A DNA strand (purple) primes exponential amplification of DNA (red) as signals for directing light emission of DNA nanodeveices. [Organic and Biomolecular Chemistry]

Scientists in Japan say they have developed a way of amplifying DNA on a scale suitable for use in the emerging fields of DNA-based computing and molecular robotics. By enabling highly sensitive nucleic acid detection, their method could improve disease diagnostics and accelerate the development of biosensors, for example, for food and environmental applications, according to the team.

Researchers from the Tokyo Institute of Technology (Tokyo Tech), Abbott Japan, and the University of Electro-Communications, Japan, published an article (“Leak-free million-fold DNA amplification with locked nucleic acid and targeted hybridization in one pot”) in  Organic and Biomolecular Chemistry that describes a method for achieving a  million-fold DNA amplification and targeted hybridization that works at body temperature (37°C/98.6°F).

The scientists note that the technique, named L-TEAM (Low-TEmperature AMplification), is the result of more than five years of research and offers several advantages over traditional PCR, the dominant technique used to amplify DNA segments of interest.

“An isothermal cascade reaction that exponentially amplifies pre-designed, single-stranded DNA as a sensor and signal amplifier module for DNA-based computing and molecular robotics was developed. Taking advantage of the finding that locked nucleic acid can suppress problematic ab initio DNA synthesis, up to million-fold amplification rates and concurrent hybridization were achieved at a physiological temperature in a single reactor. Although the effect of locked nucleic acid introduction to the templates was complicated, undesired leak DNA amplification was generally suppressed in the amplification reaction for distinct DNA sequences,” write the investigators.

“The present reaction that senses one DNA as an input and generates a large amount of another DNA as an output, exhibiting a high correlation between the molecular concentration and the amplification time, is applicable for nucleic acid quantification.”

With its “one-pot” design, L-TEAM avoids the need for heating and cooling steps and specialized equipment usually associated with PCR, explains Ken Komiya, PhD, assistant professor at Tokyo Tech’s School of Computing. That means it is an efficient, inexpensive method that can importantly prevent protein denaturation, thereby opening a new route to real-time analysis of living cells, he continues.

In their study the researchers introduced synthetic molecules called locked nucleic acids (LNAs) into the DNA strands, as these molecules are known to help achieve greater stability during hybridization. The addition of LNA led to an unexpected, but beneficial, outcome. The team observed a reduced level of “leak” amplification, a type of non-specific amplification that has long been an issue in DNA amplification studies as it can lead to an error in disease diagnosis, that is, a false positive.

“We were surprised to discover the novel effect of LNA in overcoming the common leak problem in DNA amplification reactions,” says Komiya. “We plan to investigate the mechanisms behind leak amplification in detail and further improve the sensitivity and speed of L-TEAM.”

In the near future, the method could be used to detect short nucleic acids such as microRNA for medical diagnostics. In particular, it could facilitate point-of-care testing and early disease detection. MicroRNAs are now increasingly recognized as promising biomarkers for cancer detection and may hold the key to uncovering many other aspects of human health and environmental science.

In addition, Komiya says that L-TEAM paves the way to practical use of DNA computing and DNA-controlled molecular robotics. “The original motivation behind this work was the construction of a novel amplified module that is essential to build advanced molecular systems,” he points out. “Such systems could provide insights into the operational principle behind living things.”

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