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1.
Nat Commun ; 14(1): 543, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36725852

RESUMO

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.


Assuntos
RNA Catalítico , Tetrahymena thermophila , RNA/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Sequência de Bases , Splicing de RNA , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Conformação de Ácido Nucleico
2.
J Chem Phys ; 158(4): 045101, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36725513

RESUMO

Triplex DNA structure has potential therapeutic application in inhibiting the expression of genes involved in cancer and other diseases. As a DNA-targeting antitumor and antibiotic drug, coralyne shows a remarkable binding propensity to triplex over canonical duplex and thus can modulate the stability of triplex structure, providing a prospective gene targeting strategy. Much less is known, however, about coralyne-binding interactions with triplex. By combining multiple steady-state spectroscopy with ultrafast fluorescence spectroscopy, we have investigated the binding behaviors of coralyne with typical triplexes. Upon binding with a G-containing triplex, the fluorescence of coralyne is markedly quenched owing to the photoinduced electron transfer (PET) of coralyne with the G base. Systematic studies show that the PET rates are sensitive to the binding configuration and local microenvironment, from which the coexisting binding modes of monomeric (full and partial) intercalation and aggregate stacking along the sugar-phosphate backbone are distinguished and their respective contributions are determined. It shows that coralyne has preferences for monomeric intercalation within CGG triplex and pure TAT triplex, whereas CGC+ triplex adopts mainly backbone binding of coralyne aggregates due to charge repulsion, revealing the sequence-specific binding selectivity. The triplex-DNA-induced aggregation of coralyne could be used as a probe for recognizing the water content in local DNA structures. The strong π-π stacking of intercalated coralyne monomer with base-triplets plays an important role in stabilizing the triplex structure. These results provide mechanistic insights for understanding the remarkable propensity of coralyne in selective binding to triplex DNA and shed light on the prospective applications of coralyne-triplex targeted anti-gene therapeutics.


Assuntos
DNA , Espectrometria de Fluorescência , Desnaturação de Ácido Nucleico , Conformação de Ácido Nucleico , DNA/química
3.
Molecules ; 28(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36677900

RESUMO

Despite structural differences between the right-handed conformations of A-RNA and B-DNA, both nucleic acids adopt very similar, left-handed Z-conformations. In contrast to their structural similarities and sequence preferences, RNA and DNA exhibit differences in their ability to adopt the Z-conformation regarding their hydration shells, the chemical modifications that promote the Z-conformation, and the structure of junctions connecting them to right-handed segments. In this review, we highlight the structural and chemical properties of both Z-DNA and Z-RNA and delve into the potential factors that contribute to both their similarities and differences. While Z-DNA has been extensively studied, there is a gap of knowledge when it comes to Z-RNA. Where such information is lacking, we try and extend the principles of Z-DNA stability and formation to Z-RNA, considering the inherent differences of the nucleic acids.


Assuntos
DNA Forma Z , Ácidos Nucleicos , RNA , Conformação de Ácido Nucleico , DNA/química
5.
Methods Enzymol ; 679: 97-129, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36682874

RESUMO

The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.


Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Edição de Genes/métodos , DNA/metabolismo , RNA
6.
Sci Rep ; 13(1): 288, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690669

RESUMO

To realize nucleic acid-targeting photodynamic therapy, a photosensitizer should be attached at the optimal position on a complementary oligonucleotide, where a guanine photooxidation is maximized. Here we show the photooxidation of 22 DNA duplexes with varied lengths between a 1O2-generating biphenyl photosensitizer attached at a midchain thymine in a strand and the single guanine reactant in the other strand. The best photooxidation efficiencies are achieved at 9, 10, and 21 base intervals, which coincides with the pitch of 10.5 base pairs per turn in a DNA duplex. The low efficiencies for near and far guanines are due to quenching of the biphenyl by guanine and dilution of 1O2 by diffusion, respectively. The 1O2-diffusion mapping along DNA duplex provides clues to the development of efficient and selective photosensitizer agents for nucleic acid-targeting photodynamic therapy, as well as an experimental demonstration of diffusion of a particle along cylindrical surface in molecular level.


