Cargando…

Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans-...

Descripción completa

Detalles Bibliográficos
Autores principales: Ivanov, Aleksandr V., Safenkova, Irina V., Biketov, Sergey F., Zherdev, Anatoly V., Dzantiev, Boris B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10003267/
https://www.ncbi.nlm.nih.gov/pubmed/36901914
http://dx.doi.org/10.3390/ijms24054484
_version_ 1784904565412331520
author Ivanov, Aleksandr V.
Safenkova, Irina V.
Biketov, Sergey F.
Zherdev, Anatoly V.
Dzantiev, Boris B.
author_facet Ivanov, Aleksandr V.
Safenkova, Irina V.
Biketov, Sergey F.
Zherdev, Anatoly V.
Dzantiev, Boris B.
author_sort Ivanov, Aleksandr V.
collection PubMed
description Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs’ surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120–300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP’s surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.
format Online
Article
Text
id pubmed-10003267
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-100032672023-03-11 Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors Ivanov, Aleksandr V. Safenkova, Irina V. Biketov, Sergey F. Zherdev, Anatoly V. Dzantiev, Boris B. Int J Mol Sci Article Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs’ surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120–300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP’s surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures. MDPI 2023-02-24 /pmc/articles/PMC10003267/ /pubmed/36901914 http://dx.doi.org/10.3390/ijms24054484 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ivanov, Aleksandr V.
Safenkova, Irina V.
Biketov, Sergey F.
Zherdev, Anatoly V.
Dzantiev, Boris B.
Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title_full Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title_fullStr Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title_full_unstemmed Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title_short Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors
title_sort engineering of dna structures attached to magnetic particles for effective trans- and cis-cleavage in cas12-based biosensors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10003267/
https://www.ncbi.nlm.nih.gov/pubmed/36901914
http://dx.doi.org/10.3390/ijms24054484
work_keys_str_mv AT ivanovaleksandrv engineeringofdnastructuresattachedtomagneticparticlesforeffectivetransandciscleavageincas12basedbiosensors
AT safenkovairinav engineeringofdnastructuresattachedtomagneticparticlesforeffectivetransandciscleavageincas12basedbiosensors
AT biketovsergeyf engineeringofdnastructuresattachedtomagneticparticlesforeffectivetransandciscleavageincas12basedbiosensors
AT zherdevanatolyv engineeringofdnastructuresattachedtomagneticparticlesforeffectivetransandciscleavageincas12basedbiosensors
AT dzantievborisb engineeringofdnastructuresattachedtomagneticparticlesforeffectivetransandciscleavageincas12basedbiosensors