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RNA molecules adopt three-dimensional structures that are critical to their function and of interest in drug discovery. Few RNA structures are known, however, and predicting them computationally has proven challenging. We introduce a machine learning approach that enables identification of accurate structural models without assumptions about their defining characteristics, despite being trained with only 18 known RNA structures. The resulting scoring function, the Atomic Rotationally Equivariant Scorer (ARES), substantially outperforms previous methods and consistently produces the best results in community-wide blind RNA structure prediction challenges. By learning effectively even from a small amount of data, our approach overcomes a major limitation of standard deep neural networks. Because it uses only atomic coordinates as inputs and incorporates no RNA-specific information, this approach is applicable to diverse problems in structural biology, chemistry, materials science, and beyond. [ABSTRACT FROM AUTHOR]

We study the behaviour of double-stranded RNA under twist and tension using oxRNA, a recently developed coarse-grained model of RNA. Introducing explicit salt-dependence into the model allows us to directly compare our results to data from recent single-molecule experiments. The model reproduces extension curves as a function of twist and stretching force, including the buckling transition and the behaviour of plectoneme structures. For negative supercoiling, we predict denaturation bubble formation in plectoneme end-loops, suggesting preferential plectoneme localisation in weak base sequences. OxRNA exhibits a positive twist-stretch coupling constant, in agreement with recent experimental observations. [ABSTRACT FROM AUTHOR]

To bridge the gap between the sequences and 3-dimensional (3D) structures of RNAs, some computational models have been proposed for predicting RNA 3D structures. However, the existed models seldom consider the conditions departing from the room/body temperature and high salt (1M NaCl), and thus generally hardly predict the thermodynamics and salt effect. In this study, we propose a coarse-grained model with implicit salt for RNAs to predict 3D structures, stability, and salt effect. Combined with Monte Carlo simulated annealing algorithm and a coarse-grained force field, the model folds 46 tested RNAs (<45 nt) including pseudoknots into their native-like structures from their sequences, with an overall mean RMSD of 3.5 Â and an overall minimum RMSD of 1.9 Â from the experimental structures. For 30 RNA hairpins, the present model also gives the reliable predictions for the stability and salt effect with the mean deviation ~ 1.0 °C of melting temperatures, as compared with the extensive experimental data. In addition, the model could provide the ensemble of possible 3D structures for a short RNA at a given temperature/salt condition. [ABSTRACT FROM AUTHOR]

Background: RNA structure is the crucial basis for RNA function in various cellular processes. Over the last decade, high throughput structure profiling (SP) experiments have brought enormous insight into RNA secondary structure. Results: In this review, we first provide an overview of approaches for RNA secondary structure prediction, including free energy-based algorithms and comparative sequence analysis. Then we introduce SP technologies, databases to document SP data, and pipelines/algorithms to normalize and interpret SP data. Computational frameworks that incorporate SP data in RNA secondary structure prediction are also presented. Conclusions: We finally discuss potential directions for improvement in the prediction and differential analysis of RNA secondary structure. [ABSTRACT FROM AUTHOR]

The HIV-1 Rev Response Element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The "early" RRE was less functionally active than the "late" RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE sequence diversity varied over the course of infection with evidence of selection pressure that led to sequence convergence as disease progressed. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding. Importance. HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev Response Element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account. [ABSTRACT FROM AUTHOR]

RNA higher-order structures play an important role for control of the gene expression, and the small molecules binding to these structures have potential to act as interfering agents in the RNA-mediated-pathway. In this study, we synthesized new RNA binding molecules based on the G-clamp structure and evaluated their binding properties using the model RNA. The monomeric G-clamp ligand exhibited a fluorescence quenching with RNA-binding. The dimeric G-clamp ligand showed a significant fluorescence OFF/ON response to the RNA hairpin structure containing the guanines, indicating a high affinity of the G-clamp dimer to two neighboring guanines located in the RNA hairpin loop. [ABSTRACT FROM AUTHOR]

Previously, we have introduced an improved version of jViz.RNA which enabled faster and more stable RNA visualization by employing compressed tree graphs. However, the new RNA representation and visualization method required a sophisticated mechanism of pseudoknot visualization. In this work, we present our novel pseudoknot classification and implementation of pseudoknot visualization in the context of the new RNA graph model. We then compare our approach with other RNA visualization software, and demonstrate jViz.RNA 4.0’s benefits compared to other software. Additionally, we introduce interactive editing functionality into jViz.RNA and demonstrate its benefits in exploring and building RNA structures. The results presented highlight the new high degree of utility jViz.RNA 4.0 now offers. Users are now able to visualize pseudoknotted RNA, manipulate the resulting automatic layouts to suit their individual needs, and change both positioning and connectivity of the RNA molecules examined. Care was taken to limit overlap between structural elements, particularly in the case of pseudoknots to ensure an intuitive and informative layout of the final RNA structure. Availability: The software is freely available at: . [ABSTRACT FROM AUTHOR]