Assuntos
Guanina , Fármacos Fotossensibilizantes , Conformação de Ácido Nucleico , DNA
7.
Nat Commun ; 14(1): 382, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36693871

RESUMO

Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterize secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.


Assuntos
Nanoestruturas , Nanotecnologia , Nanotecnologia/métodos , RNA , Microscopia Crioeletrônica , Conformação de Ácido Nucleico , Nanoestruturas/química , RNA Mensageiro
8.
Biomolecules ; 13(1)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36671514

RESUMO

DNA is an attractive material for a range of applications in nanoscience and nanotechnology, and it has recently been demonstrated that the electronic properties of DNA are uniquely sensitive to its sequence and structure, opening new opportunities for the development of electronic DNA biosensors. In this report, we examine the origin of multiple conductance peaks that can occur during single-molecule break-junction (SMBJ)-based conductance measurements on DNA. We demonstrate that these peaks originate from the presence of multiple DNA conformations within the solutions, in particular, double-stranded B-form DNA (dsDNA) and G-quadruplex structures. Using a combination of circular dichroism (CD) spectroscopy, computational approaches, sequence and environmental controls, and single-molecule conductance measurements, we disentangle the conductance information and demonstrate that specific conductance values come from specific conformations of the DNA and that the occurrence of these peaks can be controlled by controlling the local environment. In addition, we demonstrate that conductance measurements are uniquely sensitive to identifying these conformations in solutions and that multiple configurations can be detected in solutions over an extremely large concentration range, opening new possibilities for examining low-probability DNA conformations in solutions.


Assuntos
Quadruplex G , Nanotecnologia , Conformação de Ácido Nucleico , Nanotecnologia/métodos , DNA/química , Dicroísmo Circular
9.
Int J Mol Sci ; 24(2)2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36674728

RESUMO

DNAzyme is a class of DNA molecules that can perform catalytic functions with high selectivity towards specific metal ions. Due to its potential applications for biosensors and medical therapeutics, DNAzyme has been extensively studied to characterize the relationships between its biochemical properties and functions. Similar to protein enzymes and ribozymes, DNAzymes have been found to undergo conformational changes in a metal-ion-dependent manner for catalysis. Despite the important role the conformation plays in the catalysis process, such structural and dynamic information might not be revealed by conventional approaches. Here, by using the single-molecule fluorescence resonance energy transfer (smFRET) technique, we were able to investigate the detailed conformational dynamics of a uranyl-specific DNAzyme 39E. We observed conformation switches of 39E to a folded state with the addition of Mg2+ and to an extended state with the addition of UO22+. Furthermore, 39E can switch to a more compact configuration with or without divalent metal ions. Our findings reveal that 39E can undergo conformational changes spontaneously between different configurations.


Assuntos
DNA Catalítico , DNA Catalítico/metabolismo , Metais/química , Transferência Ressonante de Energia de Fluorescência , Conformação de Ácido Nucleico , Íons/química
10.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675317

RESUMO

Four-stranded folded structures, such as G-quadruplexes and i-motifs in the genome, have attracted a growing interest nowadays since they have been discovered in the telomere and in several oncogene promoter regions. Their biological relevance is undeniable since their existence in living cells has been observed. In vivo they take part in the regulation of gene expression, in vitro they are used in the analytical biochemistry. They are attractive and promising targets for cancer therapy. Pressure studies can reveal specific aspects of the molecular processes. Pressure tuning experiments allow the determination of the volumetric parameters of the folded structures and of the folding-unfolding processes. Here, we review the thermodynamic parameters with a special focus on the volumetric ones, which were determined using pressure tuning spectroscopic experiments on the G-quadruplex and i-motif nucleic acid forms.


Assuntos
Quadruplex G , Ácidos Nucleicos , Termodinâmica , Telômero , Conformação de Ácido Nucleico
11.
Biochem Biophys Res Commun ; 644: 55-61, 2023 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-36630735

RESUMO

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.


