Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.
A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the conjugates. Graphical abstract DNA sensing platform based on an immobilized ssDNA capture probe and a hybrid ssDNA-dsDNA detection probe that specifically hybridize with the ssDNA target. The hybrid ssDNA-dsDNA detection probe also provides the binding sites for the enzyme-DNA binding protein conjugates (HRP-scCro and GOx-dHP) that generate the colorimetric signal.
Bioengineering and Bioelectrochemistry Group, Interfibio, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
Gülsen Betül
GB
Aktas
Bioengineering and Bioelectrochemistry Group, Interfibio, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
Vasso
V
Skouridou
Bioengineering and Bioelectrochemistry Group, Interfibio, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain. lluis.masip@urv.cat.
Lluis
http://orcid.org/0000-0002-3938-4633
L
Masip
D052685:Alphapapillomavirus / Q000302:isolation & purification*; D031215:Armoracia / Q000201:enzymology; D001234:Aspergillus niger / Q000201:enzymology; D001435:Bacteriophages / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis*; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D061925:Immobilized Nucleic Acids / Q000737:chemistry*; D056968:Lac Repressors / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D030361:Papillomavirus Infections / Q000821:virology; D012097:Repressor Proteins / Q000737:chemistry; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D061925:Immobilized Nucleic Acids; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase
DNA binding proteins; Dimeric headpiece domain of Escherichia coli LacI repressor (dHP); Glucose oxidase (GOx); Horseradish peroxidase (HRP); Nucleic acid detection; Single-chain bacteriophage lambda Cro repressor (scCro)