Background: Traditional cancer treatments such as surgery, radiation, and chemotherapy destroy both cancer and normal cells, which limit their clinical application. It is difficult to achieve the best results for any liver cancer patients using any single treatment method. Gene therapy for HCC demands non-invasive, efficient, targeted and safe gene transfection strategies.Objective: In this study, a nonviral shRNA gene delivery system utilizing a combination of PEI, US, and NBs was developed for targeting survivin in liver Cancer.Methods and Results: The PEI-shRNA-NBs cumulated in the tumor tissue because of the EPR effect. By exposure to the US, micelles shRNA may be released from PEI-shRNA-NBs in tumor tissues and the shRNA then transmitted efficiently to cancer cells. Considerably enhanced therapeutic outcome was obtained with the gene silencing effect enhanced.Conclusions: PEI-shRNA-NBs possess the potential to become promising tools intended for shRNA delivery. [ABSTRACT FROM AUTHOR]

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com–RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form. [ABSTRACT FROM AUTHOR]

We identified 29 G-quadruplex binding proteins by affinity purification and quantitative LC-MS/MS. We demonstrated that the DEAD-box RNA helicases Dbp2, Ded1 and Mss116 preferentially bind to G-quadruplex nucleic acids in vitro and destabilize RNA quadruplexes, suggesting new potential roles for these helicases in disruption of quadruplex structures in RNA. [ABSTRACT FROM AUTHOR]

Abstract RNA is accurately entangled in virtually all pathways that maintain cellular homeostasis. To name but a few, RNA is the "messenger" between DNA encoded information and the resulting proteins. Furthermore, RNAs regulate diverse processes by forming DNA::RNA or RNA::RNA interactions. Finally, RNA itself can be the scaffold for ribonucleoprotein complexes, for example, ribosomes or cellular bodies. Consequently, disruption of any of these processes can lead to disease. This review describes known and emerging RNA-based disease mechanisms like interference with regular splicing, the anomalous appearance of RNA–protein complexes and uncommon RNA species, as well as non-canonical translation. Due to the complexity and entanglement of the above-mentioned pathways, only few drugs are available that target RNA-based disease mechanisms. However, advances in our understanding how RNA is involved in and modulates cellular homeostasis might pave the way to novel treatments. Graphical Abstract Unlabelled Image Highlights • RNA is involved in virtually all pathways that maintain cellular homeostasis. • Dysregulation of RNA levels results in altered protein homeostasis. • Abnormal RNA::RNA or RNA–protein interactions lead to functional deficits. • Novel toxic RNA species arise from repeat expansions and might contribute to disease. • Altered RNA structure leads to changes in translation. [ABSTRACT FROM AUTHOR]

Background: RNA secondary structure comparison is a fundamental task for several studies, among which are RNA structure prediction and evolution. The comparison can currently be done efficiently only for pseudoknot-free structures due to their inherent tree representation. Results: In this work, we introduce an algebraic language to represent RNA secondary structures with arbitrary pseudoknots. Each structure is associated with a unique algebraic RNA tree that is derived from a tree grammar having concatenation, nesting and crossing as operators. From an algebraic RNA tree, an abstraction is defined in which the primary structure is neglected. The resulting structural RNA tree allows us to define a new measure of similarity calculated exploiting classical tree alignment. Conclusions: The tree grammar with its operators permit to uniquely represent any RNA secondary structure as a tree. Structural RNA trees allow us to perform comparison of RNA secondary structures with arbitrary pseudoknots without taking into account the primary structure. [ABSTRACT FROM AUTHOR]

Motivation RNA secondary structure is a useful representation for studying the function of RNA, which captures most of the free energy of RNA folding. Using empirically determined energy parameters, secondary structures of nucleic acids can be efficiently computed by recursive algorithms. Several software packages supporting this task are readily available. As RNA secondary structures are outerplanar graphs, they can be drawn without intersection in the plane. Interpretation by the practitioner is eased when these drawings conform to a series of additional constraints beyond outerplanarity. These constraints are the reason why RNA drawing is difficult. Many RNA drawing algorithms therefore do not always produce intersection-free (outerplanar) drawings. Results To remedy this shortcoming we propose here the RNApuzzler algorithm which is guaranteed to produce intersection-free drawings. It is based on a drawing algorithm respecting constraints based on nucleotide distances (RNAturtle). We investigate relaxations of these constraints allowing for intersection-free drawings. Based on these relaxations, we implemented a fully automated, simple, and robust algorithm that produces aesthetic drawings adhering to previously established guidelines. We tested our algorithm using the RFAM database and found that we can compute intersection-free drawings of all RNAs therein efficiently. Availability and implementation The software can be accessed freely at: https://github.com/dwiegreffe/RNApuzzler. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent de novo SAD phasing. PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography. [ABSTRACT FROM AUTHOR]

Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 (C. eth-2.0), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0, corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits. [ABSTRACT FROM AUTHOR]

