Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Methods Mol Biol ; 2822: 443-469, 2024.
Article in English | MEDLINE | ID: mdl-38907934

ABSTRACT

In vitro selection of allosteric ribozymes has many challenges, such as complex and time-consuming experimental procedures, uncertain results, and the unwanted functionality of the enriched sequences. The precise computational design of allosteric ribozymes is achievable using RNA secondary structure folding principles. The computational design of allosteric ribozymes is based on experimentally validated EAs, random search algorithms, and a partition function for RNA folding. The in silico design achieves an accuracy exceeding 90%. Various algorithms with different logic gates have been automated via computer programs that can quickly create many allosteric sequences. This can eliminate the need for in vitro selection of allosteric ribozymes, thus vastly reducing the time and cost required.


Subject(s)
Algorithms , Computational Biology , Nucleic Acid Conformation , RNA, Catalytic , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Computational Biology/methods , Allosteric Regulation , RNA Folding , Software , Computer Simulation
2.
J Biotechnol ; 373: 82-89, 2023 Aug 20.
Article in English | MEDLINE | ID: mdl-37499876

ABSTRACT

High-speed allosteric hammerhead ribozymes can be engineered to distinguish well between a perfectly matching effector and the nucleic acid sequences with a few mismatches under physiologically relevant conditions. Such ribozymes can be designed to control the expression of exogenous mRNAs and can be used to develop new gene therapies, including anticancer treatments. The in vivo selection of such ribozymes is a complicated and lengthy procedure with no guarantee of success. Thus, in silico selection of high-speed ribozymes can be employed using secondary RNA structure computation based on the partition function of the RNA folding in combination with random search algorithms. This approach has already been proven very accurate in designing allosteric hammerhead ribozymes. Herein, we present two programs for the computational design of allosteric ribozymes sensing randomized oligonucleotides based on the extended version of the hammerhead ribozyme. A Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with NOT logic function (GHOST-NOT) and a Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with YES logic function (GHOST-YES) for computational design of high-speed allosteric ribozymes are described. The allosteric hammerhead ribozymes had a high self-cleavage rate of about 1.8 per minute and were very selective in sensing an effector sequence.


Subject(s)
Biosensing Techniques , RNA, Catalytic , RNA, Catalytic/genetics , RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Oligonucleotides , Base Sequence , Binding Sites , Algorithms , Nucleic Acid Conformation
3.
Comput Biol Med ; 145: 105469, 2022 06.
Article in English | MEDLINE | ID: mdl-35398809

ABSTRACT

Designing oligonucleotide-sensing ribozymes using computational approaches is advantageous to in vitro selection methods for efficiency and accuracy. Allosteric ribozymes can be computationally designed for various applications in gene therapy, designer gene control systems, biosensors, and molecular computation. Here we present two programs, the allosteric Ribozyme Generator (RG) and the Inverse Folding Ribozyme Generator (IFRG), engineered to generate allosteric ribozymes with YES logic. The RG computes allosteric ribozyme sequences' secondary structure using the minimal sequence of the hammerhead ribozyme by inserting oligonucleotide binding site (OBS) elements in the second stem. The IFRG program uses inverse folding to generate allosteric ribozyme sequences with OBS bearing distinct sequences and similar folding. For the generation of the OBS sequences, random search algorithms are employed. Allosteric ribozyme sequences generated by the RG can be used as a matrix for the IFRG program. This approach applies RNA-folding algorithms based on applying thermodynamic parameters using the partition function of the RNAfold, and the RNAinverse source codes from the Vienna RNA folding package. The two algorithms apply dynamic programming and random search algorithms to generate in silico allosteric ribozymes with predefined properties within minutes using a personal computer with over 90% accuracy, without high computation power as experimentally validated and published by us previously.


Subject(s)
RNA, Catalytic , Algorithms , Nucleic Acid Conformation , Oligonucleotides/metabolism , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Software
4.
Article in English | MEDLINE | ID: mdl-32248122

ABSTRACT

The RSwitch is a MySQL database implemented on a PHP-based server, which also provides various useful tools for analyses of DNA, RNA, and protein sequences applying a user-friendly interface. The RSwitch database currently contains information and annotations of 215 bacterial riboswitches from 16 different types found in 50 human pathogenic bacteria. The riboswitch classes include those sensing FMN, glmS, Cobalamin, Lysine, SAH, SAM, Purine, TPP, c-di-GMPI, c-di-GMPII, Moco, PreQ1, Fluoride, Glycine, Mg2+, and Mn2+ type of bacterial riboswitches. The database provides information about the riboswitch aptamer sequences, the thermodynamic ensemble of the RNA structures on partition function and on free minimum energy function. Additionally, the database presents the centroid structure and the positional entropy for each position of the aptamer sequences. The database also provides the biochemical pathways in which the riboswitches are involved in, as well as multiple sequence alignments, multi-drug resistance bacterial strains and consensus motifs for each type of the switches. The RSwitch database is permanently available online without any restrictions. This bioinformatics database provides for the first time all information needed for assessing the suitability of the presented riboswitches as antibacterial drug targets.


