DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors.

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

Surface passivation improves the synthesis of highly stable and specific DNA-functionalized gold nanoparticles with variable DNA density.

We report a novel and multifaceted approach for the quick synthesis of highly stable single-stranded DNA (ssDNA) functionalized gold nanoparticles (AuNPs). The method is based on the combined effect of surface passivation by (1-mercaptoundec-11-yl)hexa(ethylene glycol) and low pH conditions, does not require any salt pretreatment or high excess of ssDNA, and can be generalized for oligonucleotides of any length or base sequence. The synthesized ssDNA-coated AuNPs conjugates are stable at salt concentrations as high as 3.0 M, and also functional and specific toward DNA-DNA hybridization, as shown from UV-vis spectrophotometry, scanning electron microscopy, gel electrophoresis, fluorescence, and small angle X-ray scattering based analyses. The method is highly flexible and shows an additional advantage of creating ssDNA-AuNP conjugates with a predefined number of ssDNA strands per particle. Its simplicity and tenability make it widely applicable to diverse biosensing applications involving ssDNA functionalized AuNPs.

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles.

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of alpha-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.

Probing Au nanoparticle uptake by enzyme following the digestion of a starch-Au-nanoparticle composite.

In this letter, we report on the digestion of starch, when present as a composite with Au nanoparticles (NPs), by alpha-amylase. It has been observed that the rate of digestion of free starch and that in the composite were identical. Also, the well-established iodine test could be carried out to investigate the kinetics in the presence of Au NPs. The investigations revealed that following the digestion of starch in the composite the NPs were released and subsequently attached to the enzyme only and not to the degraded products of starch. Also, the enzyme attached to NPs, following digestion, retained its catalytic activity. The particle sizes of the NPs were not affected in the process because no agglomeration was observed. Experimental observations indicated the possibility of oriented attachment of alpha-amylase to the NPs in comparison to amyloglucosidase, another digestive enzyme. Finally, we observed a change in the surface plasmon resonance (SPR) of the NPs following the digestion of starch in the composite, and thus we could demonstrate that the SPR of the NPs could be used as a direct probe for monitoring the digestion of the composite by the enzyme.

Retention of enzymatic activity of alpha-amylase in the reductive synthesis of gold nanoparticles.

In this paper, we report the generation of Au nanoparticles (NPs), using a pure enzyme for the reduction of AuCl4(-), with the retention of enzymatic activity in the complex. As a model system, alpha-amylase was used to readily synthesize and stabilize Au NPs in aqueous solution. Although several other enzymes were also pursued for the synthesis, it was interesting to observe that only alpha-amylase and EcoRI could produce Au NPs. Following NP synthesis, the activity of the enzyme was retained in the Au NP-alpha-amylase complex. The presence of Au NPs and alpha-amylase in the complex was established by UV-visible and FT-IR spectroscopy, X-ray diffraction (XRD) and transmission electron microscopic (TEM) measurements. Our observations suggest that the presence of free and exposed S-H groups is essential in the reduction of AuCl4(-) to Au NPs. Structural analysis of the enzymes showed that both alpha-amylase and EcoRI enzymes have free and exposed S-H groups in their native form and thus are suitable for the generation of NPs, whereas the other ones used here do not have such groups. Fortuitously, the enzymatic functional group of alpha-amylase is positioned opposite to that of the free and exposed S-H group, which makes it ideal for the production of Au NPs; binding of the enzyme to Au NPs via Au-S bond and also retention of the biological activity of the enzyme.