Abstract The existence of evolutionary conservation in base pairing is strong evidence for functional elements of RNA structure, although available tools for rigorous identification of structural conservation are limited. R-scape is a recently developed program for statistical prediction of pairwise covariation from sequence alignments, but it initially showed limited utility on long RNAs, especially those of eukaryotic origin. Here we show that R-scape can be adapted for a more powerful analysis of structure conservation in long RNA molecules, including mammalian lncRNAs. Graphical Abstract Unlabelled Image Highlights • Identifying conserved RNA structures in alignments with less diverse sequences is a challenging task. • The detection of conserved structures in well-studied mammalian lncRNAs has been challenging with existing statistical tools. • Here, we report strategies and improved parameters that allow for identification of conserved structures in lncRNAs. [ABSTRACT FROM AUTHOR]

ABSTRACT Research Question Is there any genetic association between oestrogen receptor alpha [ ERα ] -PvuII polymorphism and idiopathic male infertility? Design A total of 226 infertile and 213 fertile men participated in the present case-control study. ERα-PvuII genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] method. Meta-analysis was also performed by pooling data collected from seven other eligible studies identified by searches of PubMed, Embase, Google Scholar, and Science Direct databases. Summary odds ratios were estimated by fixed- or random-effects models. The molecular effects of ERα-PvuII polymorphism were evaluated by bioinformatics tools. Results A significant protective association was reported between ERα-PvuII and male infertility in the homozygote model [OR=0.54, 95%CI=0.3–0.98, p =0.042]. Also, a similar association was observed in asthenozoospermia subgroup [OR=0.4, 95%CI=0.18–0.9, p =0.025]. Meta-analysis also revealed that the ER-PvuII polymorphism was significantly associated with the decreased risk of male infertility in the heterozygote co-dominant model [OR=0.80, 95%CI=0.64–0.99, p =0.042]. Moreover, similar protective results were reported in stratified analyses in Caucasian subgroup in the dominant genetic model [OR=0.66, 95%CI=0.45–0.96, p =0.029] and in the heterozygote co-dominant model [OR=0.62, 95%CI=0.41–0.93, p =0.021]. A significant association was also found in studies with sample size of less than 400 subjects in heterozygote co-dominant model [OR=0.69, 95%CI=0.50–0.95, p =0.023]. The bioinformatics data indicated that ER-PvuII polymorphism could significantly affect RNA structure of ERα [ p =0.004]. Conclusion The ERα-PvuII polymorphism could be considered as a possible protective factor against male infertility. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) in 5′ UTRs of eukaryotic mRNAs are increasingly recognized as important elements that regulate cellular protein synthesis. Since uORFs can start from non-AUG codons, an enormous number of potential uORF initiation sites exists in 5′UTRs. However, only a subset of these sites is used and it has been unclear how actual start sites are selected. Studies of the DEAD-box helicase Ded1p from S. cerevisiae show that translation of uORFs with non-AUG initiation codons occurs upstream of mRNA structures that emerge with defective Ded1p. The data designate mRNA structure as important determinant for non-AUG initiation sites of uORFs. Ded1p can control this RNA structure and thereby regulate uORF translation. [ABSTRACT FROM AUTHOR]

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3′ to 5′ exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease. [ABSTRACT FROM AUTHOR]

Extensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Differential expression from the two gene copies in diploid cells could potentially contribute, yet our ability to measure from which gene copy individual RNAs originated remains limited, particularly in the context of tissues. Here, we demonstrate quantitative, single molecule allele-specific RNA FISH adapted for use on tissue sections, allowing us to determine the chromosome of origin of individual RNA molecules in formaldehyde-fixed tissues. We used this method to visualize the allele-specific expression of Xist and multiple autosomal genes in mouse kidney. By combining these data with mathematical modeling, we evaluated models for allele-specific heterogeneity, in particular demonstrating that apparent expression from only one of the alleles in single cells can arise as a consequence of low-level mRNA abundance and transcriptional bursting. [ABSTRACT FROM AUTHOR]

Motivation In the study of 3D RNA structure, information about non-canonical interactions between nucleobases is increasingly important. Specialized databases support investigation of this issue based on experimental data, and several programs can annotate non-canonical base pairs in the RNA 3D structure. However, predicting the extended RNA secondary structure which describes both canonical and non-canonical interactions remains difficult. Results Here, we present RNAvista that allows predicting an extended RNA secondary structure from sequence or from the list enumerating canonical base pairs only. RNAvista is implemented as a publicly available webserver with user-friendly interface. It runs on all major web browsers. Availability and implementation http://rnavista.cs.put.poznan.pl [ABSTRACT FROM AUTHOR]

