Researchers at Ludwig-Maximilians-Universität München in Germany have developed a light-activated form of the enzymes that power the polymerase chain reaction (PCR) assay. The assay has gained recent fame among the general public because of its use as a COVID-19 diagnostic tool. The technique could lead to alternatives to current heat-activated enzymes, which are difficult to design and create, and are not suitable for enzymes that are easily damaged by high temperatures. The method may help to expand the scope of what is possible with PCR.
PCR has been around for quite a while, but recently we have come to rely heavily on it as a COVID-19 diagnostic test. The assay uses various enzymes to amplify tiny amounts of DNA so that we can detect the presence of the SARS-CoV-2 virus. However, the technology is also a cornerstone of medical research, providing invaluable data on gene expression.
The enzymes that power the PCR assay are a little finicky. During sample preparation, the enzymes can become active and begin to work, potentially affecting the results or ruining the sample. To get around this, researchers typically design enzymes that will only become active once they reach a certain temperature. These are known as “hot start” enzymes.
However, hot start enzymes are difficult to design, and the concept doesn’t work for enzymes that are particularly sensitive to heat. “For diagnostics tests based on PCR, such as the[…] test for COVID-19, the solution is the development of a hot-start enzyme, which shows no activity until a high activation temperature is reached,” said Andrés Vera, a researcher involved in the study. “The main drawback of these hot-start approaches is that they cannot be used for enzymes that are damaged by heat. Besides, the design of a hot-start enzyme is tedious and the exhausting design process has to be repeated for each new enzyme that we want to engineer.”
To make it easier to create enzymes for PCR assays, Vera and colleagues turned to light-activated enzymes as an alternative. “Light-controlled enzymes have been around for quite a while, but what makes our approach unique is that it can be applied to virtually any DNA processing enzyme,” said Vera. “In the past you always needed very detailed information on how your enzyme works and you were never sure that you would come with a smart way to block the enzyme and reactivate it with light.”
To achieve light activation, the researchers bound a piece of DNA to the enzyme, resulting in an inactive enzyme that can be activated with a pulse of UV light that frees the enzyme to begin acting on its substrate. So far, the team has shown that their light-activated enzymes show similar or improved performance as hot start equivalents.
“This is definitely going to help to produce better enzymes for biotechnological and diagnostics use,” said Philip Tinnefeld, another researcher involved in the study. “Besides, current real-time PCR machines already incorporate light sources and they could be easily modified to bring these enzymes to the market anytime soon.”
Study in Nucleic Acids Research: A simple and general approach to generate photoactivatable DNA processing enzymes