scholarly journals A convolutional neural network for the prediction and forward design of ribozyme-based gene-control elements

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin M Schmidt ◽  
Christina D Smolke
Keyword(s):  
Machine Learning ◽  
Rational Design ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Regulatory Activity ◽  
Rna Sequences ◽  
Genetic Switch ◽  
Gene Regulatory ◽  
Functional Rnas

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large datasets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the dataset that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.

scholarly journals Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

2016 ◽  
Vol 113 (13) ◽  
pp. E1835-E1843 ◽  
Author(s):  
Mina Fazlollahi ◽  
Ivor Muroff ◽  
Eunjee Lee ◽  
Helen C. Causton ◽  
Harmen J. Bussemaker
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Nonsynonymous Mutation ◽  
Gene Regulatory ◽  
Genetic Modulators

Regulation of gene expression by transcription factors (TFs) is highly dependent on genetic background and interactions with cofactors. Identifying specific context factors is a major challenge that requires new approaches. Here we show that exploiting natural variation is a potent strategy for probing functional interactions within gene regulatory networks. We developed an algorithm to identify genetic polymorphisms that modulate the regulatory connectivity between specific transcription factors and their target genes in vivo. As a proof of principle, we mapped connectivity quantitative trait loci (cQTLs) using parallel genotype and gene expression data for segregants from a cross between two strains of the yeast Saccharomyces cerevisiae. We identified a nonsynonymous mutation in the DIG2 gene as a cQTL for the transcription factor Ste12p and confirmed this prediction empirically. We also identified three polymorphisms in TAF13 as putative modulators of regulation by Gcn4p. Our method has potential for revealing how genetic differences among individuals influence gene regulatory networks in any organism for which gene expression and genotype data are available along with information on binding preferences for transcription factors.

scholarly journals Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

2017 ◽  
Vol 114 (37) ◽  
pp. 9779-9784 ◽  
Author(s):  
Allison H. Squires ◽  
W. E. Moerner
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Rational Design ◽  
Light Adaptation ◽  
De Novo ◽  
Photosynthetic Apparatus ◽  
Emission Spectra ◽  
Free Solution ◽  
Surface Attachment

Phycobilisomes are highly organized pigment–protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment–protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

scholarly journals Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

1998 ◽  
Vol 9 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
Peter Lorenz ◽  
Brenda F. Baker ◽  
C. Frank Bennett ◽  
David L. Spector
Keyword(s):  
Antisense Oligonucleotides ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Phosphorothioate Oligodeoxynucleotides ◽  
Nuclear Structures ◽  
In Cells

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

scholarly journals In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

1999 ◽  
Vol 73 (3) ◽  
pp. 2410-2419 ◽  
Author(s):  
Ramaswamy Raju ◽  
Mustapha Hajjou ◽  
Kristie R. Hill ◽  
Vandana Botta ◽  
Sisir Botta
Keyword(s):  
Sindbis Virus ◽  
De Novo ◽  
Genome Replication ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Cytoplasmic Polyadenylation ◽  
Nontranslated Region ◽  
Rna Genome ◽  
Negative Sense

ABSTRACT Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

scholarly journals Guiding biomolecular interactions in cells using de novo protein-protein interfaces

2018 ◽  
Author(s):  
Abigail J Smith ◽  
Franziska Thomas ◽  
Deborah Shoemark ◽  
Derek N Woolfson ◽  
Nigel J Savery
Keyword(s):  
Rational Design ◽  
De Novo ◽  
Gene Activation ◽  
Low Complexity ◽  
Coiled Coils ◽  
Biomolecular Interactions ◽  
Biomolecular Systems ◽  
Binding Domains ◽  
In Cells

An improved ability to direct and control biomolecular interactions in living cells would impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here we show that low-complexity, de novo designed protein-protein-interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and hetero-dimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase; to recruit RNA polymerase to a promoter and activate gene expression; and to oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

scholarly journals Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and contributes to its activity

2019 ◽  
Vol 47 (13) ◽  
pp. 6726-6736 ◽  
Author(s):  
Elias Akoury ◽  
Guoli Ma ◽  
Segolene Demolin ◽  
Cornelia Brönner ◽  
Manuel Zocco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromosome Segregation ◽  
Genome Stability ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
H3k9 Methylation ◽  
Set Domain ◽  
Nucleosome Core

Abstract Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.

scholarly journals 99mTc Labelling Strategies for the Development of Potential Nitroimidazolic Hypoxia Imaging Agents

Inorganics ◽  
2019 ◽  
Vol 7 (11) ◽  
pp. 128 ◽  
Author(s):  
Giglio ◽  
Rey
Keyword(s):  
Rational Design ◽  
Biological Properties ◽  
Fine Tuning ◽  
Oxidation States ◽  
Hypoxia Imaging ◽  
Biological Target ◽  
99Mtc Radiopharmaceuticals ◽  
99Mtc Labelling

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

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