The interferon (IFN)-mediated innate immune response is the first line of defense against viruses. However, an IFN-stimulated gene, the adenosine deaminase acting on RNA 1 (ADAR1), favors the replication of several viruses. ADAR1 binds double-stranded RNA and converts adenosine to inosine by deamination. This form of editing makes duplex RNA unstable, thereby preventing IFN induction. To better understand how ADAR1 works at the cellular level, we generated cell lines that express exclusively either the IFN-inducible, cytoplasmic isoform ADAR1p150, the constitutively expressed nuclear isoform ADAR1p110, or no isoform. By comparing the transcriptome of these cell lines, we identified more than 150 polymerase II transcripts that are extensively edited, and we attributed most editing events to ADAR1p150. Editing is focused on inverted transposable elements, located mainly within introns and untranslated regions, and predicted to form duplex RNA structures. Editing of these elements occurs also in primary human samples, and there is evidence for cross-species evolutionary conservation of editing patterns in primates and, to a lesser extent, in rodents. Whereas ADAR1p150 rarely edits tightly encapsidated standard measles virus (MeV) genomes, it efficiently edits genomes with inverted repeats accidentally generated by a mutant MeV. We also show that immune activation occurs in fully ADAR1-deficient (ADAR1KO) cells, restricting virus growth, and that complementation of these cells with ADAR1p150 rescues virus growth and suppresses innate immunity activation. Finally, by knocking out either protein kinase R (PKR) or mitochondrial antiviral signaling protein (MAVS)—another protein controlling the response to duplex RNA—in ADAR1KO cells, we show that PKR activation elicits a stronger antiviral response. Thus, ADAR1 prevents innate immunity activation by cellular transcripts that include extensive duplex RNA structures. The trade-off is that viruses take advantage of ADAR1 to elude innate immunity control. [ABSTRACT FROM AUTHOR]

Riboswitches modulate gene expression in response to smallmolecule ligands. Switching is generally thought to occur via the stabilization of a specific RNA structure conferred by binding the cognate ligand. However, it is unclear whether any such stabilization occurs for riboswitches whose ligands also play functional roles, such as the glmS ribozyme riboswitch, which undergoes selfcleavage using its regulatory ligand, glucosamine 6-phosphate, as a catalytic cofactor. To address this question, it is necessary to determine both the conformational ensemble and its ligand dependence. We used optical tweezers to measure folding dynamics and cleavage rates for the core glmS ribozyme over a range of forces and ligand conditions. We found that the folding of a specific structural element, the P2.2 duplex, controls active-site formation and catalysis. However, the folded state is only weakly stable, regardless of cofactor concentration, supplying a clear exception to the ligand-based stabilization model of riboswitch function. [ABSTRACT FROM AUTHOR]

Motivation The computational prediction of RNA secondary structure by free energy minimization has become an important tool in RNA research. However in practice, energy minimization is mostly limited to pseudoknot-free structures or rather simple pseudoknots, not covering many biologically important structures such as kissing hairpins. Algorithms capable of predicting sufficiently complex pseudoknots (for sequences of length n) used to have extreme complexities, e.g. Pknots has O (n 6) time and O (n 4) space complexity. The algorithm CCJ dramatically improves the asymptotic run time for predicting complex pseudoknots (handling almost all relevant pseudoknots, while being slightly less general than Pknots), but this came at the cost of large constant factors in space and time, which strongly limited its practical application (∼200 bases already require 256 GB space). Results We present a CCJ -type algorithm, Knotty, that handles the same comprehensive pseudoknot class of structures as CCJ with improved space complexity of Θ (n 3 + Z) —due to the applied technique of sparsification, the number of 'candidates', Z, appears to grow significantly slower than n 4on our benchmark set (which include pseudoknotted RNAs up to 400 nt). In terms of run time over this benchmark, Knotty clearly outperforms Pknots and the original CCJ implementation, CCJ 1.0 ; Knotty 's space consumption fundamentally improves over CCJ 1.0, being on a par with the space-economic Pknots. By comparing to CCJ 2.0, our unsparsified Knotty variant, we demonstrate the isolated effect of sparsification. Moreover, Knotty employs the state-of-the-art energy model of 'HotKnots DP09', which results in superior prediction accuracy over Pknots. Availability and implementation Our software is available at https://github.com/HosnaJabbari/Knotty. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Abstract RGG/RG motifs are RNA binding segments found in many proteins that can partition into membraneless organelles. They occur in the context of low-complexity disordered regions and often in multiple copies. Although short RGG/RG-containing regions can sometimes form high-affinity interactions with RNA structures, multiple RGG/RG repeats are generally required for high-affinity binding, suggestive of the dynamic, multivalent interactions that are thought to underlie phase separation in formation of cellular membraneless organelles. Arginine can interact with nucleotide bases via hydrogen bonding and π-stacking; thus, nucleotide conformers that provide access to the bases provide enhanced opportunities for RGG interactions. Methylation of RGG/RG regions, which is accomplished by protein arginine methyltransferase enzymes, occurs to different degrees in different cell types and may regulate the behavior of proteins containing these regions. Graphical abstract Unlabelled Image Highlights • RGG/RG regions are disordered protein regions with multiple RGG/RG repeats. • They bind RNA and DNA likely via weak multivalent interactions. • RGG/RG regions undergo LLPS with or without RNA. • RNA binding and LLPS are modulated by methylation of arginines in RGG/RG motifs. [ABSTRACT FROM AUTHOR]

