Updated: May 11, 2004
N A N O P R O B E S E - N E W S
Vol. 5, No. 5 May 11, 2004
In this Issue:
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This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.
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To achieve successful gold labeling of oligonucleotides, there are several issues you should consider in planning and optimizing the conjugation and separation. The negative charge of the phosphate backbone means that these molecules may react differently to proteins and peptides, and in addition, their predominantly linear tertiary structure means that they often behave differently in separations.
Conjugation
The most successful method for labeling oligonucleotides has been to incorporate a thiol group at the position you wish to label, then conjugate using Monomaleimido Nanogold®. This is not affected by the backbone charge because Monomaleimido Nanogold is uncharged. Thiols may usually be incorporated during oligonucleotide synthesis using thiol-modified or disulfide-modified phosphoramidites from sources such as Glen Research. This approach has been used successfully by Alivisatos and by Dubertret:
- Alivisatos, A. P., Johnsson, K. P., Peng, X., Wilson, T. E., Loweth, C. J., Bruchez, M. P., Jr., and Schultz, P. G.: Organization of 'Nanocrystal Molecules' using DNA. Nature, 382, 609 (1996).
- Dubertret, B., Calame, M., and Libchaber, A.; Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol., 19, 365-370 (2001).
However, successful labeling may also be achieved using Mono-Sulfo-NHS-Nanogold to label at an amine site. This is particularly useful for incorporating gold within the oligonucleotide rather than at the ends, since the synthesis or appropriately thiol-modified oligonucleotides may not be possible. This approach was used by Hamad-Schifferli and co-workers:
Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 415, 152-155 (2002).
Other approaches are feasible, such as phosphate activation using carbonyldiimidazole followed by reaction with Monoamino Nanogold. Nucleosides containing a 1,2-dihydroxy unit (and other carbohydrates) may be labeled by oxidation to a dialdehyde followed by reaction with Monoamino Nanogold and reduction of the resulting Schiff base. Detailed protocols for both procedures are given in the technical help and applications sections of our web site:
Separation
To ensure an efficient separation of the Nanogold-labeled oligonucleotide, you need to consider not just the separation method, but also the reaction stoichiometry: choose a ratio of gold label to oligonucleotide such that out of the two reagents, the one that is more easily separated if it is unreacted is used in excess, and the less easily separated one is the limiting reagent and is completely conjugated.
We usually recommend liquid chromatography over a gel filtration or size fractionation column for conjugate separation. Using this method, the more easily separated component will be the smaller of the two, and therefore this should be present in excess. Small excesses (two-fold to five-fold) work best because they allow better resolution of multiple peaks. If you are labeling a large oligonucleotide (20 bases or more) that is larger than Nanogold, the gold label should be present in excess; if you are labeling a smaller oligonucleotide that is smaller that the gold label (10 bases or fewer), use an excess of the oligonucleotide. Appropriate media for this separation include Superdex-75 and Superdex PG30 (Amersham Pharmacia Biotech).
However, we have also found that reverse-phase chromatography can be helpful for separating labeled oligonucleotides, particularly if the oligonucleotide and gold particle are similar in size. Separation of unlabeled oligonucleotide, unconjugated gold, and conjugate may be achieved using a butyl column, eluted with a gradient of 0 to 70% acetonitrile in 0.1 M triethylammonium acetate buffer at pH 7. Other methods that may be useful include hydrophobic interaction chromatography (HIC) and ion exchange.
Gel electrophoresis has also been used to separate labeled oligonucleotides, as described by Dubertret in the reference above. This is helpful in applications such as molecular beacon preparation, in which it is critical that one component of the mixture be limiting whether or not it is the smaller of the two. For effective detection of labeled oligoucleotides, divide the reaction mixture in two, and run two gels in parallel. Treat one with a standard DNA stain such as ethidium bromide: this will visualize the location of DNA-containing species. Develop the second using silver enhancement with LI Silver. This will visualize the Nanogold. Compare the silver enhanced and ethidium bromide stained gels: if the same band is stained using both treatments, it is labeled oligonucleotide. An application note on our web site describes the detection of Nanogold-labeled molecules in gel in more detail. In addition, our Guide to Gold Cluster Labeling includes detailed discussions of how to get the best out of conjugation and separation procedures.
