol of disaccharides that were isolated from as few as 500 cells. According to the authors, Glyco-qPCR has a detection limit that is a million times more sensitive than the 1-ng detection limit of Western blotting, and the technique is many orders of magnitude more sensitive than detection using mass spectrometry or fluorescence approaches.

Moreover, Linhardt and his team demonstrated that Glyco-qPCR can quantify carbohydrate-protein interactions, such as the interaction between heparin—a medication used to prevent blood clotting associated with cardiovascular conditions and certain types of surgery—and antithrombin III—a protein that regulates blood clotting.

In the clinical realm, Glyco-qPCR could be used to study virus-carbohydrate interactions, and as a high-throughput screening method for the discovery of carbohydrate-based drugs, for example, drugs that interfere with the carbohydrate-mediated binding of the influenza virus to host cells. Linhardt also foresees the technique being useful for cancer diagnostics—predicting whether cancer will be metastatic based on the glycome, which reveals the disease state of tissue.

“If that would allow you to make a prognosis that the patient was going to recover after the tumor was removed by surgery, and didn't need chemotherapy or additional radiation therapy, then you could go with the less aggressive treatment. If the cancer looked metastatic based on its glycome, then you would go with more aggressive therapy,” says Linhardt.

Moving forward, Linhardt hopes to achieve single-cell resolution and single-molecule glycan detection. This could be achieved by improving DNA marker conjugation and incorporating Glyco-qPCR into an automated microfluidic platform that runs both qPCR and capillary electrophoresis, which can be used to separate small quantities of oligosaccharides and allows for more complete compositional analysis. Because PCR amplification allows the detection of a single molecule of DNA, Linhardt believes that it could be possible to detect individual carbohydrate–DNA conjugates in the future.

Fundamental Flaw

The technology of attaching DNA to a non-amplifiable molecule and using PCR for detection is not new. It is based on a concept—known as encoded combinatorial chemistry—that was introduced in a paper published in Proceedings of the National Academy of Sciences in 1992, says Markus Aebi, a microbial glycobiologist at ETH Zurich, the Swiss Federal Institute of Technology (2).

Glyco-qPCR is also similar to immuno-PCR, an antigen detection system—reported in Science two decades ago—that also relies on biotin–streptavidin interaction combined with PCR amplification (3). “The protein people worked this out a long time ago,” says Pamela Stanley, a glycobiologist at Albert Einstein College of Medicine. “So, in terms of novelty, Dr. Linhardt has conjugated a piece of DNA to a sugar or glycan.”

Currently, Glyco-qPCR is limited to glycans with a free reducing end and a carboxy group. Although these properties are common in N-glycans, O-glycans, and glycosaminoglycans, many glycans lack carboxyl groups. “The technology will have to be expanded in order to really be able to detect all kinds of glycans, including those without reducing ends and those without carboxyl groups,” says Cummings. “I think it can be done. That will be the next step, obviously.”

Another limitation is that the approach doesn't provide information about the structure of the glycan. It is impossible to specifically amplify a defined glycan structure out of a mixture. “It doesn’t tell you what type of sugar you have if you start from an unknown sample ,” says Daniel Kolarich, a glycoproteomics expert at the Max Planck Institute of Colloids and Interfaces. “If you don’t know what sugar you have in the first place, you don’t really know what you're amplifying. If you have the sugar defined, then it might be useful for a couple of different types of interaction studies.”

For example, the technique could provide a sensitive means for detecting low levels of a known glycan associated with a particular disease. “It's a very interesting approach that can be applied to particular questions without doubt. Like amplifying interaction signals might be one option, as long as you have one part known,” says Kolarich.

But it may be possible to get around this problem. “Starting with a mixture of different glycans that are tagged differentially, it is possible to detect specifically one component out of a defined mixture,” says Aebi.

Indeed, the study authors explain that multiple DNA markers—serving as molecular barcodes—could be used to tag individual glycans released from a glycoconjugate to facilitate ultrasensitive glycan identification. “However, this type of application is then very similar to the well-known technology of DNA-encoded chemical libraries that are successfully used in drug discovery. Its specific application on glycan structures is a nice extension of the principle but not a totally new technology,” says Aebi.

In the end, Glyco-qPCR will not replace current glycomics technologies, says Kolarich. “The fact that glycans by themselves are not templates for PCR and can't be amplified directly in that particular context—I think this is just a fundamental flaw of biology that we won't be able to overcome.”

References

1. Kwon, S. J., K. B. Lee, K. Solakyildirim, S. Masuko, M. Ly, F. Zhang, L. Li, J. S. Dordick, and R. J. Linhardt. 2012. Signal amplification by Glyco-qPCR for ultrasensitive detection of carbohydrates: Applications in glycobiology. Angew Chem Int Ed Engl 51(47):11800-4.

2. Brenner, S., and R. A. Lerner. 1992. Encoded combinatorial chemistry. Proc Natl Acad Sci USA 89: 5381-5383.

3. Sano, T., C. L. Smith, and C. R. Cantor. 1992. Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258(5079):120-122.

Keywords:  pcr glycobiology