DNA quadruplexes have been the subject of investigation because of their biological relevance and because of their potential application in supramolecular chemistry. Similarly, RNA quadruplexes are now gaining increasing attention. Although DNA and RNA quadruplexes are structurally very similar, the latter show higher stability. In this study we report dispersion‐corrected density functional theory (DFT‐D) quantum chemical calculations that were undertaken to understand the difference in stabilities of RNA and DNA quadruplexes. The smallest meaningful model of a stack of quartets, interacting with alkali metal cations, was simulated in an aqueous environment. The energy decomposition analysis allows for in‐depth examination of the interaction energies, emphasising the role of noncovalent interactions and better electrostatics in determining RNA‐GQs higher stabilities, particularly pinpointing the role of the extra 2′‐OH groups. Furthermore, our computations present new insights on why the cation is required for self‐assembly: unexpectedly the cation is not necessary to relieve the repulsion between the oxygen atoms in the central cavity, but it is needed to overcome the entropic penalty. Why so stable? Dispersion‐corrected (DFT‐D) quantum chemical calculations on the stabilities of RNA and DNA quadruplexes show electrostatic interactions and hydrogen bonds make the decisive difference (see figure). [ABSTRACT FROM AUTHOR]

Scientific breakthroughs in recent decades have uncovered the capability of RNA molecules to fulfill a wide array of structural, functional, and regulatory roles in living cells, leading to a concomitantly significant increase in both the number and diversity of experimentally determined RNA three-dimensional (3D) structures. Atomic coordinates from a representative training set of solved RNA structures, displaying low sequence and structure similarity, facilitate derivation of knowledge-based energy functions. Here we develop an all-atom four-body statistical potential and evaluate its capacity to distinguish native RNA 3D structures from nonnative folds based on calculated free energy scores. Atomic four-body nearest-neighbors are objectively identified by their occurrence as tetrahedral vertices in the Delaunay tessellations of RNA structures, and rates of atomic quadruplet interactions expected by chance are obtained from a multinomial reference distribution. Our four-body energy function, referred to as RAMP (ribonucleic acids multibody potential), is subsequently derived by applying the inverted Boltzmann principle to the frequency data, yielding an energy score for each type of atomic quadruplet interaction. Several well-known benchmark datasets reveal that RAMP is comparable with, and often outperforms, existing knowledge- and physics-based energy functions. To the best of our knowledge, this is the first study detailing an RNA tertiary structure-based multibody statistical potential and its comparative evaluation. [ABSTRACT FROM AUTHOR]

Background: Computational RNA 3D structure prediction and modeling are rising as complementary approaches to high-resolution experimental techniques for structure determination. They often apply to substitute or complement them. Recently, researchers’ interests have directed towards in silico methods to fit, remodel and refine RNA tertiary structure models. Their power lies in a problem-specific exploration of RNA conformational space and efficient optimization procedures. The aim is to improve the accuracy of models obtained either computationally or experimentally. Results: Here, we present RNAfitme, a versatile webserver tool for remodeling of nucleobase- and nucleoside residue conformations in the fixed-backbone RNA 3D structures. Our approach makes use of dedicated libraries that define RNA conformational space. They have been built upon torsional angle characteristics of PDB-deposited RNA structures. RNAfitme can be applied to reconstruct full-atom model of RNA from its backbone; remodel user-selected nucleobase/nucleoside residues in a given RNA structure; predict RNA 3D structure based on the sequence and the template of a homologous molecule of the same size; refine RNA 3D model by reducing steric clashes indicated during structure quality assessment. RNAfitme is a publicly available tool with an intuitive interface. It is freely accessible at http://rnafitme.cs.put.poznan.pl/ Conclusions: RNAfitme has been applied in various RNA 3D remodeling scenarios for several types of input data. Computational experiments proved its efficiency, accuracy, and usefulness in the processing of RNAs of any size. Fidelity of RNAfitme predictions has been thoroughly tested for RNA 3D structures determined experimentally and modeled in silico. [ABSTRACT FROM AUTHOR]

Gromacs is one of the most popular molecular simulation suites currently available. In this contribution we present streaMD, the first interface between Gromacs trajectory files and the statistical language R. The amount of data created due to ever increasing computational power renders fast and efficient analysis of trajectories into a challenge. Especially as standard approaches such as root‐mean square fluctuations and the like provide only limited physical insight. In our streaMD package integration of the Gromacs I/O libraries with advanced, graph‐based analysis methods as the java library Stream leads to both: improved speed and analysis depth. We benchmark our results and highlight the applicability of the package by an interesting problem in RNA design, namely the interaction of tetracycline with an aptamer. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Pairwise ordered tree alignment are combinatorial objects that appear in important applications, such as RNA secondary structure comparison. However, the usual representation of tree alignments as supertrees is ambiguous, i.e. two distinct supertrees may induce identical sets of matches between identical pairs of trees. This ambiguity is uninformative, and detrimental to any probabilistic analysis. In this work, we consider tree alignments up to equivalence. Our first result is a precise asymptotic enumeration of tree alignments, obtained from a context-free grammar by mean of basic analytic combinatorics. Our second result focuses on alignments between two given ordered trees S and T. By refining our grammar to align specific trees, we obtain a decomposition scheme for the space of alignments, and use it to design an efficient dynamic programming algorithm for sampling alignments under the Gibbs-Boltzmann probability distribution. This generalizes existing tree alignment algorithms, and opens the door for a probabilistic analysis of the space of suboptimal alignments. [ABSTRACT FROM AUTHOR]

