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Journal of Nanobiotechnology May 2024Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid,...
Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.
Topics: RNA, Ribosomal; Nucleic Acid Conformation; Nanostructures; Humans; RNA Folding; DNA; Polylysine; Polyethylene Glycols
PubMed: 38698435
DOI: 10.1186/s12951-024-02489-2 -
The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription.Molecular Cell Jan 2020The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of...
The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.
Topics: Escherichia coli; Protein Binding; RNA; RNA Folding; Ribosomes; Signal Recognition Particle; Transcription, Genetic
PubMed: 31706702
DOI: 10.1016/j.molcel.2019.10.006 -
Methods in Enzymology 2022Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene...
Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.
Topics: Aptamers, Nucleotide; DNA-Directed RNA Polymerases; Fluorescent Dyes; Green Fluorescent Proteins; RNA; RNA Folding
PubMed: 36220271
DOI: 10.1016/bs.mie.2022.07.010 -
Bioinformatics (Oxford, England) Jan 2016The function of an RNA molecule is not only linked to its native structure, which is usually taken to be the ground state of its folding landscape, but also in many...
MOTIVATION
The function of an RNA molecule is not only linked to its native structure, which is usually taken to be the ground state of its folding landscape, but also in many cases crucially depends on the details of the folding pathways such as stable folding intermediates or the timing of the folding process itself. To model and understand these processes, it is necessary to go beyond ground state structures. The study of rugged RNA folding landscapes holds the key to answer these questions. Efficient coarse-graining methods are required to reduce the intractably vast energy landscapes into condensed representations such as barrier trees or basin hopping graphs : BHG) that convey an approximate but comprehensive picture of the folding kinetics. So far, exact and heuristic coarse-graining methods have been mostly restricted to the pseudoknot-free secondary structures. Pseudoknots, which are common motifs and have been repeatedly hypothesized to play an important role in guiding folding trajectories, were usually excluded.
RESULTS
We generalize the BHG framework to include pseudoknotted RNA structures and systematically study the differences in predicted folding behavior depending on whether pseudoknotted structures are allowed to occur as folding intermediates or not. We observe that RNAs with pseudoknotted ground state structures tend to have more pseudoknotted folding intermediates than RNAs with pseudoknot-free ground state structures. The occurrence and influence of pseudoknotted intermediates on the folding pathway, however, appear to depend very strongly on the individual RNAs so that no general rule can be inferred.
AVAILABILITY AND IMPLEMENTATION
The algorithms described here are implemented in C++ as standalone programs. Its source code and Supplemental material can be freely downloaded from http://www.tbi.univie.ac.at/bhg.html.
CONTACT
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Algorithms; Kinetics; Nucleic Acid Conformation; RNA; RNA Folding
PubMed: 26428288
DOI: 10.1093/bioinformatics/btv572 -
Methods in Molecular Biology (Clifton,... 2024A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest...
A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest neighbor parameters that models the folding stability of a RNA secondary structure as the sum of folding stabilities of the structural elements that comprise the secondary structure. In the software suite RNAstructure, the free energy change calculation is implemented in the program efn2. The efn2 program estimates the folding free energy change and the experimental uncertainty in the folding free energy change. It can be run through the graphical user interface for RNAstructure, from the command line, or a web server. This chapter provides detailed protocols for using efn2.
Topics: RNA; RNA Folding; Software; Nucleic Acid Conformation; Thermodynamics; Computational Biology; Models, Molecular
PubMed: 38780725
DOI: 10.1007/978-1-0716-3519-3_1 -
Nucleic Acids Research Jul 2016We introduce a method for predicting RNA folding pathways, with an application to the most important RNA tetraloops. The method is based on the idea that ensembles of...
We introduce a method for predicting RNA folding pathways, with an application to the most important RNA tetraloops. The method is based on the idea that ensembles of three-dimensional fragments extracted from high-resolution crystal structures are heterogeneous enough to describe metastable as well as intermediate states. These ensembles are first validated by performing a quantitative comparison against available solution nuclear magnetic resonance (NMR) data of a set of RNA tetranucleotides. Notably, the agreement is better with respect to the one obtained by comparing NMR with extensive all-atom molecular dynamics simulations. We then propose a procedure based on diffusion maps and Markov models that makes it possible to obtain reaction pathways and their relative probabilities from fragment ensembles. This approach is applied to study the helix-to-loop folding pathway of all the tetraloops from the GNRA and UNCG families. The results give detailed insights into the folding mechanism that are compatible with available experimental data and clarify the role of intermediate states observed in previous simulation studies. The method is computationally inexpensive and can be used to study arbitrary conformational transitions.
Topics: Diffusion; Kinetics; Magnetic Resonance Spectroscopy; Markov Chains; Molecular Dynamics Simulation; Motion; Nucleic Acid Conformation; Oligoribonucleotides; RNA; RNA Folding; Thermodynamics
PubMed: 27091499
DOI: 10.1093/nar/gkw239 -
Methods in Molecular Biology (Clifton,... 2020We have previously described (Geffroy et al. Methods Mol Biol 1665:25-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap...
