Epigenetherapy, a new concept

2011 ◽  
Vol 2 (3) ◽  
pp. 127-134
Author(s):  
Tiia Husso ◽  
Mikko P. Turunen ◽  
Nigel Parker ◽  
Seppo Ylä-Herttuala
Keyword(s):  
Gene Expression ◽  
Small Rnas ◽  
Basic Research ◽  
Clinical Applications ◽  
Regulate Gene Expression ◽  
Endogenous Gene ◽  
Rna Molecules ◽  
Regulate Gene

AbstractSmall RNAs have been shown to regulate gene transcription by interacting with the promoter region and modifying the histone code. The exact mechanism of function is still unclear but the feasibility to activate or repress endogenous gene expression with small RNA molecules has already been demonstrated in vitro and in vivo. In traditional gene therapy non-mutated or otherwise useful genes are inserted into patient's cells to treat a disease. In epigenetherapy the action of small RNAs is utilized by delivering only the small RNAs to patient's cells where they then regulate gene expression by epigenetic mechanisms. This method could be widely useful not only for basic research but also for clinical applications of small RNAs.

scholarly journals Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

2020 ◽  
Author(s):  
Clara D. Wang ◽  
Rachel Mansky ◽  
Hannah LeBlanc ◽  
Chandra M. Gravel ◽  
Katherine E. Berry
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Binding Energetics ◽  
Response To Stress ◽  
First Time ◽  
Regulate Gene

ABSTRACTNon-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

scholarly journals In Vivo Behavior of the Tandem Glycine Riboswitch in Bacillus subtilis

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Arianne M. Babina ◽  
Nicholas E. Lea ◽  
Michelle M. Meyer
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Regulate Gene Expression ◽  
Binding Domains ◽  
Expression Platform ◽  
The Impact ◽  
Organismal Fitness ◽  
Regulate Gene

ABSTRACT In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous in vitro studies, the in vivo behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in Bacillus subtilis using β-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the B. subtilis genome. Our results indicate that glycine does not need to bind both aptamers for regulation in vivo and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in B. subtilis, the glycine riboswitch-regulated gcvT operon is important for glycine detoxification. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets.

scholarly journals Enhancers Predominantly Regulate Gene Expression in vivo Via Transcription Initiation

2019 ◽  
Author(s):  
Martin Stephen Charles Larke ◽  
Takayuki Nojima ◽  
Jelena Telenius ◽  
Jacqueline A. Sharpe ◽  
Jacqueline A. Sloane-Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Regulate Gene Expression ◽  
Regulate Gene

scholarly journals Small RNAs with 5′-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica

2008 ◽  
Vol 4 (11) ◽  
pp. e1000219 ◽  
Author(s):  
Hanbang Zhang ◽  
Gretchen M. Ehrenkaufer ◽  
Justine M. Pompey ◽  
Jason A. Hackney ◽  
Upinder Singh
Keyword(s):  
Gene Expression ◽  
Entamoeba Histolytica ◽  
Small Rnas ◽  
Related Protein ◽  
Regulate Gene Expression ◽  
Regulate Gene

scholarly journals 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Polychlorinated Biphenyl Coexposure Alters the Expression Profile of MicroRNAs in the Liver Associated with Atherosclerosis

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Qiuli Shan ◽  
Fan Qu ◽  
Ningning Chen
Keyword(s):  
Small Rnas ◽  
Polychlorinated Biphenyl ◽  
System Development ◽  
Complex Mixtures ◽  
Regulate Gene Expression ◽  
Significant Expression ◽  
And Function ◽  
Function Network ◽  
Regulate Gene

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent organic pollutants that exist as complex mixtures in vivo. When humans are simultaneously exposed to these compounds, the development of atherosclerosis is known to be enhanced. However, the roles of miRNA in TCDD- and PCB-induced atherosclerosis are largely unknown. Therefore, the present study is aimed at elucidating the possible dysregulation of miRNAs in atherogenesis induced by coexposure to TCDD and PCBs. Eight-week-old male ApoE-/- mice were coexposed to TCDD (15 μg/kg) and Aroclor1254 (55 mg/kg, a representative mixture of PCBs) by intraperitoneal injection four times over a 6-week period. Microarray analysis of miRNAs and mRNAs in the liver of ApoE-/- mice with or without TCDD and Aroclor1254 coexposure was performed. We discovered that 68 miRNAs and 1312 mRNAs exhibited significant expression changes in response to TCDD and PCB coexposure and revealed that both changed miRNAs and mRNAs are involved in cardiovascular disease processes. An integrated miRNA-mRNA approach indicated that miRNA-26a-5p, miRNA-193a-3p, and miRNA-30c-5p participated in specific TCDD and Aroclor1254 coresponsive networks which are relevant to the cardiovascular system development and function network. Furthermore, our results also indicated that miRNA-130a-3p and miRNA-376a-3p were novel players in the regulation of TCDD- and Aroclor1254-induced atherosclerosis pathways. In summary, our finding provided new insights into the mechanism of atherosclerosis in response to TCDD and PCB coexposure.

