title: Detection of the strand exchange reaction using DNAzyme and Thermotoga maritima recombinase A.
abstract: We have designed multiple detection systems for the DNA strand exchange process. Thermostable Thermotoga maritima recombinase A (TmRecA), a core protein in homologous recombination, and DNAzyme, a catalytic DNA that can cleave a specific DNA sequence, are introduced in this work. In a colorimetric method, gold nanoparticles (AuNPs) modified with complementary DNAs (cDNAs) were assembled by annealing. Aggregated AuNPs were then separated irreversibly by TmRecA and DNAzyme, leading to a distinct color change in the particles from purple to red. For the case of fluorometric detection, fluorescein isothiocyanate (FITC)-labeled DNA as a fluorophore and black hole quencher 1 (BHQ1)-labeled DNA as a quencher were used; successful strand exchange was clearly detected by variations in fluorescence intensity. In addition, alterations in the impedance of a gold electrode with immobilized DNA were employed to monitor the regular exchange of DNA strands. All three methods provided sufficient evidence of efficient strand exchange reactions and have great potential for applications in the monitoring of recombination, discovery of new DNAzymes, detection of DNAzymes, and measurement of other protein activities.
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology, Pohang, Gyungbuk 790-784, South Korea.
authors.forename: Hunho
authors.initials: H
authors.lastname: Jo
authors.forename: Seonghwan
authors.initials: S
authors.lastname: Lee
authors.forename: Kyoungin
authors.initials: K
authors.lastname: Min
authors.forename: Changill
authors.initials: C
authors.lastname: Ban
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D021881:DNA, Catalytic; D055664:Electrochemical Techniques; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry; D006046:Gold; D053768:Metal Nanoparticles; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D006046:Gold; D004247:DNA; D011938:Rec A Recombinases; D016650:Fluorescein-5-isothiocyanate