Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices

Powell, Richard D.; Hainfeld, James F.

doi:10.1016/j.micron.2010.08.007

Cited by 14 publications

(14 citation statements)

References 76 publications

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“…Additionally, gold nanoparticles may be used as point labels to mark polymer objects under EM study with very high resolution. [9] Scanning electron microscopy devices are nowadays applied to resolve even single macromolecules. Yaffee et al [10] applied cryo SEM for direct observation of individual DNA molecules complexed with colloidal particles of Fe(III) gluconate.…”

Section: Comparison With Scanning Electron Microscopy (Sem)mentioning

confidence: 99%

Scanning Force Microscopy as Applied to Conformational Studies in Macromolecular Research

Gallyamov

2011

Macromol. Rapid Commun.

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The modern state of SFM research on polymer nano-objects including single chains is discussed in comparison with other similar high-resolution microscopy techniques. The range of problems to be solved preferentially by SFM is highlighted. Promising methodology to describe quantitatively the morphology of macromolecular objects is proposed. The main benefits of this algorithm seem to be the apparent mathematical correctness as well as the possibility to estimate errors and the confidence of the numbers obtained. Special attention is paid to the dynamic observations of conformational transitions on a substrate in real time regime. This approach allows one to realise direct control of the adsorbed macromolecules by means of exposure to different vapours. Driving forces of the vapour-induced reorganisation are discussed.

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Section: Comparison With Scanning Electron Microscopy (Sem)mentioning

confidence: 99%

Scanning Force Microscopy as Applied to Conformational Studies in Macromolecular Research

Gallyamov

2011

Macromol. Rapid Commun.

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“…Commercial preparations of a 1.4 nm cluster compound, known as Nanogold (NG), are now available . One advantage of NG compared to CG is that a variety of molecules can be covalently linked to NG [36], including antibody Fab' [34] or even ScFv fragments [80], peptides [96], oligonucleotides [79], lipids [35,37], enzyme substrates [61], and toxins [43]-molecules that do not conjugate well to CG. On the other hand, molecules that adsorb to CG do so through less precise electrostatic or hydrophobic interactions.…”

Section: Nanogoldmentioning

confidence: 99%

FluoroNanogold: an important probe for correlative microscopy

et al. 2015

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Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.

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“…Due to their amenability of synthesis, unique surface, high electron density and strong optical absorption, gold nanoparticles (AuNPs) are an obvious choice in many biomedical research (8). For example, AuNPs have been used widely for labeling proteins for molecular localization in electron microscopy (9), or as markers to directly visualize the structure and conformational changes of biomolecules by atomic force microscopy (AFM) imaging (10).…”

Section: Introductionmentioning

confidence: 99%

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

Wang¹,

Chen

Hu³

2020

Preprint

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Classification Biological Sciences/Biochemistry; Physical Sciences/ChemistryKeywords Site-specific nanoparticle labeling, expanded genetic alphabet, TPT3-NaM, large RNAs, X-ray scattering interferometry AbstractConjugation of RNAs with nanoparticles is of significant importance for its numerous applications in biology and medicine, which however remains challenging, especially for large ones. So far, the majority of RNA labeling rely on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nts. We here present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with gold nanoparticle (AuNP) empowered by expanded genetic alphabet transcription. We synthesize an amine-derivatized TPT3 (TPT3 A ), which are site-specifically incorporated into a 97-nt 3'SL RNA and a 719-nt mini genomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by standard in vitro transcription with expanded genetic alphabet containing the A-T, G-C natural base pairs and the TPT3-NaM unnatural base pair. TPT3 modification cause minimal structural perturbations to the RNAs by small angle X-ray scattering. The purified TPT3 A -modified RNAs are covalently conjugated with mono-Sulfo-NHS-Nanogold nanoparticles via the highly selective amine-NHS ester reaction and purified under non-denaturing conditions. We demonstrate the application of the AuNP-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The inter-nanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus validate the applicability of this novel labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies, and is expected to have wide applications in large RNA structural biology and RNA nanotechnology. Significance StatementWe present a site-specific labeling strategy for large RNAs by T7 transcription with expanded genetic alphabet containing TPT3-NaM unnatural base pair. The applicability of this labeling strategy is validated by X-ray scattering interferometry measurements on a 97-nt and a 719-nt RNAs. This strategy can be applicable to natural RNAs or artificial RNA nanostructures with sizes from tens up to thousands of nucleotides, or covalent conjugation of RNAs with other metal nanoparticles. The usage of a far upstream forward primer during PCR enables easy purification of RNA from DNA templates, the non-denaturing conditions for conjugation reactions and purification avoids potential large RNA misfolding. This labeling strategy expands our capability to site-specifically conjugate RNA with nanoparticles for many applications. Main Text

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Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices

Cited by 14 publications

References 76 publications

Scanning Force Microscopy as Applied to Conformational Studies in Macromolecular Research

Scanning Force Microscopy as Applied to Conformational Studies in Macromolecular Research

FluoroNanogold: an important probe for correlative microscopy

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

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