A simple and general approach to generate photoactivatable DNA processing enzymes. Issue 6 (14th December 2021)

• Record Type: Journal Article
• Authors: Kesici, Merve-Zeynep; Tinnefeld, Philip; Vera, Andrés Manuel
• Is Part Of: Nucleic acids research. Volume 50:Issue 6(2022)
• Page Start: e31
• Page End: e31
• Publication Date: 2021-12-14
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkab1212
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case-specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq, and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease, and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays and showed comparable performance to commercial hot-start enzymes.