Assuntos
COVID-19 , Riboswitch , Humanos , Poliaminas , Hidroxicloroquina , RNA Viral , SARS-CoV-2/genética , Aminoquinolinas/farmacologia , RNA Bacteriano/genética , Conformação de Ácido Nucleico
12.
Bioinformatics ; 39(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36655786

RESUMO

MOTIVATION: Folding during transcription can have an important influence on the structure and function of RNA molecules, as regions closer to the 5' end can fold into metastable structures before potentially stronger interactions with the 3' end become available. Thermodynamic RNA folding models are not suitable to predict structures that result from cotranscriptional folding, as they can only calculate properties of the equilibrium distribution. Other software packages that simulate the kinetic process of RNA folding during transcription exist, but they are mostly applicable for short sequences. RESULTS: We present a new algorithm that tracks changes to the RNA secondary structure ensemble during transcription. At every transcription step, new representative local minima are identified, a neighborhood relation is defined and transition rates are estimated for kinetic simulations. After every simulation, a part of the ensemble is removed and the remainder is used to search for new representative structures. The presented algorithm is deterministic (up to numeric instabilities of simulations), fast (in comparison with existing methods), and it is capable of folding RNAs much longer than 200 nucleotides. AVAILABILITY AND IMPLEMENTATION: This software is open-source and available at https://github.com/ViennaRNA/drtransformer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Heurística , Dobramento de RNA , Conformação de Ácido Nucleico , RNA/química , Software , Algoritmos
13.
J Am Chem Soc ; 145(4): 2142-2151, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36651186

RESUMO

A significant barrier to biological applications of DNA structures is their instability to nucleases. UV-mediated thymine dimerization can crosslink and stabilize DNA nanostructures, but its effect on DNA strand hybridization fidelity and function is unclear. In this work, we first compare a number of methods for DNA irradiation with different wavelengths of light and different photosensitizers. We demonstrate that all approaches can achieve nuclease protection; however, the levels of DNA off-target crosslinking and damage vary. We then describe mild irradiation conditions intended to safeguard DNA against nuclease degradation. We demonstrate up to 25× increase in serum stability while minimizing off-target damage and maintaining functions such as hybridization efficiency, gene silencing, aptamer binding, and DNA nanostructure formation. Our methodology requires no complex instruments beyond a UV light source and no synthetic modification of the DNA itself, allowing for applications in numerous areas of nucleic acid therapy and nanotechnology.


Assuntos
DNA , Nanoestruturas , DNA/química , Nanoestruturas/química , Nanotecnologia/métodos , Oligonucleotídeos/química , Hibridização de Ácido Nucleico , Conformação de Ácido Nucleico
14.
J Am Chem Soc ; 145(4): 2455-2460, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36657115

RESUMO

Mesojunctions were introduced as a basic type of crossover configuration in the early development of structural DNA nanotechnology. However, the investigations of self-assembly from multiple mesojunction complexes have been overlooked in comparison to their counterparts based on regular junctions. In this work, we designed standardized component strands for the construction of complex mesojunction lattices. Three typical mesojunction configurations with three and four arms were showcased in the self-assembly of 1-, 2-, and 3-dimensional lattices constructed from both a scaffold-free tiling approach and a scaffolded origami approach.


Assuntos
Nanoestruturas , Nanoestruturas/química , Conformação de Ácido Nucleico , DNA/química , Nanotecnologia/métodos
15.
Methods Mol Biol ; 2586: 15-34, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705896

RESUMO

RNA secondary structure prediction is widely used to understand RNA function. Existing dynamic programming-based algorithms, both the classical minimum free energy (MFE) methods and partition function methods, suffer from a major limitation: their runtimes scale cubically with the RNA length, and this slowness limits their use in genome-wide applications. Inspired by incremental parsing for context-free grammars in computational linguistics, we designed linear-time heuristic algorithms, LinearFold and LinearPartition, to approximate the MFE structure, partition function and base pairing probabilities. These programs are orders of magnitude faster than Vienna RNAfold and CONTRAfold on long sequences. More interestingly, LinearFold and LinearPartition lead to more accurate predictions on the longest sequence families for which the structures are well established (16S and 23S Ribosomal RNAs), as well as improved accuracies for long-range base pairs (500 + nucleotides apart). This chapter provides protocols for using LinearFold and LinearPartition for secondary structure prediction.


