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Monitoring RAYT activity by surface plasmon resonance biosensor

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Abstract

The process of DNA transposition involves the binding, cleavage, and recombination of specific DNA segments (transposable elements, TE) and is catalyzed by special enzymes encoded by the TE transposases. REP-associated tyrosine transposases (RAYTs) are a class of Y1 nucleases related to the IS200/IS605 transposases associated with a bacterial TE known as repetitive extragenic palindrome elements (REPs). Although RAYT has been subject of numerous studies, where DNA binding and cleavage by RAYT have been confirmed for Escherichia coli, the molecular mechanism of DNA insertion has not been fully understood. In this work, it is demonstrated that surface plasmon resonance (SPR) biosensor technology combined with a system of DNA hairpin probes (mimicking the natural REP sequence) and short oligonucleotides (ONs) can provide a rapid and real-time platform for monitoring and quantification of RAYT activity. We utilized RAYT from E. coli (strain MG1655) as a model system, where we evaluated its activity towards both a natural REP sequence as well as REP sequences having modifications targeting specific features of the DNA crucial for the DNA binding and cleavage. The characteristics of the RAYT-DNA interaction obtained by means of the SPR approach were compared with the results of SDS-PAGE analysis.

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Acknowledgments

This research was supported by Praemium Academiae of the Academy of Sciences of the Czech Republic, the Czech Science Foundation under contract # GAP305/12/1801, by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF and by RVO 86652036 and RVO 67985882.

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Authors and Affiliations

  1. Institute of Photonics and Electronics AS CR, v. v. i., Chaberská 57, 182 51, Prague, Czech Republic

    Markéta Bocková, Tomáš Špringer & Jiří Homola

  2. Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20, Prague, Czech Republic

    Iva Nečasová, Jaroslav Nunvar & Bohdan Schneider

Authors
  1. Markéta Bocková
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  2. Tomáš Špringer
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  3. Iva Nečasová
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  4. Jaroslav Nunvar
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  5. Bohdan Schneider
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  6. Jiří Homola
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Corresponding author

Correspondence to Jiří Homola.

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Published in the topical collection Direct Optical Detection with guest editors Guenter Gauglitz and Jiří Homola.

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Bocková, M., Špringer, T., Nečasová, I. et al. Monitoring RAYT activity by surface plasmon resonance biosensor. Anal Bioanal Chem 407, 3985–3993 (2015). https://doi.org/10.1007/s00216-015-8491-y

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  • DOI: https://doi.org/10.1007/s00216-015-8491-y

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