Highly selective preparation of N-terminus Horseradish peroxidase-DNA conjugate with fully retained enzymatic activity: HRP-DNA structure - activity relation

Željka Ban, Antun Barišić, Ivo Crnolatac, Saša Kazazić, Sanja Škulj, Filippo Savini, Branimir Bertoša, Ivan Barisic, Ivo Piantanida

Publikation: Beitrag in FachzeitschriftArtikelBegutachtung

Abstract

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein-oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure-activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).

OriginalspracheEnglisch
Aufsatznummer110257
Seitenumfang11
FachzeitschriftEnzyme and Microbial Technology
Volume168
DOIs
PublikationsstatusVeröffentlicht - Aug. 2023

Research Field

  • Molecular Diagnostics

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