scholarly journals A natural riboswitch scaffold with self-methylation activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Laurin Flemmich ◽  
Sarah Heel ◽  
Sarah Moreno ◽  
Kathrin Breuker ◽  
Ronald Micura
Keyword(s):  
Rational Design ◽  
In Vitro Selection ◽  
Rna World ◽  
Organic Substances ◽  
Methyl Group ◽  
Non Coding Rna ◽  
Metabolic Reactions ◽  
Protein Methyltransferases ◽  
Methylation Activity

AbstractMethylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand’s methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.

Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

2004 ◽  
Vol 76 (7-8) ◽  
pp. 1547-1561 ◽  
Author(s):  
R. Nutiu ◽  
Shirley Mei ◽  
Zhongjie Liu ◽  
Y. Li
Keyword(s):  
Dna Sequences ◽  
Rational Design ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Pair Potential ◽  
Dna Aptamers ◽  
Dna Molecules ◽  
Dna Enzymes ◽  
Fluorescence Signaling

Single-stranded DNA molecules with ligand-binding ability and catalytic function, referred to as DNA aptamers and DNA enzymes, respectively, are special DNA sequences isolated from random-sequence DNA libraries by “in vitro selection”. These two new classes of artificial DNA molecules have the potential of being used as molecular tools in a variety of innovative applications ranging from biosensing to gene regulation. Our laboratory is interested in engineering fluorescence-signaling DNA aptamers and DNA enzymes that can be widely exploited for detection-directed applications. In this article, we will first discuss our recent efforts on the rational design of a new class of signaling aptamers denoted “structure- switching signaling aptamers”, which report target binding by switching structures from DNA/DNA duplex to DNA/target complex. We will then describe the in vitro selection of fluorescence-signaling DNA enzymes that exhibit a synchronized catalysis-signaling capability by cleaving a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by closely spaced fluorophore-quencher pair. Potential utilities of these signaling DNA molecules will also be discussed.

scholarly journals RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

2022 ◽  
Author(s):  
Shan Qi ◽  
Javier Mota ◽  
Siu-Hong Chan ◽  
Johanna Villarreal ◽  
Nan Dai ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rna Binding ◽  
Methyl Group ◽  
High Affinity ◽  
Methylation Of Dna ◽  
Dna And Rna ◽  
Close Proximity ◽  
Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

2010 ◽  
Vol 1 (1) ◽  
pp. 3
Author(s):  
Ivan B. Brukner ◽  
Anne-Marie Larose ◽  
Izabella Gorska-Flipot ◽  
Maja Krajinovic ◽  
Damian Labuda
Keyword(s):  
Rational Design ◽  
In Vitro Selection ◽  
Clinical Performance ◽  
Initial Step ◽  
Detection Methods ◽  
Ambient Temperatures ◽  
Hybridization Probes ◽  
Specificity And Sensitivity ◽  
Hpv Types

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through i<em>n vitro </em>selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

scholarly journals oRNAment: a database of putative RNA binding protein target sites in the transcriptomes of model species

2019 ◽  
Author(s):  
Louis Philip Benoit Bouvrette ◽  
Samantha Bovaird ◽  
Mathieu Blanchette ◽  
Eric Lécuyer
Keyword(s):  
In Vitro Selection ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Model Organisms ◽  
Rna Motifs ◽  
Non Coding Rna ◽  
Distribution Features ◽  
Available Information ◽  
User Friendly

AbstractProtein–RNA interactions are essential for controlling most aspects of RNA metabolism, including synthesis, processing, trafficking, stability and degradation. In vitro selection methods, such as RNAcompete and RNA Bind-n-Seq, have defined the consensus target motifs of hundreds of RNA-binding proteins (RBPs). However, readily available information about the distribution features of these motifs across full transcriptomes was hitherto lacking. Here, we introduce oRNAment (o RNA motifs enrichment in transcriptomes), a database that catalogues the putative motif instances of 223 RBPs, encompassing 453 motifs, in a transcriptome-wide fashion. The database covers 525 718 complete coding and non-coding RNA species across the transcriptomes of human and four prominent model organisms: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster and Mus musculus. The unique features of oRNAment include: (i) hosting of the most comprehensive mapping of RBP motif instances to date, with 421 133 612 putative binding sites described across five species; (ii) options for the user to filter the data according to a specific threshold; (iii) a user-friendly interface and efficient back-end allowing the rapid querying of the data through multiple angles (i.e. transcript, RBP, or sequence attributes) and (iv) generation of several interactive data visualization charts describing the results of user queries. oRNAment is freely available at http://rnabiology.ircm.qc.ca/oRNAment/.

The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

2020 ◽  
Vol 65 (9-10) ◽  
pp. 3-7
Author(s):  
V. V. Gostev ◽  
Yu. V. Sopova ◽  
O. S. Kalinogorskaya ◽  
M. E. Velizhanina ◽  
I. V. Lazareva ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
High Concentration ◽  
Short Term ◽  
High Concentrations ◽  
Selection Of ◽  
Selection Of Resistance ◽  
Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

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