NEW YORK (GenomeWeb) – Restriction enzyme cascades can be used to drive isothermal DNA detection, according to a publication in Scientific Reports.
Scientists led by Maria Smith and Andrey Ghindilis of Cascade Biosystems, described in the paper an assay that uses cascades of restriction endonucleases to detect target DNA. It is highly specific, cost-efficient, and performs about as well as real-time PCR, they said.
"The original idea came from multiple discussions on how to make a system for enzymatic signal amplification," Smith said. "At that time, in 2007, we were aware of only one exponential amplification process, namely PCR. However, PCR amplifies a DNA target."
Cascade's technology, instead, amplifies a signal related to the event of the target DNA hybridizing to a DNA probe.
There are two steps to the process, known as Restriction Cascade Exponential Amplification (RCEA): a DNA recognition step and an amplification step, both of which are dependent on restriction endonucleases. The key molecule throughout is called the amplification restriction endonuclease (Ramp).
In the recognition step, Ramp is bonded to an oligonucleotide probe complementary and specific to the target DNA strand. If the target DNA hybridizes to the Ramp-probe complex, a "recognition" restriction endonuclease will cleave it, releasing the Ramp enzyme, which is fully functional.
As opposed to PCR, where the target DNA sequence and also the molecule are being amplified, it's the Ramp enzyme activity that becomes amplified in the next step. The solution, which contains free and active Ramp if the target is detected, is transferred to a separate chamber by pipette that features double-stranded DNA bonded to both another Ramp molecule and horseradish peroxidase (HRP), which will become the signal.
Any available Ramp will begin to cleave the Ramp-HRP complex, releasing both molecules, and kicking off a cascade of cleaving activity. As the amount of free Ramp grows exponentially, the HRP signal grows with it and is detected colorimetrically.
The main challenge in designing the probe was to preserve the enzymatic activity of the amplification restriction endonuclease. "We are using mutant restriction endonuclease enzymes with a surface Cys available for coupling without loss of enzymatic activity. These mutant REases can be coupled to any oligonucleotide probe of interest," Smith said.
She added that the probe sequence required a suitable restriction site for the recognition restriction enzyme to cleave, but with probes of about 1,000 nucleotides, that wasn't a problem.
Compared to qPCR, RCEA offers several advantages, the authors said. For example, Ghindilis said RCEA had a detection limit as low as about 200 molecules in a sample. It's isothermal, which could allow it to be done faster if automated, and it could be performed at the point of care or in the field. It's very specific, Ghindilis said, because it requires both hybridization and restriction; if non-target DNA somehow hybridizes, it's unlikely to present the correct restriction site to the cleaving enzyme. "It shouldn't have any false positives," he said.
Ghindilis does not see RCEA competing directly with qPCR, but instead sees it filling niches where qPCR is either too expensive or where quantification is overkill and a qualitative technology would suffice, such as in food safety and environmental testing.
The technology is still very much in development. Cascade Biosystems is a small startup with only three employees, started in 2010 to work on this technology. Ghindilis said he did most of the science by himself. Though the firm received a grant worth about $300,000 from the government of Qatar, it doesn't have its own permanent laboratory space. Smith added that Cascade has obtained seven US patents related to RCEA.
The firm's next goal is to optimize the technology. "It's a lot of pipetting right now," Ghindilis said. "Sensitivity is good enough, but the time of the assay can shorten drastically. Theoretically it should be much faster than PCR."