News : Light-activated PCR tests 17th January 2022 – Posted in: Uncategorised – Tags: , ,

In the last year there have been many developments and news around PCR tests, these days a new approach has emerged by LMU chemists could help to significantly improve PCR-based diagnostic tests. The enzymes used are activated by pulses of light.

DNA polymerases and other DNA-modifying enzymes are essential tools in biotechnology and diagnostics. They are the key component for PCR diagnosis of COVID-19. As useful as they are, DNA-processing enzymes often have significant defects. Some of them show significant activity during sample preparation, while others have unpleasant side-activities. Both can lead to loss of specificity and sensitivity, which should be avoided in a diagnostic test.

The trick is to block any kind of enzyme activity until the assay is started. In the case of PCR-based diagnostic tests, such as the aforementioned COVID-19 test, the solution is the development of a hot-start enzyme, which does not show activity until a high activation temperature is reached. The main drawback of these hot-start methods is that they cannot be used for enzymes that are damaged by heat, says LMU biochemist Andrés Vera. “In addition, the layout of a hot-begin enzyme is tedious and the laborious layout method needs to be repeated for every new enzyme we need to layout.”

Together with Merve-Zeynep Kesici, from Professor Philip Tinnefeld’s group in the Department of Chemistry at LMU, Andrés found a way around these problems by designing hot-start enzymes. His light-starting enzymes are blocked until a pulse of ultraviolet light reactivates them. “Light-controlled enzymes have been around for a long time, but what makes our approach unique is that it can be applied to virtually any DNA processing enzyme. In the past, you always needed very detailed information about how the enzyme works, and also you had been in no way positive if you may provide you with a smart manner to dam the enzyme and reactivate it with light,” says Vera, the undertaking leader.

In their approach, the researchers attached a piece of DNA to the enzyme itself, which competes excessively with any other enzyme substrate, effectively rendering the enzyme inactive (including its secondary activities).

The light pulse is used to cut the DNA bound to the enzyme, resulting in a 100% active enzyme. The main advantage is that the mechanism should work for a wide range of DNA-binding enzymes, regardless of their specific mode of action.

To demonstrate this, the researchers produced four light-activatable versions of different DNA-processing enzymes. Among them was the so-called Phi29 DNA polymerase, an enzyme widely used to amplify whole genomes, but too heat-sensitive to be suitable for hot-start methods. In addition, the team demonstrated light-start PCR and showed that their DNA polymerases were as good or better compared to commercial hot-start PCR enzymes.

Philip Tinnefeld welcomes this new development: “This will definitely help to produce better enzymes for biotech and diagnostic use. In addition, existing real-time PCR machines already incorporate light sources and could easily be modified to bring these enzymes to market at any time.

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