High resolution atomic force microscopy of double-stranded RNA

Ares, Pablo; Fuentes-Pérez, M.; Herrero-Galán, Elías; Valpuesta, José M.; Gil, Adriana; Gomez-Herrero, Julio; Moreno‐Herrero, Fernando

doi:10.1039/c5nr07445b

Cited by 47 publications

(51 citation statements)

References 44 publications

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“…RNA nanostructures showed a double-stranded RNA mean periodicity of 3.1 ± 0.3 nm ( Fig. 3, right): this measurement was consistent with the A-form helical pitch of dsRNA previously reported [52]. The split Broccoli aptamer was sometimes visible as a slightly elevated side on the origami surface ( Fig.…”

Section: Rna Origami Co-transcriptional Folding and Afm Imagingsupporting

confidence: 89%

“…The estimated RNA nanostructures dimensions were approx. 27 nm x 5 nm, as the A-form RNA helix revealed a rise per base pair of 0.28 nm [52].…”

Section: Rna Origami Co-transcriptional Folding and Afm Imagingmentioning

confidence: 98%

“…Samples were diluted and deposited on a passivated mica surface using a silane solution instead of Mg 2+ . Indeed, it has been suggested that the different phosphate groups orientation in dsRNA compared to dsDNA can be a possible reason for the difficult dsRNA adsorption on mica using magnesium ions [52]. The estimated RNA nanostructures dimensions were approx.…”

Section: Rna Origami Co-transcriptional Folding and Afm Imagingmentioning

confidence: 99%

“…The images were taken in solution immediately after co-transcription. The white arrows show the dsRNA helical pitch (3.2 ± 0.3 nm,[52]). Scale bar: 10 nm.…”

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confidence: 99%

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Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Torelli

Kozyra

Shirt-Ediss

et al. 2019

Preprint

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The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time.Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications. KEYWORDSCo-transcriptional folding, scaffolded RNA origami, bio-orthogonal, split Broccoli aptamer, oxRNA simulation 1 Introduction RNA plays sophisticated roles with different and essential coding and noncoding functions. mRNAs, tRNAs, rybozymes, aptamers and CRISPR RNAs are just few examples of RNA species in a vast functional repertoire. Considering the diversity in functional and structural motifs, the emerging field of RNA nanotechnology has been developing rapidly over the past years. As a consequence, a variety of RNA nanostructures with different functionalities, sizes and shapes have

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Section: Rna Origami Co-transcriptional Folding and Afm Imagingsupporting

confidence: 89%

“…The estimated RNA nanostructures dimensions were approx. 27 nm x 5 nm, as the A-form RNA helix revealed a rise per base pair of 0.28 nm [52].…”

Section: Rna Origami Co-transcriptional Folding and Afm Imagingmentioning

confidence: 98%

Section: Rna Origami Co-transcriptional Folding and Afm Imagingmentioning

confidence: 99%

“…The images were taken in solution immediately after co-transcription. The white arrows show the dsRNA helical pitch (3.2 ± 0.3 nm,[52]). Scale bar: 10 nm.…”

mentioning

confidence: 99%

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Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Torelli

Kozyra

Shirt-Ediss

et al. 2019

Preprint

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“…Atomic force microscopy (AFM) is a scanning probe microscopy method that enables the collection of three-dimensional topographic image data, and has been widely applied to characterisation of biological and non-biological macromolecules. AFM encompasses a range of techniques for structural studies of biological molecules, including the study of inter- and intramolecular interactions [1], molecular dynamics [2], molecular remodelling under force [3] and imaging of molecules in air [4] or in liquid [5,6]. AFM operating in non-contact mode is capable of reaching atomic resolution on samples of small molecules [7], whereas AFM imaging of biomolecules routinely reaches nanometre resolutions in single high signal-to-noise images, and is able to characterise biological populations at a true single molecule level.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

Lutter

Serpell²,

Tuite

et al. 2020

Preprint

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ABSTRACTAtomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes ‘magnification’ of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.

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High Electrical Conductivity of Single Metal–Organic Chains

et al. 2018

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Molecular wires are essential components for future nanoscale electronics. However, the preparation of individual long conductive molecules is still a challenge. MMX metal-organic polymers are quasi-1D sequences of single halide atoms (X) bridging subunits with two metal ions (MM) connected by organic ligands. They are excellent electrical conductors as bulk macroscopic crystals and as nanoribbons. However, according to theoretical calculations, the electrical conductance found in the experiments should be even higher. Here, a novel and simple drop-casting procedure to isolate bundles of few to single MMX chains is demonstrated. Furthermore, an exponential dependence of the electrical resistance of one or two MMX chains as a function of their length that does not agree with predictions based on their theoretical band structure is reported. This dependence is attributed to strong Anderson localization originated by structural defects. Theoretical modeling confirms that the current is limited by structural defects, mainly vacancies of iodine atoms, through which the current is constrained to flow. Nevertheless, measurable electrical transport along distances beyond 250 nm surpasses that of all other molecular wires reported so far. This work places in perspective the role of defects in 1D wires and their importance for molecular electronics.

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High resolution atomic force microscopy of double-stranded RNA

Cited by 47 publications

References 44 publications

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

High Electrical Conductivity of Single Metal–Organic Chains

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