Subject(s)
Anti-Bacterial Agents/pharmacology , Computational Biology/methods , Databases, Genetic , RNA, Bacterial , Riboswitch , Bacteria/drug effects , Bacteria/genetics , Bacterial Infections/microbiology , Humans , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Riboswitch/drug effects , Riboswitch/genetics
5.
Gene ; 708: 38-48, 2019 Aug 05.
Article in English | MEDLINE | ID: mdl-31128223

ABSTRACT

Riboswitches are gene control elements that directly bind to specific ligands to regulate gene expression without the need for proteins. They are found in all three domains of life, including Bacteria, Archaea, and Eukaryota. Riboswitches are mostly spread in bacteria and archaea. In this paper, we discuss the general distribution, structure, and function of 28 different riboswitch classes as we focus our attention on riboswitches in bacteria. Bacterial riboswitches regulate gene expression by four distinct mechanisms. They regulate the expression of a limited number of genes. However, most of these genes are responsible for the synthesis of essential metabolites without which the cell cannot function. Therefore, riboswitch distribution is also important for antibacterial drug development.


Subject(s)
Bacteria/genetics , Gene Expression Regulation, Bacterial/genetics , RNA, Bacterial/genetics , Riboswitch/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Drug Development , Gene Expression Regulation, Bacterial/drug effects , RNA, Bacterial/antagonists & inhibitors , Riboswitch/drug effects
6.
Front Plant Sci ; 10: 348, 2019.
Article in English | MEDLINE | ID: mdl-30984217

ABSTRACT

The tomato pedicel abscission zone (AZ) is considered a model system for flower and fruit abscission development, activation, and progression. O-glycosylated proteins such as the Arabidopsis IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) peptide and Arabinogalactan proteins (AGPs) which undergo proline hydroxylation were demonstrated to participate in abscission regulation. Considering that the frequency of occurrence of proline hydroxylation might determine the structure as well the function of such proteins, the expression of a tomato prolyl 4 hydroxylase, SlP4H3 (Solanum lycopersicum Prolyl 4 Hydroxylase 3) was suppressed in order to investigate the physiological significance of this post-translational modification in tomato abscission. Silencing of SlP4H3 resulted in the delay of abscission progression in overripe tomato fruits 90 days after the breaker stage. The cause of this delay was attributed to the downregulation of the expression of cell wall hydrolases such as SlTAPGs (tomato abscission polygalacturonases) and cellulases as well as expansins. In addition, minor changes were observed in the mRNA levels of two SlAGPs and one extensin. Moreover, structural changes were observed in the silenced SlP4H3AZs. The fracture plane of the AZ was curved and not along a line as in wild type and there was a lack of lignin deposition in the AZs of overripe fruits 30 days after breaker. These results suggest that proline hydroxylation might play a role in the regulation of tomato pedicel abscission.

7.
Article in English | MEDLINE | ID: mdl-29993817

ABSTRACT

The Essential Bioinformatics Web Services (EBWS) are implemented on a new PHP-based server that provides useful tools for analyses of DNA, RNA, and protein sequences applying a user-friendly interface. Nine Web-based applets are currently available on the Web server. They include reverse complementary DNA and random DNA/RNA/peptide oligomer generators, a pattern sequence searcher, a DNA restriction cutter, a prokaryotic ORF finder, a random DNA/RNA mutation generator. It also includes calculators of melting temperature (TM) of DNA/DNA, RNA/RNA, and DNA/RNA hybrids, a guide RNA (gRNA) generator for the CRISPR/Cas9 system and an annealing temperature calculator for multiplex PCR. The pattern-searching applet has no limitations in the number of motif inputs and applies a toolbox of Regex quantifiers that can be used for defining complex sequence queries of RNA, DNA, and protein sequences. The DNA enzyme digestion program utilizes a large database of 1502 restriction enzymes. The gRNA generator has a database of 25 bacterial genomes searchable for gRNA target sequences and has an option for searching in any genome sequence given by the user. All programs are permanently available online at http://penchovsky.atwebpages.com/applications.php without any restrictions.

