Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach

Zhang, Kaiming; Keane, Sarah C.; Su, Zhaoming; Irobalieva, Rossitza N.; Chen, Muyuan; Van, Verna; Sciandra, Carly A.; Marchant, Jan; Heng, Xiao; Schmid, Michael F.; Case, David A.; Ludtke, Steven J.; Summers, Michael F.; Chiu, Wah

doi:10.1016/j.str.2018.01.001

Cited by 64 publications

(69 citation statements)

References 87 publications

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“…Single-particle cryo-EM and 3D reconstruction. As a rapidly growing technique in structural biology, cryo-EM single-particle analysis has been used for the validation of many DNA or RNA structures [31][32][33][34] . This technique was used to evaluate the structures of 4WJ-X and 4WJ-X-24 PTXs nanoparticles in vitrified solution.…”

Section: Resultsmentioning

confidence: 99%

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

et al. 2020

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Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the watersolubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD 50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.

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Section: Resultsmentioning

confidence: 99%

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

et al. 2020

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“…Additionally, these maps exhibit several more complex features that were not present in the previously determined 9 Å HIV-1 DIS map ( Fig. 2S) (9). The maps for each distinct RNA sequence reveal intricate 3D folds with tertiary features such as pockets and holes idiosyncratic to each RNA.…”

Section: Cryo-em Easily Resolves the Global Architectures Of Rna Molementioning

confidence: 59%

“…is only one published sub-nanometer resolution cryo-EM map of an RNA molecule produced without protein partners, a 9 Å map of the 30 kDa HIV-1 dimerization initiation signal (DIS) (9). However, to reconstruct atomic coordinates into this map, detailed supplementary NMR measurements were required.…”

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confidence: 99%

Ribosolve: Rapid determination of three-dimensional RNA-only structures

Kappel

Zhang

et al. 2019

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The discovery and design of biologically important RNA molecules is dramatically outpacing three-dimensional structural characterization. To address this challenge, we present Ribosolve, a hybrid method integrating moderate-resolution cryo-EM maps, chemical mapping, and Rosetta computational modeling, and demonstrate its application to thirteen previously unknown 119-to 338-nucleotide protein-free RNA-only structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length V. cholerae and F. nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and computer-designed spinach-TTR-3, eterna3D-JR_1, and ATP-TTR-3 with and without AMP. Blind challenges, prospective compensatory mutagenesis, internal controls, and simulation benchmarks validate the Ribosolve models and establish that modeling convergence is quantitatively predictive of model accuracy. These results demonstrate that RNA-only 3D structure determination can be rapid and routine.3 Main Text:RNA molecules fold into intricate three-dimensional structures to perform essential biological and synthetic functions including regulating gene expression, sensing small molecules, and catalyzing reactions, often without the aid of proteins or other partners (1, 2). It is estimated that more than eighty percent of the human genome is transcribed to RNA, while just 1.5 percent codes for proteins, but our knowledge of RNA structure lags far behind our knowledge of protein structure (3). The Protein Data Bank, the repository for three-dimensional structures, currently contains fewer than 1,400 RNA structures, compared to ~143,000 protein structures. Accurate all-atom models of RNAs could dramatically enhance our understanding of functional similarities between distantly related RNA sequences, enable visualization of the conformational rearrangements that accompany substrate and ligand binding, and accelerate our ability to design and evolve synthetic structured RNA molecules. However, the conformational heterogeneity of RNA molecules, particularly in the absence of protein partners, challenges conventional structure determination techniques such as X-ray crystallography and NMR (4, 5). Even when such techniques are applied, the process is often laborious, time-consuming, and requires extensive construct-specific optimization and, typically, publications have reported only one or two 3D RNA structures at a time ((4, 6) and references therein).Single-particle cryo-EM may provide an orthogonal approach to RNA structure determination. Recent advances in the technique have enabled high-resolution structure determination of proteins and large RNA-protein complexes that previously could not be solved with X-ray crystallography or NMR (7,8). However, it has been widely assumed that most functional noncoding RNA molecules that are not part of large RNA-protein complexes are either too small or conformationally heterogeneous to characterize with cryo-EM. To date there

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“…Here for the benchmark of small RNA-protein systems, we simulated this situation by assuming specific RNA-protein contacts, and for the three large RNA-protein tests, we used FRET or crosslinking data, or information about highly conserved residues. However this method could be further generalized to include other types of experimental information such as NMR restraints (Zhang et al, 2018), contacts derived from evolutionary couplings (Weinreb et al, 2016), or SAXS data (Schneidman-Duhovny et al, 2012;Schwieters et al, 2018), as we have accomplished recently for cryoEM data, achieving models with near-atomic accuracy in blind challenges (Kappel et al, 2018). Second, improving the accuracy of this method and discriminating the top model (rather than the top 100) will require new high-resolution refinement methods that can be applied to RNA-protein complexes.…”

Section: Discussionmentioning

confidence: 99%

Sampling native-like structures of RNA-protein complexes through Rosetta folding and docking

Kappel

Das

2018

Preprint

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RNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of these complexes are crucial to their functions but often elude high-resolution structure determination. Computational methods are needed that can integrate low-resolution data for RNA-protein complexes while modeling de novo the large conformational changes of RNA components upon complex formation. To address this challenge, we describe a Rosetta method called RNP-denovo to simultaneously fold and dock RNA to a protein surface. On a benchmark set of structurally diverse RNA-protein complexes that are not solvable with prior strategies, this fold-and-dock method consistently sampled native-like structures with better than nucleotide resolution. We revisited three past blind modeling challenges in which previous methods gave poor results:human telomerase, an RNA methyltransferase with a ribosomal RNA domain, and the spliceosome. When coupled with the same sparse FRET, cross-linking, and functional data used in previous work, RNP-denovo gave models with significantly improved accuracy. These results open a route to computationally modeling global folds of RNA-protein complexes from low-resolution data.

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Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach

Cited by 64 publications

References 87 publications

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

Ribosolve: Rapid determination of three-dimensional RNA-only structures

Sampling native-like structures of RNA-protein complexes through Rosetta folding and docking

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