Cryo-EM structure and functional landscape of an RNA polymerase ribozyme.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs.

RNA origami scaffolds facilitate cryo-EM characterization of a Broccoli-Pepper aptamer FRET pair.

Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA structures and complexes. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight and a high signal-to-noise ratio. By placing RNA aptamers on larger RNA scaffolds, the contrast for cryo-EM can be increased to allow the determination of the tertiary structure of the aptamer. Here we use the RNA origami method to scaffold two fluorescent aptamers (Broccoli and Pepper) in close proximity and show that their cognate fluorophores serve as donor and acceptor for FRET. Next, we use cryo-EM to characterize the structure of the RNA origami with the two aptamers to a resolution of 4.4 Å. By characterizing the aptamers with and without ligand, we identify two distinct modes of ligand binding, which are further supported by selective chemical probing. 3D variability analysis of the cryo-EM data show that the relative position between the two bound fluorophores on the origami fluctuate by only 3.5 Å. Our results demonstrate a general approach for using RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision.

RNA target highlights in CASP15: Evaluation of predicted models by structure providers.

The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x-ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA-protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.

An RNA Paranemic Crossover Triangle as A 3D Module for Cotranscriptional Nanoassembly.

RNA nanotechnology takes advantage of structural modularity to build self-assembling nano-architectures with applications in medicine and synthetic biology. The use of paranemic motifs, that form without unfolding existing secondary structure, allows for the creation of RNA nanostructures that are compatible with cotranscriptional folding in vitro and in vivo. In previous work, kissing-loop (KL) motifs have been widely used to design RNA nanostructures that fold cotranscriptionally. However, the paranemic crossover (PX) motif has not yet been explored for cotranscriptional RNA origami architectures and information about the structural geometry of the motif is unknown. Here, a six base pair-wide paranemic RNA interaction that arranges double helices in a perpendicular manner is introduced, allowing for the generation of a new and versatile building block: the paranemic-crossover triangle (PXT). The PXT is self-assembled by cotranscriptional folding and characterized by cryogenic electron microscopy, revealing for the first time an RNA PX interaction in high structural detail. The PXT is used as a building block for the construction of multimers that form filaments and rings and a duplicated PXT motif is used as a building block to self-assemble cubic structures, demonstrating the PXT as a rigid self-folding domain for the development of wireframe RNA origami architectures.

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21.

DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we used label-free proteomic MS/MS to identify 26 proteins that are expressed at significantly different levels in cells expressing an rG4-binding deficient DDX21 (M4). MS data are available via ProteomeXchange with identifier PXD013501. From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5'-UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in MCF-7 cells, rendering them sensitive to TRAIL-mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by the potential to form rG4 in their 5'-UTRs.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

Human DDX21 binds and unwinds RNA guanine quadruplexes.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

Insights into the RNA quadruplex binding specificity of DDX21.

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3'-Tail of the Long Non-coding RNA BC200 (BCYRN1).

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3'-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3'-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.

An RNA origami robot that traps and releases a fluorescent aptamer.

RNA nanotechnology aims to use RNA as a programmable material to create self-assembling nanodevices for application in medicine and synthetic biology. The main challenge is to develop advanced RNA robotic devices that both sense, compute, and actuate to obtain enhanced control over molecular processes. Here, we use the RNA origami method to prototype an RNA robotic device, named the "Traptamer," that mechanically traps the fluorescent aptamer, iSpinach. The Traptamer is shown to sense two RNA key strands, acts as a Boolean AND gate, and reversibly controls the fluorescence of the iSpinach aptamer. Cryo-electron microscopy of the closed Traptamer structure at 5.45-angstrom resolution reveals the mechanical mode of distortion of the iSpinach motif. Our study suggests a general approach to distorting RNA motifs and a path forward to build sophisticated RNA machines that through sensing, computing, and actuation modules can be used to precisely control RNA functionalities in cellular systems.

Tertiary structure of single-instant RNA molecule reveals folding landscape.

