The solution turned from colorless to deep red, indicating the synthesis of AuNPs. Instead, our approach involves easy and fast steps for the preparation of the DNA-AuNP based substrate, which is stable in terms of time and buffer conditions e.
Full size image To monitor the RecQ4-catalysed enzymatic reaction, helicase assay buffer was added to the AuNP aggregate hereafter referred to as substrate that was then transferred to a Perkin Elmer UV-vis spectrophotometer cuvette, followed by the addition of the enzyme and ATP. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The enzyme was able to unwind both substrates.
More importantly, we demonstrated that AuNPs of fixed valences could form well-defined heterogenous oligomeric nanostructures with precise, atom-like control. This shift in the absorbance can be used to monitor the change in the aggregation state of the AuNPs, due to the DNA unwinding catalysed by a helicase. Not surprisingly, given their essential tasks in living organisms, they are emerging as an important class of targets for antiviral, antibiotic and anti-cancer drugs 1 , 2. Bioconjug Chem. Helicase assay As RecQ4 includes a long, partially unstructured N-terminal domain, which is apparently not involved in the helicase activity 32 , the enzyme used in the assay corresponds to a deletion mutant, which includes the helicase core and the putative RQC domain of the human RecQ4 helicase Fig.
Helicase assay As RecQ4 includes a long, partially unstructured N-terminal domain, which is apparently not involved in the helicase activity 32 , the enzyme used in the assay corresponds to a deletion mutant, which includes the helicase core and the putative RQC domain of the human RecQ4 helicase Fig. The percentage of unwinding against the respective time interval for various concentrations of the enzyme is shown in Fig. DNA functionalized gold-nanoparticles Au-nps have been broadly used as labeling reagents in the development of molecular diagnostics as well as building blocks in nanotechnology. The method was validated by comparison with a conventional fluorescence-based assay and the results from the two methods were found to be in good agreement. The amino acid residues shown to play a role in enzyme function 33 , were mutated to alanine shown in Fig.
Au-nps covered with a thermally tunable stabilization layer through mononucleotide adsorption were shown to readily conjugate with thiol-DNAs in 0. The red color arises from the plasmonic adsorption of nm AuNPs at nm. Reduce the DNA into thiols by adding 0.
The protein was expressed and purified to homogeneity in bacterial cells Previously reported methods all rely on the use of thiolated DNA to functionalize AuNPs, resulting in relatively low yields. As expected, the Walker A mutant KA displays no detectable unwinding activity showing that the disaggregation of the substrate is specifically due to the RecQ4 unwinding activity and is not an artefact. The time dependent UV-vis graphs for the experiment, with an enzyme concentration of 60 nM, are shown in Fig. The solution was centrifuged in an eppendorf tube at a speed of 14, rpm for 10 minutes using a table top Beckman Coulter centrifuge and the supernatant was discarded to remove the unbound ssDNA and TOEG6 molecules from the functionalized AuNPs.
Full size image Melting experiments with the assembled substrates were carried out to confirm that the aggregate formation was based on DNA-DNA recognition and not due to non-specific interactions. Instead, our approach involves easy and fast steps for the preparation of the DNA-AuNP based substrate, which is stable in terms of time and buffer conditions e.