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

2020 ◽  
Vol 117 (37) ◽  
pp. 22823-22832
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3′SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle 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 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.

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

2020 ◽  
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Base Pair ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

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 (TPT3A), 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 TPT3A-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.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

Small-Angle X-Ray Scattering for the Discerning Macromolecular Crystallographer

2014 ◽  
Vol 67 (12) ◽  
pp. 1786 ◽  
Author(s):  
Lachlan W. Casey ◽  
Alan E. Mark ◽  
Bostjan Kobe
Keyword(s):  
Structural Biology ◽  
Small Angle ◽  
Significant Risk ◽  
Macromolecular Crystallography ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

The role of small-angle X-ray scattering (SAXS) in structural biology is now well established, and its usefulness in combination with macromolecular crystallography is clear. However, the highly averaged SAXS data present a significant risk of over-interpretation to the unwary practitioner, and it can be challenging to frame SAXS results in a manner that maximises the reliability of the conclusions drawn. In this review, a series of recent examples are used to illustrate both the challenges for interpretation and approaches through which these can be overcome.

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