We have previously described (Geffroy et al. Methods Mol Biol 1665:25-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap setup. In this chapter, we complementarily describe how to analyze the corresponding data and how to interpret it in terms of RNA three-dimensional structure. As with all single-molecule methods, single RNA molecule force data often exhibit several discrete states where state-to-state transitions are blurred in a noisy signal. In order to cope with this limitation, we have implemented a novel strategy to analyze the data, which uses a hidden Markov modeling procedure. A representative example of such an analysis is presented.
Topics: Markov Chains; Models, Molecular; Nucleic Acid Conformation; Optical Tweezers; RNA; RNA Folding; Single Molecule Imaging; Software
PubMed: 32006309
DOI: 10.1007/978-1-0716-0278-2_7 -
BMC Bioinformatics Oct 2019The understanding of the importance of RNA has dramatically changed over recent years. As in the case of proteins, the function of an RNA molecule is encoded in its...
BACKGROUND
The understanding of the importance of RNA has dramatically changed over recent years. As in the case of proteins, the function of an RNA molecule is encoded in its tertiary structure, which in turn is determined by the molecule's sequence. The prediction of tertiary structures of complex RNAs is still a challenging task.
RESULTS
Using the observation that RNA sequences from the same RNA family fold into conserved structure, we test herein whether parallel modeling of RNA homologs can improve ab initio RNA structure prediction. EvoClustRNA is a multi-step modeling process, in which homologous sequences for the target sequence are selected using the Rfam database. Subsequently, independent folding simulations using Rosetta FARFAR and SimRNA are carried out. The model of the target sequence is selected based on the most common structural arrangement of the common helical fragments. As a test, on two blind RNA-Puzzles challenges, EvoClustRNA predictions ranked as the first of all submissions for the L-glutamine riboswitch and as the second for the ZMP riboswitch. Moreover, through a benchmark of known structures, we discovered several cases in which particular homologs were unusually amenable to structure recovery in folding simulations compared to the single original target sequence.
CONCLUSION
This work, for the first time to our knowledge, demonstrates the importance of the selection of the target sequence from an alignment of an RNA family for the success of RNA 3D structure prediction. These observations prompt investigations into a new direction of research for checking 3D structure "foldability" or "predictability" of related RNA sequences to obtain accurate predictions. To support new research in this area, we provide all relevant scripts in a documented and ready-to-use form. By exploring new ideas and identifying limitations of the current RNA 3D structure prediction methods, this work is bringing us closer to the near-native computational RNA 3D models.
Topics: Algorithms; Models, Molecular; RNA; RNA Folding; Riboswitch; Sequence Homology; Software
PubMed: 31640563
DOI: 10.1186/s12859-019-3120-y -
PLoS Computational Biology Aug 2022We propose a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. This...
We propose a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. This heuristic is inspired by the kinetic partitioning mechanism, by which molecules follow alternative folding pathways to their native structure, some much faster than others. Similarly, our algorithm RAFFT starts by generating an ensemble of concurrent folding pathways ending in multiple metastable structures, which is in contrast with traditional thermodynamic approaches that find single structures with minimal free energies. When we constrained the algorithm to predict only 50 structures per sequence, near-native structures were found for RNA molecules of length ≤ 200 nucleotides. Our heuristic has been tested on the coronavirus frameshifting stimulation element (CFSE): an ensemble of 68 distinct structures allowed us to produce complete folding kinetic trajectories, whereas known methods require evaluating millions of sub-optimal structures to achieve this result. Thanks to the fast Fourier transform on which RAFFT (RNA folding Algorithm wih Fast Fourier Transform) is based, these computations are efficient, with complexity [Formula: see text].
Topics: Algorithms; Fourier Analysis; Nucleic Acid Conformation; RNA; RNA Folding; Thermodynamics
PubMed: 36026505
DOI: 10.1371/journal.pcbi.1010448 -
Methods in Molecular Biology (Clifton,... 2021Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols...
Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols for using inverse folding software to design a ribozyme sequence that will fold to a known ribozyme secondary structure and for testing the catalytic activity of the sequence experimentally. This protocol is able to design sequences that include pseudoknots, which is important as all naturally occurring full-length ribozymes have pseudoknots. The starting point is the known pseudoknot-containing secondary structure of the ribozyme and knowledge of any nucleotides whose identity is required for function. The output of the protocol is a set of sequences that have been tested for function. Using this protocol, we were previously successful at designing highly active double-pseudoknotted HDV ribozymes.
Topics: Base Sequence; Computational Biology; G-Quadruplexes; Hepatitis Delta Virus; In Vitro Techniques; Kinetics; Models, Molecular; Nucleic Acid Conformation; Nucleotide Motifs; RNA Folding; RNA, Catalytic; Software; Transcription, Genetic
PubMed: 32712918
DOI: 10.1007/978-1-0716-0716-9_8