A new approach by German scientists could help to significantly improve diagnostic tests based on PCR. The enzymes used are triggered by light pulses. They presented a quite general method to produce photoactivatable enzymes. They showed the successful development of three light-activatable DNA polymerases and one nuclease, and prove their applicability to classical molecular biology methods.
DNA polymerases and other enzymes that modify DNA are essential tools in biotechnology and diagnostics. They are the key component for COVID-19 diagnostics by PCR. As useful as they are, DNA processing enzymes often have important flaws. Some of them display significant activity during the preparation of the samples, while others have nasty secondary activities. Both can lead to loss of specificity and sensitivity, which has to be avoided in a diagnostic test. The trick is to block any type of enzymatic activity until the assay starts. For diagnostics tests based on PCR, such as the above mentioned 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. 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.
Together with Merve-Zeynep Kesici from the group of Prof. Philip Tinnefeld at the Department of Chemistry of LMU Andrés found a way around these problems designing light-start enzymes. Their light-start enzymes are blocked until a pulse of UV light reactivates them. 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. 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. In their approach, the researchers bound a piece of DNA to the enzyme itself, which over-compete any other enzymatic substrates rendering the enzyme effectively inactive (including their secondary activities). The light pulse is used to cut the DNA attached to the enzyme resulting in a 100% active enzyme. The main advantage is that the mechanism should work for a broad range of DNA biding enzymes regardless of their specific way of action.
To prove their point the researchers produced four light-activatable versions of different DNA processing enzymes. Among them was the so called Phi29 DNA polymerase, an enzyme broadly used to amplify whole genomes but too heat-sensitive to be adapted to hot-start methods. Moreover, the team showed light-start PCR and proved that their DNA polymerases were as good or better compared to commercial hot-start enzymes for PCR. Philip Tinnefeld is positive about this new development: This is definitely going to help to produce better enzymes for biotechnological and diagnostics use. 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.
The application of the presented methodology to four different DNA processing enzymes, with diverse type of enzymatic activity, including DNA polymerase, exonuclease (5′-3′ and 3′-5′ exonuclease) and endonuclease activity shows promise as a general method to control the activity of these enzymes. In addition, producing the light-activatable version of the enzymes proves to be straightforward, as it sufficed to bind the photocleavable oligonucleotide in an extra stretch of amino acids at the c-teminus of the enzyme. The latter is of particular practical interest because no specific design or knowledge of the enzymes needs to be taken into account. The authors are confident that this approach offers an alternative option before engaging in more complex and demanding strategies. Furthermore, the strategy would be potentially transferable to enzymes with strict dependence on dsDNA, by means of including a stem–loop structure in the blocking oligo.
Merve-Zeynep Kesici, Philip Tinnefeld, Andrés Manuel Vera. A simple and general approach to generate photoactivatable DNA processing enzymes. Nucleic Acids Research, gkab1212, https://doi.org/10.1093/nar/gkab1212Go To Nucleic Acids Research