More information:
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We recently introduced a series of new FluoroNanogold combined fluorescent and gold-labeled probes, based on the bright, highly stable Alexa Fluor®* 488 and 594 fluorophores developed by Molecular Probes. With Alexa Fluor® 594, you can differentiate a FluoroNanogold-labeled target from a second target labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other fluorophores. These new, brighter combined fluorescent and gold probes offer new performance levels and additional features:
- Increased fluorescence brightness and higher quantum yield.
- Improved solubility means lower background and higher signal-to-noise ratios.
- Fluorescence remains high and consistent across wider pH range.
- Uses fluorescein (Alexa Fluor 488) or Texas Red (Alexa Fluor 594) filter sets.
- Available in 1 mL or affordable 0.5 mL sizes.
References:
- Takizawa, T., and Robinson, J. M.: Correlative Microscopy of Ultrathin Cryosections is a Powerful Tool for Placental Research. Placenta, 24, 557-565 (2003).
- Abstract (courtesy of Science Direct):
LINK
- Takizawa, T., and Robinson, J. M.: Ultrathin Cryosections. An important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem., 51, 707-714 (2003).
We have been asked about the possibility of preparing custom FluoroNanogold-labeled peptides, oligonucleotides, or even small molecule probes such as phalloidin. When considering the preparation of such probes, it is important to consider the quenching effect that the gold particles have on the fluorescence. Gold particles are excellent acceptors for fluorescence resonance energy transfer, or FRET, and absorb strongly at the emission wavelengths of commonly used fluorophores. We describe this process in our paper:
- Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.: Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech., 42, 2-12 (1998).
In order for fluorescence to be retained, the fluorescent tag has to be positioned far enough away from the gold particle that a significant fraction is emitted directly rather than lost through energy transfer. This means that the separation between the two must be greater than the Frster distance (the distance at which 50 % of absorbed energy is lost through energy transfer, and 50 % emitted as fluorescence). For Nanogold$#174; and fluorescein, the Frster distance was calculated to be between 6 and 7 nm; this means that in order for a combined fluorescein and Nanogold probe to be functional, the two labels should be at least this far apart. For this reason, FluoroNanogold is not a single label but two labels attached separately to the conjugate biomolecule: the preparation of FluoroNanogold probes is only feasible for probes of sufficient size to give adequate separation of the two labels.
More information:
*Alexa Fluor is a registered trademark of Molecular Probes, Inc.
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Miyazaki and co-workers extended the scope of enzymatic and immunogold double labeling in their studies of the development and roles of climbing fibers (CFs) and parallel fibers (PFs) in the developing mouse, by combining silver-enhanced immunogold with DAB visualization of a neuronal tracer by both light and electron microscopy. The developmental wiring of PFs and that of CFs are competitive with each other: the glutamate receptor GluR{delta}2 supports PF synaptogenesis, and reciprocal changes (expanded PF innervation and regressed CF innervation) are induced in the adult cerebellum when afferent activities are silenced by tetrodotoxin. These results suggest that counter mechanisms that support CFs over PFs in an activity-dependent manner should exist to ensure properly organized innervation. This was tested by examination of cerebellum lacking the alpha-1A subunit of P/Q-type Ca2+ channels in knockout mice.
