Cargando…

A simple and general approach to generate photoactivatable DNA processing enzymes

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 appl...

Descripción completa

Detalles Bibliográficos
Autores principales: Kesici, Merve-Zeynep, Tinnefeld, Philip, Vera, Andrés Manuel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8989547/
https://www.ncbi.nlm.nih.gov/pubmed/34904657
http://dx.doi.org/10.1093/nar/gkab1212
_version_ 1784683199593447424
author Kesici, Merve-Zeynep
Tinnefeld, Philip
Vera, Andrés Manuel
author_facet Kesici, Merve-Zeynep
Tinnefeld, Philip
Vera, Andrés Manuel
author_sort Kesici, Merve-Zeynep
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8989547
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-89895472022-04-08 A simple and general approach to generate photoactivatable DNA processing enzymes Kesici, Merve-Zeynep Tinnefeld, Philip Vera, Andrés Manuel Nucleic Acids Res Methods Online 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. Oxford University Press 2021-12-14 /pmc/articles/PMC8989547/ /pubmed/34904657 http://dx.doi.org/10.1093/nar/gkab1212 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Kesici, Merve-Zeynep
Tinnefeld, Philip
Vera, Andrés Manuel
A simple and general approach to generate photoactivatable DNA processing enzymes
title A simple and general approach to generate photoactivatable DNA processing enzymes
title_full A simple and general approach to generate photoactivatable DNA processing enzymes
title_fullStr A simple and general approach to generate photoactivatable DNA processing enzymes
title_full_unstemmed A simple and general approach to generate photoactivatable DNA processing enzymes
title_short A simple and general approach to generate photoactivatable DNA processing enzymes
title_sort simple and general approach to generate photoactivatable dna processing enzymes
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8989547/
https://www.ncbi.nlm.nih.gov/pubmed/34904657
http://dx.doi.org/10.1093/nar/gkab1212
work_keys_str_mv AT kesicimervezeynep asimpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes
AT tinnefeldphilip asimpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes
AT veraandresmanuel asimpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes
AT kesicimervezeynep simpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes
AT tinnefeldphilip simpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes
AT veraandresmanuel simpleandgeneralapproachtogeneratephotoactivatablednaprocessingenzymes