The folding of RNA and protein molecules during their synthesis is a crucial self-assembly process that nature employs to convert genetic information into the complex molecular machinery that supports life. Misfolding events are the cause of several diseases, and the folding pathway of central biomolecules, such as the ribosome, is strictly regulated by programmed maturation processes and folding chaperones. However, the dynamic folding processes are challenging to study because current structure determination methods heavily rely on averaging, and existing computational methods do not efficiently simulate non-equilibrium dynamics. Here we utilize individual-particle cryo-electron tomography (IPET) to investigate the folding landscape of a rationally designed RNA origami 6-helix bundle that undergoes slow maturation from a "young" to "mature" conformation. By optimizing the IPET imaging and electron dose conditions, we obtain 3D reconstructions of 120 individual particles at resolutions ranging from 23-35 Å, enabling us first-time to observe individual RNA helices and tertiary structures without averaging. Statistical analysis of 120 tertiary structures confirms the two main conformations and suggests a possible folding pathway driven by helix-helix compaction. Studies of the full conformational landscape reveal both trapped states, misfolded states, intermediate states, and fully compacted states. The study provides novel insight into RNA folding pathways and paves the way for future studies of the energy landscape of molecular machines and self-assembly processes.

Structure, folding and flexibility of co-transcriptional RNA origami.

RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.

RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds.

RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.

Monitoring Enzymatic RNA G-Quadruplex Unwinding Activities by Nuclease Sensitivity and Reverse Transcription Stop Assays.

Multiple different methods have been employed to investigate the unwinding of RNA G-quadruplexes by various helicase proteins. Each has their own pitfalls, namely, looking at non-native or chemically modified RNA sequences, biasing the unwinding process with competing trap nucleotides, and a lack of context sequence to the 5' and 3' of the RNA G-quadruplex structure. Herein we present two straightforward methods that allow for quadruplex unwinding to be monitored on native RNA sequences without the use of fluorescent modifications, specialized equipment, or trap nucleotides to be employed.

2D Saturation Transfer Difference NMR for Determination of Protein Binding Sites on RNA Guanine Quadruplexes.

Saturation transfer difference (STD) NMR is a technique that provides information on the intermolecular interfaces of heterogenous complexes by cross-saturation from one molecule to the other. In this case, selective saturation of protein protons is applied, and the cross-relaxation to the RNA sample results in a reduction of the peak intensities in the measured H1-H1 NOESY spectrum. This allows for a relatively rapid and simple method of identifying the protein binding interface of an RNA with assigned chemical shift data.

Binding and photodynamic action of the cationic zinc phthalocyanines with different types of DNA toward understanding of their cancer therapy activity.

Two cationic zinc phthalocyanines have been tested for their interactions with several DNA secondary structures. Despite different aggregation properties, both phthalocyanines bind to DNA in monomeric forms. The strong photodynamic activity of phthalocyanines was demonstrated by in vitro experiments and correlate well with high singlet oxygen yields determined experimentally with 1,3-diphenylisobenzofurane. Both phthalocyanines accumulate in the cell cytoplasm prior to radiation; however, only the octacationic photosensitizer was observed in the cell nuclei after irradiation.

On Characterizing the Interactions between Proteins and Guanine Quadruplex Structures of Nucleic Acids.

Guanine quadruplexes (G4s) are four-stranded secondary structures of nucleic acids which are stabilized by noncanonical hydrogen bonding systems between the nitrogenous bases as well as extensive base stacking, or pi-pi, interactions. Formation of these structures in either genomic DNA or cellular RNA has the potential to affect cell biology in many facets including telomere maintenance, transcription, alternate splicing, and translation. Consequently, G4s have become therapeutic targets and several small molecule compounds have been developed which can bind such structures, yet little is known about how G4s interact with their native protein binding partners. This review focuses on the recognition of G4s by proteins and small peptides, comparing the modes of recognition that have thus far been observed. Emphasis will be placed on the information that has been gained through high-resolution crystallographic and NMR structures of G4/peptide complexes as well as biochemical investigations of binding specificity. By understanding the molecular features that lead to specificity of G4 binding by native proteins, we will be better equipped to target protein/G4 interactions for therapeutic purposes.

Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU.

G4 quadruplexes are stable secondary structures prevalent in DNA and RNA that exhibit diverse regulatory functions. Herein, we describe an in vitro technique using the purified RNA helicase RHAU to unwind a G4 quadruplex identified near the 5' end of the human telomerase RNA (hTR). A synthetic RNA corresponding to the quadruplex forming region of hTR (hTR10-43), as well as a predicted complementary strand (25P1), are combined in a reaction containing the purified helicase and ATP. Reaction products and appropriate controls are resolved by native gel electrophoresis. Gels can be stained using a combination of total RNA and quadruplex-specific dyes to observe the expected quadruplex to duplex conversion. This straightforward method can be extended to study structural changes in other inter- or intramolecular quadruplex containing DNA/RNA molecules with the RHAU helicase or other RNA/DNA remodeling enzymes.