Anterograde labeling was performed under anesthesia. A glass pipette filled with 2-3 microliters of 10% solution of biotinylated dextran amine (BDA; 10,000 molecular weight) or dextran Texas red (DTR; 3000 MW) in PBS, pH 7.4, was inserted stereotaxically to the inferior olive by the dorsal approach. The tracer was injected by air pressure at 20 psi with 5 second intervals for 1 minute. After 2 days, mice were anesthetized and fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2. Excised brains were immersed overnight in the same fixative and processed for preparation of 50 micrometer parasagittal microslicer sections. For brightfield light microscopy, BDA-labeled CFs were visualized by overnight incubation with avidin-biotin-peroxidase complex (Elite ABC kit; Vector Laboratories) and colored in black using DAB and cobalt. Unmodified DAB immunoperoxidase for calbindin was used in some sections to label PCs with brown products. Combined anterograde and immunofluorescence labeling was also used to localize additional targets: DTR-labeled microsclicer sections were incubated with calbindin antiserum or with a mixture of calbindin antiserum and guinea pig vesicular glutamate transporter 2 (VGluT2) antibody (1 g/ml) followed by incubation with fluorescent secondary antibodies for 2 hr. Images of double labeling were taken with a confocal laser-scanning microscope, and those of triple labeling were taken with a fluorescence microscope and deconvoluted.
For electron microscopy, mice were perfused with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Microslicer sections (400 micrometers) were postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour, dehydrated in graded alcohols, and embedded in Epon 812 for the preparation of ultrathin sections (70 nm). For electron microscopy by combined anterograde and immunoelectron labeling, BDA-labeled microslicer sections were incubated overnight with a mixture of VGluT2 antibody and avidin-biotin-peroxidase complex diluted with Tris-buffered saline containing 1% BSA and 0.004% saponin. Sections were then incubated with Nanogold® anti-guinea pig antibody (1 : 200) for 3 hr. Silver enhancement was performed with HQ Silver, and then BDA was visualized with DAB. Sections were postfixed with 1% osmium tetroxide for 15 min, dehydrated in graded alcohols, and embedded in Epon 812. Serial ultrathin sections were prepared in the transverse plane (i.e., parallel to the pial surface to reconstruct innervation patterns from the base of Purkinje cell dendrites upwards).
In the alpha-1A knock-out mouse, CF innervation was regressed proximally and PF innervation expanded reciprocally, and multiple CFs persisted in the majority of mutant Purkinje cells by innervating the same somatodendritic compartments. VDCC alpha-1A subunit consolidates innervation territory by a single main CF, and expels surplus CFs and PFs from its territory. Therefore, we hypothesize that postsynaptic P/Q-type Ca2+ channels in PCs fuel heterosynaptic competition for extending the CF innervation territory along the PC dendritic tree and also fuel homosynaptic competition for establishing the monoinnervation by a main single CF of each PC.
Reference:
Miyazaki, T.; Hashimoto, K.; Shin, H. S.; Kano, M., and Watanab, M.: P/Q-type Ca2+ channel alpha1A regulates synaptic competition on developing cerebellar Purkinje cells. J. Neurosci., 24, 1734-1743 (2004).
More information:
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Gold enhancement is a novel and robust process in which gold, rather than silver, is deposited onto gold nanoparticles to enlarge them for optical or electron microscope observation. Advantages include resistance to osmium etching, compatibility with many commonly used and physiological buffers, improved backscatter detection in SEM, and in many cases lower background and non-specific development. Wolfgang and co-workers used these advantages to help show that signaling through the heterotrimeric G protein, Gs, plays a role in synapse growth and retraction over synapse lifetime that coincides with increases in intracellular Ca+2 in the regulation of adenylyl cyclases (ACs) during synaptic growth function. In Drosophila larvae containing a hypomorphic mutation in the dgs gene encoding the Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization and defects in the facilitation of synaptic transmission.