The aim of this study was to understand the spatial distribution of microbial communities (18S and 16S rRNA genes) across one of the harshest terrestrial landscapes on Earth. We carried out Illumina sequencing using samples from two expeditions to the high slopes (up to 6050 m.a.s.l.) of Volcán Socompa and Llullaillaco to describe the microbial communities associated with the extremely dry tephra compared to areas that receive water from fumaroles and ice fields made up of nieves penitentes. There were strong spatial patterns relative to these landscape features with the most diverse (alpha diversity) communities being associated with fumaroles. Penitentes did not significantly increase alpha diversity compared to dry tephra at the same elevation (5825 m.a.s.l.) on Volcán Socompa, but the structure of the 18S community (beta diversity) was significantly affected by the presence of penitentes on both Socompa and Llullaillaco. In addition, the 18S community was significantly different in tephra wetted by penitentes versus dry tephra sites across many elevations on Llullaillaco. Traditional phototrophs (algae and cyanobacteria) were abundant in wetter tephra associated with fumaroles, and algae (but not cyanobacteria) were common in tephra associated with penitentes. Dry tephra had neither algae nor cyanobacteria but did host potential phototrophs in the Rhodospirillales on Volcán Llullaillaco, but not on Socompa. These results provide new insights into the distribution of microbes across one of the most extreme terrestrial environments on Earth and provide the first ever glimpse of life associated with nieves penitentes, spire-shaped ice structures that are widespread across the mostly unexplored high-elevation Andean Central Volcanic Zone. [ABSTRACT FROM AUTHOR]

The article offers information on various research works in the field of science and medicine. Topics discussed include a research by Biyan Zhang et al., published in May 2020 regarding the use of mice as models to understand food allergies, the use of cryo-electron microscopy by Gao et al. to determine the RNA structure of COVID-19, and the development of a robot rover called Mini Rover by Shrivastava et al. which can climb steep, granular terrain on Mars and the Moon.

Most double-stranded RNA (dsRNA) viruses transcribe RNA plus strands within a common innermost capsid shell. This process requires coordinated efforts by RNA-dependent RNA polymerase (RdRp) together with other capsid proteins and genomic RNA. Here we report the near-atomic resolution structure of the RdRp protein VP2 in complex with its cofactor protein VP4 and genomic RNA within an aquareovirus capsid using 200-kV cryoelectron microscopy and symmetry-mismatch reconstruction. The structure of these capsid proteins enabled us to observe the elaborate nonicosahedral structure within the double-layered icosahedral capsid. Our structure shows that the RdRp complex is anchored at the inner surface of the capsid shell and interacts with genomic dsRNA and four of the five asymmetrically arranged N termini of the capsid shell proteins under the fivefold axis, implying roles for these N termini in virus assembly. The binding site of the RNA end at VP2 is different from the RNA cap binding site identified in the crystal structure of orthoreovirus RdRp λ3, although the structures of VP2 and λ3 are almost identical. A loop, which was thought to separate the RNA template and transcript, interacts with an apical domain of the capsid shell protein, suggesting a mechanism for regulating RdRp replication and transcription. A conserved nucleoside triphosphate binding site was localized in our RdRp cofactor protein VP4 structure, and interactions between the VP4 and the genomic RNA were identified. [ABSTRACT FROM AUTHOR]

This contribution sketches a work flow to design an RNA switch that is able to adapt two structural conformations in a ligand-dependent way. A well characterized RNA aptamer, i.e., knowing its K d and adaptive structural features, is an essential ingredient of the described design process. We exemplify the principles using the well-known theophylline aptamer throughout this work. The aptamer in its ligand-binding competent structure represents one structural conformation of the switch while an alternative fold that disrupts the binding-competent structure forms the other conformation. To keep it simple we do not incorporate any regulatory mechanism to control transcription or translation. We elucidate a commonly used design process by explicitly dissecting and explaining the necessary steps in detail. We developed a novel objective function which specifies the mechanistics of this simple, ligand-triggered riboswitch and describe an extensive in silico analysis pipeline to evaluate important kinetic properties of the designed sequences. This protocol and the developed software can be easily extended or adapted to fit novel design scenarios and thus can serve as a template for future needs. [ABSTRACT FROM AUTHOR]

There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a computational protocol for the characterization of interactions between proteins and RNA containing post-transcriptional modifications. Given an RNA-protein complex structure, potential RNA modified ribonucleoside positions, and molecular mechanics parameters for capturing energetics of RNA modifications, our protocol operates in two stages. In the first stage, a decision-making tool, comprising short simulations and interaction energy calculations, performs a fast and efficient search in a high-throughput fashion, through a list of different types of RNA modifications categorized into trees according to their structural and physicochemical properties, and selects a subset of RNA modifications prone to interact with the target protein. In the second stage, RNA modifications that are selected as recognized by the protein are examined in-detail using all-atom simulations and free energy calculations. We implement and experimentally validate this protocol in a test case involving the study of RNA modifications in complex with Escherichia coli (E. coli) protein Polynucleotide Phosphorylase (PNPase), depicting the favorable interaction between 8-oxo-7,8-dihydroguanosine (8-oxoG) RNA modification and PNPase. Further advancement of the protocol can broaden our understanding of protein interactions with all known RNA modifications in several systems. [ABSTRACT FROM AUTHOR]

The secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure–function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines. Herein, we present StructureFold2, a user-friendly set of analysis tools that makes precise data processing and detailed downstream analyses of such data sets both available and practical. StructureFold2 processes high-throughput reads sequenced from libraries prepared after experimental probing for reverse transcription (RT) stops generated by chemical modification of RNA at solvent accessible residues. This pipeline is able to analyze reads generated from a variety of structure-probing chemicals (e.g. DMS, glyoxal, SHAPE). Notably, StructureFold2 offers a new fully featured suite of utilities and tools to guide a user through multiple types of analyses. A particular emphasis is placed on analyzing the reactivity patterns of transcripts, complementing their use as folding restraints for predicting RNA secondary structure. StructureFold2 is hosted as a Github repository and is available at ( https://github.com/StructureFold2/StructureFold2 ). [ABSTRACT FROM AUTHOR]

The structure of RNA has been a natural subject for mathematical modeling, inviting many innovative computational frameworks. This single-stranded polynucleotide chain can fold upon itself in numerous ways to form hydrogen-bonded segments, imperfect with single-stranded loops. Illustrating these paired and non-paired interaction networks, known as RNA’s secondary (2D) structure, using mathematical graph objects has been illuminating for RNA structure analysis. Building upon such seminal work from the 1970s and 1980s, graph models are now used to study not only RNA structure but also describe RNA’s recurring modular units, sample the conformational space accessible to RNAs, predict RNA’s three-dimensional folds, and apply the combined aspects to novel RNA design. In this article, we outline the development of the RNA-As-Graphs (or RAG) approach and highlight current applications to RNA structure prediction and design. [ABSTRACT FROM AUTHOR]

In this paper, we analyze the length spectrum of rainbows in RNA secondary structures. A rainbow in a secondary structure is a maximal arc with respect to the partial order induced by nesting. We show that there is a significant gap in this length spectrum. We shall prove that there asymptotically almost surely exists a unique longest rainbow of length at least n-O(n1/2) and that with high probability any other rainbow has finite length. We show that the distribution of the length of the longest rainbow converges to a discrete limit law and that, for finite k, the distribution of rainbows of length k becomes for large n a negative binomial distribution. We then put the results of this paper into context, comparing the analytical results with those observed in RNA minimum free energy structures, biological RNA structures and relate our findings to the sparsification of folding algorithms. [ABSTRACT FROM AUTHOR]

The current RNA structure prediction methods cannot keep up the pace of the rapidly increasing number of sequences and the emerging new functions of RNAs. Template-based RNA three-dimensional structure prediction methods are restricted by the limited number of known RNA structures, and traditional motif-based search for the templates does not always lead to successful results. Here we report a new template search and assembly algorithm, the hierarchical loop template-assembly method (VfoldLA). The method searches for templates for single strand loop/junctions instead of the whole motifs, which often renders no available templates, or short fragments (several nucleotides), which requires a long computational time to assemble and refine. The VfoldLA method has the advantage of accounting for local and nonlocal interloop interactions. Benchmark tests indicate that this new method can provide low-resolution predictions for RNA conformations at different levels of structural complexities. Furthermore, the VfoldLA-predicted conformations may also serve as reliable putative models for further structure prediction and refinements. VfoldLA is accessible at http://rna.physics.missouri.edu/vfoldLA. [ABSTRACT FROM AUTHOR]

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes. [ABSTRACT FROM AUTHOR]

RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods.In this review we present different methods for large-scale production and purification of RNAs for structural and biophysical studies [ABSTRACT FROM AUTHOR]

Riboswitches are conserved RNA structures located in non-coding regions of mRNA and able to bind small molecules (e.g. metabolites) changing conformation upon binding. This feature enables them to function as regulators of gene expression. The thiamin pyrophosphate (TPP) riboswitch is the only type of riboswitches found not only in bacteria, but also in eukaryotes – in plants, green algae, protists, and fungi. Two main mechanisms of fungal TPP riboswitch action, involving alternative splicing, have been established so far. Here, we report a large-scale bioinformatic study of riboswitch structural features, action mechanisms, and distribution along the fungal taxonomy groups. For each putatively regulated gene, we reconstruct the riboswitch structure, identify other components of the regulation machinery, and establish mechanisms of riboswitch-mediated regulation. In addition to three genes known to be regulated by TPP riboswitches, thiazole synthase THI4, hydroxymethilpyrimidine-syntase NMT1, and putative transporter NCU01977, we identify two new genes, a putative thiamin transporter THI9 and a transporter of unknown specificity. While the riboswitch sequence and structure remain highly conserved in all species and genes, the mode of riboswitch-mediated regulation varies between regulated genes. The riboswitch usage varies strongly between fungal taxa, with the largest number of riboswitch-regulated genes found in Pezizomycotina and no riboswitch-mediated regulation established in Saccaromycotina. [ABSTRACT FROM AUTHOR]