Assuntos
Algoritmos , RNA , Humanos , RNA/química , Conformação de Ácido Nucleico , Pareamento de Bases , Entropia , Biologia Computacional/métodos , Análise de Sequência de RNA/métodos
16.
Methods Mol Biol ; 2586: 1-14, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705895

RESUMO

Predicting the secondary structures of RNA molecules is an essential step to characterize their functions, but the thermodynamic probability of any prediction is generally small. On the other hand, there are a few tools for calculating and visualizing various secondary structural information from RNA sequences. We implemented a web server that calculates in parallel various features of secondary structures: different types of secondary structure predictions, the marginal probabilities for local structural contexts, accessibilities of the subsequences, the energy changes by arbitrary base mutations, and the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp , which integrates software tools, CentroidFold, CentroidHomfold, IPknot, CapR, Raccess, Rchange, RintD, and RintW.


Assuntos
Algoritmos , RNA , Conformação de Ácido Nucleico , RNA/genética , RNA/química , Sequência de Bases , Análise de Sequência de RNA , Software , Internet
17.
Methods Mol Biol ; 2586: 79-88, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705899

RESUMO

RNA secondary structure comparison is one of the important analyses for elucidating individual functions of RNAs since it is widely accepted that their functions and structures are strongly correlated. However, although the RNA secondary structures with pseudoknot play important roles in vivo, it is difficult to deal with such structures in silico due to their structural complexity, which is a major obstacle to the analysis of RNA functions.Here, we introduce an algorithm and a metric for comparing pseudoknotted RNA secondary structures based on topological centroid identification and tree edit distance and describe the usage protocol of a software enabling us to run the comparison. This software is publicly available and works on both Microsoft Windows and Apple macOS.


Assuntos
Algoritmos , RNA , RNA/genética , RNA/química , Conformação de Ácido Nucleico , Software , Análise de Sequência de RNA/métodos
18.
Methods Mol Biol ; 2586: 89-105, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705900

RESUMO

This chapter introduces the RNA secondary structure prediction based on the nearest neighbor energy model, which is one of the most popular architectures of modeling RNA secondary structure without pseudoknots. We discuss the parameterization and the parameter determination by experimental and machine learning-based approaches as well as an integrated approach that compensates each other's shortcomings. Then, folding algorithms for the minimum free energy and the maximum expected accuracy using the dynamic programming technique are introduced. Finally, we compare the prediction accuracy of the method described so far with benchmark datasets.


Assuntos
Dobramento de RNA , RNA , RNA/química , Conformação de Ácido Nucleico , Entropia , Algoritmos , Termodinâmica
19.
Methods Mol Biol ; 2586: 35-48, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705897

RESUMO

The information of RNA secondary structure has been widely applied to the inference of RNA function. However, a classical prediction method is not feasible to long RNAs such as mRNA due to the problems of computational time and numerical errors. To overcome those problems, sliding window methods have been applied while their results are not directly comparable to global RNA structure prediction. In this chapter, we introduce ParasoR, a method designed for parallel computation of genome-wide RNA secondary structures. To enable genome-wide prediction, ParasoR distributes dynamic programming (DP) matrices required for structure prediction to multiple computational nodes. Using the database of not the original DP variable but the ratio of variables, ParasoR can locally compute the structure scores such as stem probability or accessibility on demand. A comprehensive analysis of local secondary structures by ParasoR is expected to be a promising way to detect the statistical constraints on long RNAs.


Assuntos
Algoritmos , RNA , RNA/genética , RNA/química , Conformação de Ácido Nucleico , Biologia Computacional/métodos , RNA Mensageiro
20.
Methods Mol Biol ; 2586: 49-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36705898

RESUMO

Here we detail the LandscapeFold secondary structure prediction algorithm and how it is used. The algorithm was previously described and tested in (Kimchi O et al., Biophys J 117(3):520-532, 2019), though it was not named there. The algorithm directly enumerates all possible secondary structures into which up to two RNA or single-stranded DNA sequences can fold. It uses a polymer physics model to estimate the configurational entropy of structures including complex pseudoknots. We detail each of these steps and ways in which the user can adjust the algorithm as desired. The code is available on the GitHub repository https://github.com/ofer-kimchi/LandscapeFold .


Assuntos
Algoritmos , RNA , Conformação de Ácido Nucleico , RNA/genética , Entropia , DNA de Cadeia Simples
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