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July 13, 2007

Playing tag highlights genetic disorder

Fluorescent and electrochemical labels help scientists detect genetic disorders that can cause cancer

Figure 1: An illustration of methylcytosine labeled directly with a fluorescent tag.

A team of Japanese scientists led by Akimitsu Okamoto from the RIKEN Frontier Research System, Wako, has developed a new method for tagging a particular DNA base responsible for causing cancer.

Cytosine, a common DNA base, is reacted to add a methyl group to form methylcytosine during many biological processes. This process, known as methylation, is recognized as playing many important roles including gene regulation, DNA and protein stabilization, parental imprinting and X chromosome inactivation. Further, excessive methylation of cytosine has been shown to result in cancer. The development of simple techniques to detect methylcytosine is therefore of great interest to scientists.

Positively good tests needed

Figure 2: A graphical representation of the fluorescent DNA probe in action.

Although conventional methods to detect this compound have many advantages, they also have problems. Current methods cannot differentiate between cytosine and methylcytosine; they also destroy the DNA sample and are time-consuming. The latest technique by Okamoto and co-workers, published in the Journal of the American Chemical Society, is selective for methylcytosine, fast and allows easy detection1.

Okamoto points out that there are, in fact, five key points to bear in mind when developing a new chemical assay for methylation detection. Firstly, techniques should take advantage of an easy-to-use form of chemistry, such as fluorescence or electrochemistry that is well established. Tests also need to produce results rapidly for them to be useful to patients in clinics. Next, any new assay should also give a positive result for methylcytosine and a negative result for cytosine. Ideally, being able to detect the exact location of the methylcytosine can provide further valuable information about the role of methylation each site. Lastly, techniques that do not damage the DNA sample avoid complications and errors during detection.

Since identifying the key features of a good assay, Okamoto’s team dedicated two years to incorporating each of them into their work. Their technique takes advantage of the easy oxidation of methylcytosine and uses three, specially designed, components to enable detection. When the reaction takes place, the methylcytosine forms a stable complex with an oxidant, potassium osmate, and a rate-enhancing ligand. The ligand, a bipyridine derivative, can then react further to bond with a variety of fluorescent or electrochemical tags allowing routine detection of the complex; therefore offering a test using straightforward chemistry (Fig. 1).

This conceptually new approach to detection also makes the grade by taking just six hours to complete. Importantly, the key complex only forms between the methylcytosine and the ligand. This leaves the cytosine in the sample untouched and allows a clear, ‘positive test’, distinction to be made. In addition, methylcytosines in single-stranded DNA efficiently formed the complex, whereas complexation of methylcytosines in a DNA duplex was suppressed. This result implies that the technique could be further developed to provide sequence-specific results giving detailed and accurate information of the methylated sites using untreated DNA samples.

Okamoto explains that there is still more work to be done. Currently, the information gained from the sequence-specific studies is limited as a consequence of the competing reaction with thymine, another DNA base. Also, the signal intensities and sensitivities are a little too weak to be useful on small sample sizes at this time.

Shifts in disease detection

Sequence-specific studies remain the focus of another, larger team in Okamoto’s group. In a project extending over the past five years, this team has been developing tests to reveal an individual’s general susceptibility to disease.

The most common type of variation in our genes is a single difference in one of the nucleotide building blocks of the DNA sequence, known as single nucleotide polymorphism (SNP—pronounced ‘snip’). Scientists believe that it is these differences in SNPs that reveal susceptibility, and that their accurate analysis would play a key role in diagnostics. A new method to detect small changes in human genes could also lead the way in personalized medicine.

There are existing methods to detect SNPs, but their use is often limited by the need to identify large DNA sequences. However, this second project, uses derivatives of a fluorescent dye, known as PRODAN, to correctly identify SNPs quickly and efficiently2.

PRODAN absorbs and emits light at different wavelengths depending on the polarity of its environment and when incorporated into DNA structures they can ‘report back’ differences in the microenvironment. Such differences would likely be the result of small changes in the DNA structure and allow detection of sequence variations (Fig. 2).

The researchers studied all combinations of DNA base matches and mismatches and observed the change between the wavelengths absorbed and emitted by the dyes. Only small differences were seen when the DNA base pairs were matched correctly, but large shifts were seen when there was a mismatch. “The use of this DNA probe makes it possible to judge the type of base located at a specific site on the target DNA, simply by mixing the DNA and the dye together. This method is a very powerful assay that does not require enzymes or time-consuming steps, and avoids errors,” says Okamoto.

Following in the success of this second project, Okamoto and his team are now striving to improve their technique for methylation detection so it can be used routinely in clinics with standard fluorescence or electronic signal analyzers. Okamoto enthuses, “Because the total process finishes in a few hours, this technique may make it possible to design machines that automate a series of processes from purification of samples to analysis.”

References

  1. Tanaka, K., Tainaka, K., Kamei, T. & Okamoto, A. Direct labeling of 5-methylcytosine and its applications. Journal of the American Chemical Society 129, 5612–5620 (2007). |  | (Link)
  2. Tainaka, K., Tanaka, K., Ikeda, S., Nishiza, K., Unzai, T., Fujiwara, Y., Saito, I. & Okamoto, A. PRODAN-conjugated DNA: Synthesis and photochemical properties. Journal of the American Chemical Society 129, 4776–4784 (2007). |  | (Link)

About the Researcher

Akimitsu Okamoto

Akimitsu Okamoto was born in Nagoya, Japan, in 1970. He graduated from the Faculty of Engineering, Kyoto University, in 1993, and obtained his PhD in 1998 from the same university. Afterwards he engaged in postdoctoral research at the Department of Chemistry, Massachusetts Institute of Technology, for a year. He returned to Japan as a research associate at the Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, where he started his career in photochemistry and synthetic nucleic acid chemistry. He moved to the Frontier Research System, RIKEN, as an initiative research scientist in 2006. Since then, he has been the unit leader of the Okamoto Initiative Research Unit. His research focuses on the design, synthesis and physical properties of new, man-made biopolymers with various functions, and the design of unprecedented organic chemical systems for recognizing, transforming and visualizing a single component or atom in biopolymers of interest.