Evaluation of restriction and Cas endonuclease kinetics using matrix-insensitive magnetic biosensors

Jisoo Im, Songeun Kim, Suhyeon Park, Shan X. Wang, Jung Rok Lee

Research output: Contribution to journalArticlepeer-review

Abstract

The enzymatic actions of endonucleases in vivo can be altered due to bound substrates and differences in local environments, including enzyme concentration, pH, salinity, ionic strength, and temperature. Thus, accurate estimation of enzymatic reactions in vivo using matrix-dependent methods in solution can be challenging. Here, we report a matrix-insensitive magnetic biosensing platform that enables the measurement of endonuclease activity under different conditions with varying pH, salinity, ionic strength, and temperature. Using biosensor arrays and orthogonal pairs of oligonucleotides, we quantitatively characterized the enzymatic activity of EcoRI under different buffer conditions and in the presence of inhibitors. To mimic a more physiological environment, we monitored the sequence-dependent star activity of EcoRI under unconventional conditions. Furthermore, enzymatic activity was measured in cell culture media, saliva, and serum. Last, we estimated the effective cleavage rates of Cas12a on anchored single-strand DNAs using this platform, which more closely resembles in vivo settings. This platform will facilitate precise characterization of restriction and Cas endonucleases under various conditions.

Original languageEnglish
Article number116017
JournalBiosensors and Bioelectronics
Volume249
DOIs
StatePublished - 1 Apr 2024

Bibliographical note

Publisher Copyright:
© 2024 Elsevier B.V.

Keywords

  • Cas12a
  • Endonuclease
  • Giant magnetoresistance
  • Kinetics
  • Magnetic biosensors
  • Surface Michaelis–Menten

Fingerprint

Dive into the research topics of 'Evaluation of restriction and Cas endonuclease kinetics using matrix-insensitive magnetic biosensors'. Together they form a unique fingerprint.

Cite this