scholarly journals Dietary microRNA—A Novel Functional Component of Food

2019 ◽  
Vol 10 (4) ◽  
pp. 711-721 ◽  
Author(s):  
Lin Zhang ◽  
Ting Chen ◽  
Yulong Yin ◽  
Chen-Yu Zhang ◽  
Yong-Liang Zhang
Keyword(s):  
Gene Expression ◽  
Small Rnas ◽  
Biological Significance ◽  
Circulatory System ◽  
Physiological Effects ◽  
Biological Processes ◽  
Regulate Gene Expression ◽  
Functional Component ◽  
Health And Disease ◽  
Regulate Gene

ABSTRACT MicroRNAs are a class of small RNAs that play essential roles in various biological processes by silencing genes. Evidence emerging in recent years suggests that microRNAs in food can be absorbed into the circulatory system and organs of humans and other animals, where they regulate gene expression and biological processes. These food-derived dietary microRNAs may serve as a novel functional component of food, a role that has been neglected to date. However, a significant amount of evidence challenges this new concept. The absorption, stability, and physiological effects of dietary microRNA in recipients, especially in mammals, are currently under heavy debate. In this review, we summarize our current understanding of the unique characteristics of dietary microRNAs and concerns about both the mechanistic and methodological basis for studying the biological significance of dietary microRNAs. Such efforts will benefit continuing investigations and offer new perspectives for the interpretation of the roles of dietary microRNA with respect to the health and disease of humans and animals.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brent Townshend ◽  
Joy S. Xiang ◽  
Gabriel Manzanarez ◽  
Eric J. Hayden ◽  
Christina D. Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2020 ◽  
Author(s):  
Brent Townshend ◽  
Joy Xiang ◽  
Gabriel Manzanarez ◽  
Eric Hayden ◽  
Christina Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Using aptamer-coupled ribozyme libraries and a novel ribozyme regeneration method, we developed de novo rapid in vitro evolution of RNA biosensors (DRIVER) that enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identified and validated biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors were applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors were also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals SIRNA-MEDIATED INHIBITION OF INTERLEUKINE-13 PRODUCTION IN VITRO

2012 ◽  
Vol 9 (6) ◽  
pp. 24-27
Author(s):  
I P Shilovskiy ◽  
D V Mazurov ◽  
N N Shershakova ◽  
M R Khaitov
Keyword(s):  
Gene Expression ◽  
Protein Product ◽  
Spleen Cells ◽  
Rna Molecules ◽  
In Vitro Methods ◽  
Gene Coding ◽  
Interfering Rna

Background. According to current views, one of the major mediators involved in the development of allergic process is IL-13. The goal of this work was to design small interfering RNA molecules to effectively inhibit il-13 gene expression of mice in experiments in vitro. Methods. For the expression of IL-13 in in vitro gene coding sequence il-13 was amplified using cDNA ConA-stimulated spleen cells from BALE / c mice as a template and cloned into the expression vector pUCHR IRES GFP. Using a computer analysis were designed six variants of siRNA, directed against mRNA-il-13. To test the efficiency of siRNA a co-transfection of 1x 105 cells HEK293T mixture (0,5 mg and 1 mg of plasmid siRNA) coupled with Lipofectamine 2000 reagent was carried out. Twenty-four hours later, the gene expression changes in il-13 recorded by flow cytometry on the fluorescence intensity of GFP+-cells. Gene expression of il-13 mRNA was assessed by quantitative PCR, and the level of the protein product by ELISA. results. As a result, siRNA molecules were obtained and three of them were able to effectively inhibit the gene expression of il-13. Conclusion. Thereby variants of siRNA, which can effectively inhibit the production of mice’s IL-13 in vitro; can be used later in experiments in vivo so to understand the role of IL-13 in the pathogenesis of allergic conditions as to develop new therapy approaches.

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