For EM studies, larvae were dissected and fixed on ice in periodatelysineparaformaldehyde for 1 hour, washed in PBS, and quenched for 5 min in PBS with 0.05 M glycine. Larvae were placed in EM blocking buffer containing 0.1% saponin and 10% horse serum in PBS for 14 h at room temperature, incubated overnight at 4°C with antibodies to Gs alpha diluted in blocking buffer (1 : 500), then washed in three changes of blocking solution over 1 hour at room temperature. Larvae were transferred to blocking solution containing Nanogold-Fab anti-rabbit IgG (1 : 100) for 1 hour, washed again for 1 hour in blocking solution, rinsed briefly in PBS, postfixed for 20 minutes in 1% glutaraldehyde, then washed in blocking solution containing 0.05 M glycine, followed by washing in water. Gold particles were enlarged to between 5 and 10 nm for electron microscopy by incubation in GoldEnhance solution for 5 min. Larvae were then rinsed briefly in cacodylate buffer, postfixed in 2% OsO4, rinsed again in cacodylate buffer, incubated in 2% uranyl acetate for 30 min, then dehydrated and embedded in epoxy for EM preparation and viewing.
Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha, either pre- or postsynaptically, rescues the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. This demonstrates that Gs alpha dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.
Reference:
Wolfgang, W. J.; Clay, C.; Parker, J.; Delgado, R.; Labarca, P.; Kidokoro, Y., and Forte, M.: Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila. Dev. Biol., 268, 295-311 (2004).
More information:
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Sandström and Åkerman investigated the electrophoretic properties of 13 nm gold-oligonucleotide conjugates particles. Conjugates were prepared both by non-specific adsorption and by thiol coordination of 12- and 25-mer oligonucleotides to the gold. Both unmodified and DNA-modified particles migrated at constant velocity in different concentrations of agarose gels. The relation of particle size to oligonucleotide length, secondary structure, and type of modification (specific or nonspecific) was modeled: thiol (specifically) modified particles incorporated a thicker DNA layer, attributed to the fact that the oligonucleotides are only anchored to the particle in one end and thus stand up from the surface more; however, the thickness of the DNA layer was smaller than the length of a corresponding stretched oligonucleotide, indicating flexibility and tilting of the strands.
Reference:
Sandström, P., and Åkerman, B.: Electrophoretic properties of DNA-modified colloidal gold Nanoparticles. Langmuir, 20, 4182-4186 (2004).
For those interested in using silver particle labels, Lahtinen and co-workers report a useful synthesis of monolayer-protected silver clusters, based on the two-phase reduction of a stable, negatively charged, silver bromide sol. Phase transfer of the colloid to toluene using tetra-n-octylammonium bromide as the phase transfer reagent was followed by reduction with aqueous borohydride in the presence of 4-bromobenzenethiol as the passivating agent.. This synthetic method uncouples the formation of the silver halide colloid from its transfer and reduction in the organic phase, thus allowing control over each reaction step independently. The resulting clusters were characterized by optical and transmission electron microscopy, energy-dispersive X-ray analysis, thermogravimetry, andUV-vis absorption spectroscopy, and were found to average 4.0±0.9 nm in diameter.
Reference:
Lahtinen, R. M.; Mertens, S. F. L.; East, E.; Kiely, C. J., and Schiffrin, D. J.: Silver halide colloid precursors for the synthesis of monolayer-protected clusters. Langmuir, 20, 3289-3296 (2004).
Landry and co-workers report a detailed study of the parameters affecting postembedding immunogold labeling in this months Journal of Histochemistry and Cytochemistry, using different sized colloidal gold particles. To improve the sensitivity of this approach, a protocol was assessed to visualize either one or the other of co-localized neuropeptides, vasopressin or galanin, after two successive rounds of immunogold with the same primary antibody performed on both faces of the grid. Different-sized gold particles were used to distinguish the respective contribution of each face of the section to the final labeling: a moderate but significant increase in both the proportion of labeled granules and the labeling intensity was found. It is possible that the use of smaller gold probes, such as Nanogold®, might enable even greater gains in sensitivity.
Reference:
Landry, M.; Vila-Porcile, E., and Calas, A.: Immunogold Detection of Co-localized Neuropeptides: Methodological Aspects. J. Histochem. Cytochem., 52, 617-628 (2004).
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