Motivation: Understanding the formation, architecture and roles of pseudoknots in RNA structures are one of the most difficult challenges in RNA computational biology and structural bioinformatics. Methods predicting pseudoknots typically perform this with poor accuracy, often despite experimental data incorporation. Existing bioinformatic approaches differ in terms of pseudoknots' recognition and revealing their nature. A few ways of pseudoknot classification exist, most common ones refer to a genus or order. Following the latter one, we propose new algorithms that identify pseudoknots in RNA structure provided in BPSEQ format, determine their order and encode in dot-bracket-letter notation. The proposed encoding aims to illustrate the hierarchy of RNA folding. Results: New algorithms are based on dynamic programming and hybrid (combining exhaustive search and random walk) approaches. They evolved from elementary algorithm implemented within the workflow of RNA FRABASE 1.0, our database of RNA structure fragments. They use different scoring functions to rank dissimilar dot-bracket representations of RNA structure. Computational experiments show an advantage of new methods over the others, especially for large RNA structures. [ABSTRACT FROM AUTHOR]

Motivation: RNA is a biopolymer with various applications inside the cell and in biotechnology. Structure of an RNA molecule mainly determines its function and is essential to guide nanostructure design. Since experimental structure determination is time-consuming and expensive, accurate computational prediction of RNA structure is of great importance. Prediction of RNA secondary structure is relatively simpler than its tertiary structure and provides information about its tertiary structure, therefore, RNA secondary structure prediction has received attention in the past decades. Numerous methods with different folding approaches have been developed for RNA secondary structure prediction. While methods for prediction of RNA pseudoknot-free structure (structures with no crossing base pairs) have greatly improved in terms of their accuracy, methods for prediction of RNA pseudoknotted secondary structure (structures with crossing base pairs) still have room for improvement. A long-standing question for improving the prediction accuracy of RNA pseudoknotted secondary structure is whether to focus on the prediction algorithm or the underlying energy model, as there is a trade-off on computational cost of the prediction algorithm versus the generality of the method. Results: The aim of this work is to argue when comparing different methods for RNA pseudoknotted structure prediction, the combination of algorithm and energy model should be considered and a method should not be considered superior or inferior to others if they do not use the same scoring model. We demonstrate that while the folding approach is important in structure prediction, it is not the only important factor in prediction accuracy of a given method as the underlying energy model is also as of great value. Therefore we encourage researchers to pay particular attention in comparing methods with different energy models. [ABSTRACT FROM AUTHOR]

Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis -acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5′-terminal RNA structural elements across coronavirus genus boundaries. [ABSTRACT FROM AUTHOR]

RNA function is intimately related to its structural dynamics. Molecular dynamics simulations are useful for exploring biomolecular flexibility but are severely limited by the accessible timescale. Enhanced sampling methods allow this timescale to be effectively extended in order to probe biologically relevant conformational changes and chemical reactions. Here, we review the role of enhanced sampling techniques in the study of RNA systems. We discuss the challenges and promises associated with the application of these methods to force-field validation, exploration of conformational landscapes and ion/ligand-RNA interactions, as well as catalytic pathways. Important technical aspects of these methods, such as the choice of the biased collective variables and the analysis of multi-replica simulations, are examined in detail. Finally, a perspective on the role of these methods in the characterization of RNA dynamics is provided. [ABSTRACT FROM AUTHOR]

In order to reduce the Cyrtotrachelus buqueti population, which is a serious pest in the bamboo industry, a range of approaches is required, which will be dependent on diverse gene expression influenced by environmental factors. In this study, samples from two regions of China, Muchuan in Sichuan Province and Chishui in Guizhou Province, were investigated through RNA-seq. Approximately 44 million high-quality reads were generated and 94.2% of the data was mapped to the transcriptome. A total of 15,641 out of the 29,406 identified genes were predicted. Moreover, 348 genes were differentially expressed between the two groups of imagoes (77 upregulated and 271 downregulated). The functional analysis showed that these genes were significantly enriched in the ribosome and metabolic pathway categories. The candidate genes contributing to the reduction in C. buqueti included 41 genes involved in the ribosome constitution category, five in the one‑carbon pool pathway by folate category, and five heat shock protein genes. The downregulation of these candidate genes seems to have impaired metabolic processes, such as protein, DNA, RNA, and purine synthesis, as well as carbon and folate metabolism, subsequently resulting in the observed population reduction of C. buqueti . Furthermore, temperature, heavy metal content, and pH might influence the population by altering the expressions of genes involved in these metabolic processes. [ABSTRACT FROM AUTHOR]

RNA transcription of mononegavirales decreases gradually from the 3′ leader promoter toward the 5′ end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M 2–1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M 2–1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M 2–1 . Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M 2–1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30. [ABSTRACT FROM AUTHOR]

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity--a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date. In this work, we describe a method for converting DNA libraries into "gene-linked RNA aptamer particles" (GRAPs) that each display ~105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescenceenhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different emission wavelengths upon binding the dye of interest. [ABSTRACT FROM AUTHOR]