Gold nanoparticles are of intense interest for nanotechnology, in addition to their use for localizing biomolecules at ultra high resolution.
Gold nanoparticles possess a number of size-dependent physical, electronic and chemical properties with great potential uses: they can amplify spectroscopic signals, absorb X-rays to reveal a biomedical target, catalyze enzymatic or chemical reactions, or burn away a tumor in which they have accumulated for radiotherapy.
When linked to molecules with biological activity, they can make hybrid materials in which the useful property of the gold is targeted, or switched on by the biological activity of the conjugate to detect or intervene in a biochemical process. This opens the door to new ways to diagnose and treat disease, and many other applications.
Gold nanoparticle-conjugated oligonucleotides are of particular interest because of the inherent programmability of nucleic acid hybridization, which enables the assembly of complex, nanostructured materials. The full potential of this approach is realized by using gold labels that may be covalently linked to specific, unique sites where their properties may be targeted or activated biochemically, such as Nanogold® and Undecagold.
Oligonucleotides are not amenable to colloidal gold labeling, and conjugation is restricted to the direct coordination of thiol-modified nucleic acids to the gold surface, an approach that provides a very limited choice of both conjugation site, usually at one end, and stoichiometry, since large numbers of thiolated molecules are required to stabilize gold nanoparticles.
However, with Nanogold® and Undecagold, you can label at any nucleotide within an oligo, and achieve 1 : 1 labeling by using reagents with close to one reactive group per nanoparticle, which afford control over reaction stoichiometry. Furthermore, because you can label selectively at amino- or thiol- (or other) modifications, you can have total control over labeling.
To label with Nanogold® labeling reagents, you first need to introduce suitable functional groups for labeling – usually thiols or amines. For synthetic oligonucleotides, this may be achieved using specially modified phosphoramidites such as those supplied by Glen Research (link to http://www.glenres.com). Most oligonucleotide suppliers will offer this type of modification as an option, and in many cases a choice of different chain lenths are available between the oligonucleotide backbone and the reactive group. Typically, you can introduce thiol modifications at the 3' or 5' end, and introduce amino- modifications at the 3' or 5' ends or at any intervening base.
If you are working with plasmids or other naturally occurring nucleic acid structures, you can introduce a reactive group by adding a modified nucleotide during plasmid preparation, such as 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP), an amino-modified form of dUTP which can be used in a 1 : 10 mixture with unmodified dUTP to prepare amino-modified DNA, or 2-thiocytidine to introduce a thiol site. If you incorporate a small amount of one of these into your plasmid preparation mixture, it will be incorporated and the reactive groups will be introduced. Trilink Biotechnologies offer a wide range of modified and functionalized nucleotides that may be suitable for this type of modification. Alternatively, you can use a photoreactive cross-linker to insert a reactive site or a hapten into the completed plasmid or oligonucleotide. We have discussed these approaches in more detail in a previous article.
Once you have introduced a modification, labeling is quite straightforward, using Monomaleimido Nanogold® to label at thiol site, or Mono-Sulfo-NHS-Nanogold® to label at amine sites. Labeling reactions typically will work well in aqueous solution, or with up to 20% isopropanol if necessary to dissolve the Nanogold® reagent. Thiol-modified oligonucleotides may be supplied in a protected form and require deprotection or reduction to activate the thiol group; if you use a thiol-based reducing agent (dithiothreitol, DTT, or mercaptoethylamine hydrochloride, MEA), it must be separated from the reduced oligonucleotide by gel filtration prior to use, otherwise it will react with the Monomaleimido Nanogold® and prevent labeling. The oligonuleotide is then mixed with reconstituted Monomaleimido Nanogold® at a pH between 6.0 and 7.0, incubated overnight at 4°C, then separated next day by gel filtration, by an alternative chromatographic method such as reverse-phase chromatography, or by gel electrophoresis. Amino-modified oligonuleotides are mixed with reconstituted Mono-Sulfo-NHS-Nanogold® at a pH between 7.5 and 8.2, incubated overnight at 4°C, then separated next day in the same manner.
With the ability to gold label oligonucleotides at any site combined with the programmability of DNA hybridization, the possibilities are vast. A variety of nanomaterials have been prepared using gold-labeled tiles, prepared by hybridization of complementary oligonucleotides, which then self-assemble to give arrays or nanogrids with controlled gold spacing and distribution, or nanotubes which can then be metallated to give conductive nanowires. Even more exciting, gold nanoparticles can be used to carry out useful functions in nanodevices. A simple example is molecular beacons, in which Nanogold® switches on a fluorophore when a conjugate hairpin oligonucleotide hybridizes to its target. Nanogold®-labeled oligonucleotides have also has functioned as nanoscale heaters, switched on or off by the application of a radiofrequency, which were used to remotely control DNA annealing. Programmed DNA assembly has been used to organize multiple biochemical or chemical events by Itamar Willner and group, who prepared a DNA template by rolling circle amplification which was used to organize a ‘cascade’ of enzymatic reactions, and as a scaffold for biocatalytic nanowire formation through the action of Nanogold®-activated glucose oxidase.