8.
Front Chem ; 6: 3, 2018.
Article in English | MEDLINE | ID: mdl-29441347

ABSTRACT

Pyridine 2,4-dicarboxylic acid is a structural analog of 2-oxoglutarate and is known to inhibit 2-oxoglutare-dependent dioxygenases. The effect of this inhibitor in tomato seedlings grown in MS media supplied with various concentrations of PDCA was investigated, resulting in shorter roots and hypocotyls in a dose-dependent manner. The partial inhibition of growth in roots was more drastic compared to hypocotyls and was attributed to a decrease in the elongation of root and hypocotyl cells. Concentrations of 100 and 250 µM of PDCA decreased hydroxyproline content in roots while only the 250 µM treatment reduced the hydroxyproline content in shoots. Seedlings treated with 100 µM PDCA exhibited enhanced growth of hypocotyl and cotyledon cells and higher hydroxyproline content resulting in cotyledons with greater surface area. However, no alterations in hypocotyl length were observed. Prolyl 4 hydroxylases (P4Hs) are involved in the O-glycosylation of AGPs and were also highly expressed during seedling growth. Moreover PDCA induced a decrease in the accumulation of HRGPs and particularly in AGPs-bound epitopes in a dose dependent-manner while more drastic reduction were observed in roots compared to shoots. In addition, bulged root epidermal cells were observed at the high concentration of 250 µM which is characteristic of root tissues with glycosylation defects. These results indicate that PDCA induced pleiotropic effects during seedling growth while further studies are required to better investigate the physiological significance of this 2-oxoglutarate analog. This pharmacological approach might be used as a tool to better understand the physiological significance of HRGPs and probably P4Hs in various growth and developmental programs in plants.

9.
Front Plant Sci ; 7: 1234, 2016.
Article in English | MEDLINE | ID: mdl-27625653

ABSTRACT

Tomato fruit ripening is a complex developmental programme partly mediated by transcriptional regulatory networks. Several transcription factors (TFs) which are members of gene families such as MADS-box and ERF were shown to play a significant role in ripening through interconnections into an intricate network. The accumulation of large datasets of expression profiles corresponding to different stages of tomato fruit ripening and the availability of bioinformatics tools for their analysis provide an opportunity to identify TFs which might regulate gene clusters with similar co-expression patterns. We identified two TFs, a SlWRKY22-like and a SlER24 transcriptional activator which were shown to regulate modules by using the LeMoNe algorithm for the analysis of our microarray datasets representing four stages of fruit ripening, breaker, turning, pink and red ripe. The WRKY22-like module comprised a subgroup of six various calcium sensing transcripts with similar to the TF expression patterns according to real time PCR validation. A promoter motif search identified a cis acting element, the W-box, recognized by WRKY TFs that was present in the promoter region of all six calcium sensing genes. Moreover, publicly available microarray datasets of similar ripening stages were also analyzed with LeMoNe resulting in TFs such as SlERF.E1, SlERF.C1, SlERF.B2, SLERF.A2, SlWRKY24, SLWRKY37, and MADS-box/TM29 which might also play an important role in regulation of ripening. These results suggest that the SlWRKY22-like might be involved in the coordinated regulation of expression of the six calcium sensing genes. Conclusively the LeMoNe tool might lead to the identification of putative TF targets for further physiological analysis as regulators of tomato fruit ripening.

10.
J Virol ; 86(20): 11163-70, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22855495

ABSTRACT

Coxsackievirus A9 (CAV9), a member of the Picornaviridae family, uses an RGD motif in the VP1 capsid protein to bind to integrin αvß6 during cell entry. Here we report that two CAV9 isolates can bind to the heparan sulfate/heparin class of proteoglycans (HSPG). Sequence analysis identified an arginine (R) at position 132 in VP1 in these two isolates, rather than a threonine (T) as seen in the nonbinding strains tested. We introduced a T132R substitution into the HSPG-nonbinding strain Griggs and recovered infectious virus capable of binding to immobilized heparin, unlike the parental Griggs strain. The known CAV9 structure was used to identify the location of VP1 position 132, 5 copies of which were found to cluster around the 5-fold axis of symmetry, presumably producing a region of positive charge which can interact with the negatively charged HSPG. Analysis of several enteroviruses of the same species as CAV9, Human enterovirus B (HEV-B), identified examples from 5 types in which blocking of infection by heparin was coincident with an arginine (or another basic amino acid, lysine) at a position corresponding to 132 in VP1 in CAV9. Together, these data show that membrane-associated HSPG can serve as a (co)receptor for some CAV9 and other HEV-B strains and identify symmetry-related clustering of positive charges as one mechanism by which HSPG binding can be achieved. This is a potentially powerful mechanism by which a single amino acid change could generate novel receptor binding capabilities, underscoring the plasticity of host-cell interactions in enteroviruses.


