Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts

Kamilla Vandsø Petersen, Cinzia Tesauro, Marianne Smedegaard Hede, Camilla Pages, Lærke Bay Marcussen, Josephine Geertsen Keller, Magnus Bugge, Kasper Holm, Lotte Bjergbæk, Magnus Stougaard, Christian Wejse, Birgitta R Knudsen*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Abstract

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

Original languageEnglish
Article number7763
JournalSensors
Volume22
Issue20
ISSN1424-8220
DOIs
Publication statusPublished - Oct 2022

Keywords

  • Cell Extracts
  • DNA/chemistry
  • DNA, Circular
  • DNA Restriction Enzymes/metabolism
  • Endonucleases/chemistry

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