scholarly journals In vitro selections with RNAs of variable length converge on a robust catalytic core

2020 ◽  
Author(s):  
Milena Popović ◽  
Alexander Q Ellingson ◽  
Theresa P Chu ◽  
Chenyu Wei ◽  
Andrew Pohorille ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Point Mutations ◽  
Rna Structures ◽  
Catalytic Core ◽  
Basic Principles ◽  
Rna Ligase ◽  
Rna Evolution ◽  
Conserved Core ◽  
Functional Rnas

Abstract In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.

scholarly journals Secondary Structural Model of Human MALAT1 Reveals Multiple Structure–Function Relationships

2019 ◽  
Vol 20 (22) ◽  
pp. 5610 ◽  
Author(s):  
Phillip J. McCown ◽  
Matthew C. Wang ◽  
Luc Jaeger ◽  
Jessica A. Brown
Keyword(s):  
Structure Function ◽  
Noncoding Rna ◽  
Structural Model ◽  
Dynamic Structure ◽  
Nucleotide Polymorphisms ◽  
Rna Structures ◽  
Rna Modifications ◽  
Functional Roles ◽  
Conserved Core

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an abundant nuclear-localized long noncoding RNA (lncRNA) that has significant roles in cancer. While the interacting partners and evolutionary sequence conservation of MALAT1 have been examined, much of the structure of MALAT1 is unknown. Here, we propose a hypothetical secondary structural model for 8425 nucleotides of human MALAT1 using three experimental datasets that probed RNA structures in vitro and in various human cell lines. Our model indicates that approximately half of human MALAT1 is structured, forming 194 helices, 13 pseudoknots, five structured tetraloops, nine structured internal loops, and 13 intramolecular long-range interactions that give rise to several multiway junctions. Evolutionary conservation and covariation analyses support 153 of 194 helices in 51 mammalian MALAT1 homologs and 42 of 194 helices in 53 vertebrate MALAT1 homologs, thereby identifying an evolutionarily conserved core that likely has important functional roles in mammals and vertebrates. Data mining revealed that RNA modifications, somatic cancer-associated mutations, and single-nucleotide polymorphisms may induce structural rearrangements that sequester or expose binding sites for several cancer-associated microRNAs. Our findings reveal new mechanistic leads into the roles of MALAT1 by identifying several intriguing structure–function relationships in which the dynamic structure of MALAT1 underlies its biological functions.

In Vitro Selection of DNA Aptamers on Chips Using a Method for Generating Point Mutations

2004 ◽  
Vol 37 (4) ◽  
pp. 645-656 ◽  
Author(s):  
Ryoichi Asai ◽  
Shin I. Nishimura ◽  
Takuyo Aita ◽  
Katsutoshi Takahashi
Keyword(s):  
In Vitro Selection ◽  
Point Mutations ◽  
Dna Aptamers ◽  
Selection Of

scholarly journals In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x.

1996 ◽  
Vol 40 (1) ◽  
pp. 152-156 ◽  
Author(s):  
F Sifaoui ◽  
M D Kitzis ◽  
L Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
In Vitro Selection ◽  
Point Mutations ◽  
Fold Increase ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Beta Lactam Antibiotics ◽  
One Step ◽  
Resistant Mutants

Many oral penicillins and cephalosporins are used to treat clinical infections caused by Streptococcus pneumoniae. Therefore, using different beta-lactams as selectors, we estimated the frequencies of one-step mutations leading to resistance. Resistant mutants were obtained from penicillin-susceptible, intermediately resistant, and penicillin resistant strains. For cefixime, cefuroxime, cefpodoxime, cefotaxime, and ceftriaxone, the frequencies of mutation ranged from 10(-6) to 10(-8) when resistant mutants were selected at 2- to 8-fold the MIC, and the MICs increased 2- to 16-fold. For ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, cefaclor, and loracarbef, the frequencies of mutation were about 10(-7) to 10(-8), and the MICs increased twofold at most. One to three resistance profiles of the resulting mutants were selected for each of the selecting antibiotics. Among those, some showed resistance to the cephalosporins associated with a 2- to 32-fold increase in susceptibility to the penicillins. Competition experiments showed a decreased affinity of PBP2x for cefpodoxime in all mutants. In some mutants that were more susceptible to amoxicillin, a decreased affinity of PBP2x for cefpodoxime was associated with an increased affinity for amoxicillin and a particular substitution of alanine for threonine at position 550 just after the KSG triad. From these results we infer (i) that among the beta-lactams tested the penicillins, cefaclor, and loracarbef selected one-step resistant mutants less frequently and that they achieved a lower level of resistance, and (ii) that mutants with different profiles may have acquired different point mutations in PBP2x.

scholarly journals New Deoxyribozymes for the Native Ligation of RNA

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3650
Author(s):  
Carolin P. M. Scheitl ◽  
Sandra Lange ◽  
Claudia Höbartner
Keyword(s):  
Catalytic Activity ◽  
In Vitro Selection ◽  
High Ligation ◽  
Rna Ligase ◽  
Dna Library ◽  
Phosphodiester Linkage ◽  
Synthetic Accessibility ◽  
Rna Ligation ◽  
Optimal Incubation

Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3′-5′-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.

scholarly journals In vitro selection and characterization of RNA aptamers binding thyroxine hormone

2007 ◽  
Vol 403 (1) ◽  
pp. 129-138 ◽  
Author(s):  
Dominique Lévesque ◽  
Jean-Denis Beaudoin ◽  
Sébastien Roy ◽  
Jean-Pierre Perreault
Keyword(s):  
In Vitro Selection ◽  
Biochemical Characterization ◽  
Rna Aptamers ◽  
Rna Structures ◽  
Chemical Probing ◽  
Guanine Quadruplex ◽  
Exponential Enrichment ◽  
Helical Region

RNA possesses the ability to bind a wide repertoire of small molecules. Some of these binding interactions have been shown to be of primary importance in molecular biology. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites (i.e. acting as direct sensors of chemical compounds). However, to date no RNA species binding a hormone has been reported. Here, we report that the use of an appropriate SELEX (systematic evolution of ligands by exponential enrichment) strategy results in the isolation of thyroxine-specific aptamers. Further biochemical characterization of these aptamers, including mutational studies, the use of transcripts with site-specific modified nucleotides, nuclease and chemical probing, binding-shift assays and CD, demonstrated that these RNA structures included a G-rich motif, reminiscent of a guanine quadruplex structure, adjacent to a helical region. The presence of the thyroxine appeared to be essential for the formation of the structural motif's scaffold. Moreover, the binding is shown to be specific to thyroxine (T4) and tri-iodothyronine (T3), the active forms of the hormone, whereas other inactive derivatives, including thyronine (T0), do not support complex formation. These results suggest that this aptamer specifically binds to the iodine moieties of the thyroxine, a previously unreported ability for an RNA molecule.

scholarly journals Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Rika Inomata ◽  
Jing Zhao ◽  
Makoto Miyagishi
Keyword(s):  
In Vitro Selection ◽  
Sequence Similarity ◽  
Sequencing Analysis ◽  
Sequence Specificity ◽  
Rna Sequences ◽  
Catalytic Core ◽  
Highly Active ◽  
Target Rna ◽  
Deep Sequencing Analysis

AbstractAlthough several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn2+-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5′-rN↓rNrPu-3′. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology.

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