Subject(s)
Capsid Proteins/metabolism , Enterovirus B, Human/genetics , Enterovirus B, Human/metabolism , Heparan Sulfate Proteoglycans/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigens, Neoplasm/metabolism , Binding Sites , Cell Line , Chlorocebus aethiops , Enterovirus Infections/metabolism , Integrins/metabolism , Molecular Sequence Data , Protein Binding , Receptors, Virus/metabolism , Sequence Analysis, RNA , Static Electricity
11.
Photosynth Res ; 103(3): 183-94, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20224939

ABSTRACT

CP12, a small intrinsically unstructured protein, plays an important role in the regulation of the Calvin cycle by forming a complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An extensive search in databases revealed 129 protein sequences from, higher plants, mosses and liverworts, different groups of eukaryotic algae and cyanobacteria. CP12 was identified throughout the Plantae, apart from in the Prasinophyceae. Within the Chromalveolata, two putative CP12 proteins have been found in the genomes of the diatom Thalassiosira pseudonana and the haptophyte Emiliania huxleyi, but specific searches in further chromalveolate genomes or EST datasets did not reveal any CP12 sequences in other Prymnesiophyceae, Dinophyceae or Pelagophyceae. A species from the Euglenophyceae within the Excavata also appeared to lack CP12. Phylogenetic analysis showed a clear separation into a number of higher taxonomic clades and among different forms of CP12 in higher plants. Cyanobacteria, Chlorophyceae, Rhodophyta and Glaucophyceae, Bryophyta, and the CP12-3 forms in higher plants all form separate clades. The degree of disorder of CP12 was higher in higher plants than in the eukaryotic algae and cyanobacteria apart from the green algal class Mesostigmatophyceae, which is ancestral to the streptophytes. This suggests that CP12 has evolved to become more flexible and possibly take on more general roles. Different features of the CP12 sequences in the different taxonomic groups and their potential functions and interactions in the Calvin cycle are discussed.


Subject(s)
Photosynthesis , Plant Proteins/chemistry , Plants/metabolism , Sequence Analysis, Protein , Algal Proteins/chemistry , Amino Acid Sequence , Eukaryota/genetics , Expressed Sequence Tags , Genome/genetics , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
12.
J Exp Bot ; 59(14): 3975-85, 2008.
Article in English | MEDLINE | ID: mdl-18974062

ABSTRACT

The chloroplast protein CP12 has been shown to regulate the activity of two Calvin cycle enzymes, phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by the reversible formation of a multiprotein complex. In Arabidopsis there are three CP12 genes, CP12-1, CP12-2, and CP12-3, and expression analysis suggested that the function of these proteins may not be restricted to the Calvin cycle. Reverse transcription-PCR analysis was used here to investigate further the expression patterns of the three CP12 Arabidopsis genes together with the genes encoding plastid GAPDH (GAPA-1 and GAPB), PRK (PRK), and plastid NAD-dependent GAPDH (GAPCp1 and GAPCp2) during development, in response to changes in light, temperature, and anaerobic conditions. Expression of the CP12-2 gene was similar to that of the Calvin cycle enzymes PRK and GAPDH. However, this was not the case for CP12-1 and -3 which were both expressed in roots. Analysis of transgenic Arabidopsis lines expressing CP12::GUS fusion constructs revealed that the CP12 genes display different spatiotemporal expression patterns. The CP12-1 gene was expressed in root tips whilst CP12-3::GUS expression was evident throughout the root tissue. The most unexpected finding was that all three CP12 genes were expressed in floral tissues; CP12-1 and CP12-2 expression was detected in the sepals and the style of the flower, while in contrast CP12-3::GUS expression was restricted to the stigma and anthers. Taken together, the data suggest that the redox-sensitive CP12 proteins may have a wider role in non-photosynthetic plastids, throughout the plant life cycle.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Carrier Proteins/genetics , Flowers/genetics , Gene Expression , Multigene Family , Plant Roots/genetics , Arabidopsis/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins/metabolism , Carrier Proteins/metabolism , Flowers/metabolism , Gene Expression/radiation effects , Genes, Reporter , Intracellular Signaling Peptides and Proteins , Light , Organ Specificity , Phylogeny , Plant Roots/metabolism , Plants/classification , Plants/genetics , Protein Transport
SELECTION OF CITATIONS
SEARCH DETAIL
...