The diagnosis of hereditary diseases and the identification of genetic fingerprints hinge on high-sensitivity DNA imaging biotechnologies. These imaging tools detect specific genes in cells using fluorophores—fluorescent tags that can illuminate DNA structures—and quenchers that interact with these tags to prevent them from emitting light, effectively working as an ‘off switch’.
In a development that expands the detection toolbox and the genetic alphabet, a team led by Ichiro Hirao from the RIKEN Systems and Structural Biology Center, Yokohama, has now designed an artificial base pair between a fluorophore (Dss) and quenchers (Pn and Px)¹. This method incorporates the pair into complementary DNA strands using polymerases and demonstrates that either Pn or Px can decrease the fluorescence of Dss upon hybridization.
Hirao and his team previously developed artificial base pairs involving Dss because of its strong fluorescence, which could illuminate DNA and RNA structures. “This time, we can put out the candle lit by Dss using the quencher as its pairing partner at will,” he says.
Hirao notes that this ability is unique because fluorescent dye Dss and quencher Pn face each other on their respective ssDNA strand, forming an artificial DNA base pair that also works in biological systems. He says that this close proximity results in strong ‘contact quenching’ of the fluorophore.
Usually, researchers have attached fluorophores and quenchers to natural bases through a linker that mediates so-called fluorescence resonance energy transfer (FRET) between dyes. However, this process lacks efficiency compared to contact quenching. Also, according to Hirao, unlike the Dss–Pn system, typical fluorophore–quencher pairs cannot be introduced at specific positions in DNA strands using polymerases, limiting their applications.
After establishing that the pairs were compatible with natural DNA synthesis techniques, Hirao’s team integrated the Dss–Pn pair in the stem of molecular beacons—hairpin-shaped single-stranded DNA (ssDNA) structures that fluoresce upon hybridization with DNA targets. They found that the beacons detected the targets with high sensitivity and differentiated ssDNA containing one mismatched base.
Next, the researchers tested the performance of Dss–Px in polymerase chain reaction (PCR)—a powerful DNA amplification technique. Dss-bearing ssDNA fragments became less fluorescent upon assimilation of Px into synthesized DNA chains, allowing the team to monitor the amplification process in real time.
“One of our present tasks is to apply this system to in vivo cell experiments,” says Hirao. “If it is possible, we will be able see the on–off of a specific gene expression.”
The corresponding author for this highlight is based at the Nucleic Acid Synthetic Biology Research Team, RIKEN Systems and Structural Biology Center
1. Kimoto, M., Mitsui, T. Yamashige, R., Sato, A., Yokoyama, S. & Hirao, I. A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology. Journal of the American Chemical Society 132, 15418–15426 (2010).
A room with a view - or how cultural differences matter in room size perception
25.04.2017 | Max-Planck-Institut für biologische Kybernetik
Studying a catalyst for blood cancers
25.04.2017 | University of Miami Miller School of Medicine
More and more automobile companies are focusing on body parts made of carbon fiber reinforced plastics (CFRP). However, manufacturing and repair costs must be further reduced in order to make CFRP more economical in use. Together with the Volkswagen AG and five other partners in the project HolQueSt 3D, the Laser Zentrum Hannover e.V. (LZH) has developed laser processes for the automatic trimming, drilling and repair of three-dimensional components.
Automated manufacturing processes are the basis for ultimately establishing the series production of CFRP components. In the project HolQueSt 3D, the LZH has...
Reflecting the structure of composites found in nature and the ancient world, researchers at the University of Illinois at Urbana-Champaign have synthesized thin carbon nanotube (CNT) textiles that exhibit both high electrical conductivity and a level of toughness that is about fifty times higher than copper films, currently used in electronics.
"The structural robustness of thin metal films has significant importance for the reliable operation of smart skin and flexible electronics including...
The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.
Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...
The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...
Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.
Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...
20.04.2017 | Event News
18.04.2017 | Event News
03.04.2017 | Event News
25.04.2017 | Earth Sciences
25.04.2017 | Life Sciences
25.04.2017 | Earth Sciences