scholarly journals Proof of concept continuous event logging in living cells

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Dna Sequences ◽  
Rna Binding ◽  
Attachment Site ◽  
Genetic Circuit ◽  
Genetic Changes ◽  
Guide Rna ◽  
Molecular Events ◽  
Long Read

AbstractCells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.

scholarly journals Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Dna Sequences ◽  
Dna Recombination ◽  
Attachment Site ◽  
Memory Systems ◽  
Rna Sequences ◽  
Guide Rna ◽  
Future Goals ◽  
Attachment Sites ◽  
Synthetic Cell ◽  
Specific Pair

AbstractSerine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.

scholarly journals Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

2016 ◽  
pp. 157-175 ◽  
Author(s):  
Colleen M. Connelly ◽  
Fardokht A. Abulwerdi ◽  
John S. Schneekloth
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Rna Binding

scholarly journals Target-Directed Approaches for Screening Small Molecules against RNA Targets

2020 ◽  
Vol 25 (8) ◽  
pp. 869-894 ◽  
Author(s):  
Hafeez S. Haniff ◽  
Laurent Knerr ◽  
Jonathan L. Chen ◽  
Matthew D. Disney ◽  
Helen L. Lightfoot
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Drug Targets ◽  
Industrial Development ◽  
Rna Binding ◽  
Cellular Functions ◽  
Rna Molecules ◽  
The Face ◽  
Rna Binders ◽  
The Right

RNA molecules have a variety of cellular functions that can drive disease pathologies. They are without a doubt one of the most intriguing yet controversial small-molecule drug targets. The ability to widely target RNA with small molecules could be revolutionary, once the right tools, assays, and targets are selected, thereby defining which biomolecules are targetable and what constitutes drug-like small molecules. Indeed, approaches developed over the past 5–10 years have changed the face of small molecule–RNA targeting by addressing historic concerns regarding affinity, selectivity, and structural dynamics. Presently, selective RNA–protein complex stabilizing drugs such as branaplam and risdiplam are in clinical trials for the modulation of SMN2 splicing, compounds identified from phenotypic screens with serendipitous outcomes. Fully developing RNA as a druggable target will require a target engagement-driven approach, and evolving chemical collections will be important for the industrial development of this class of target. In this review we discuss target-directed approaches that can be used to identify RNA-binding compounds and the chemical knowledge we have today of small-molecule RNA binders.

scholarly journals Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

2017 ◽  
Vol 114 (21) ◽  
pp. 5443-5448 ◽  
Author(s):  
Vladimir Mekler ◽  
Leonid Minakhin ◽  
Konstantin Severinov
Keyword(s):  
Dna Sequences ◽  
Rna Binding ◽  
Proximal Region ◽  
Target Sequence ◽  
Duplex Dna ◽  
Rna Molecules ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Dna Complex

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

scholarly journals Targeting RNA with Small Molecules: Identification of Selective, RNA-Binding Small Molecules Occupying Drug-Like Chemical Space

2019 ◽  
Vol 25 (4) ◽  
pp. 384-396 ◽  
Author(s):  
Noreen F. Rizvi ◽  
John P. Santa Maria ◽  
Ali Nahvi ◽  
Joel Klappenbach ◽  
Daniel J. Klein ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Small Molecules ◽  
Small Molecule ◽  
Rna Binding ◽  
Chemical Space ◽  
Chemical Properties ◽  
Identification System ◽  
Specific Chemical ◽  
Ligand Identification ◽  
Molecule Interactions

Although the potential value of RNA as a target for new small molecule therapeutics is becoming increasingly credible, the physicochemical properties required for small molecules to selectively bind to RNA remain relatively unexplored. To investigate the druggability of RNAs with small molecules, we have employed affinity mass spectrometry, using the Automated Ligand Identification System (ALIS), to screen 42 RNAs from a variety of RNA classes, each against an array of chemically diverse drug-like small molecules (~50,000 compounds) and functionally annotated tool compounds (~5100 compounds). The set of RNA–small molecule interactions that was generated was compared with that for protein–small molecule interactions, and naïve Bayesian models were constructed to determine the types of specific chemical properties that bias small molecules toward binding to RNA. This set of RNA-selective chemical features was then used to build an RNA-focused set of ~3800 small molecules that demonstrated increased propensity toward binding the RNA target set. In addition, the data provide an overview of the specific physicochemical properties that help to enable binding to potential RNA targets. This work has increased the understanding of the chemical properties that are involved in small molecule binding to RNA, and the methodology used here is generally applicable to RNA-focused drug discovery efforts.

scholarly journals Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

2014 ◽  
Vol 136 (23) ◽  
pp. 8402-8410 ◽  
Author(s):  
Joanna Sztuba-Solinska ◽  
Shilpa R. Shenoy ◽  
Peter Gareiss ◽  
Lauren R. H. Krumpe ◽  
Stuart F. J. Le Grice ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Rna Binding ◽  
Biologically Active ◽  
Tar Rna

scholarly journals Systematic analysis of the interactions driving small molecule–RNA recognition

2020 ◽  
Vol 11 (7) ◽  
pp. 802-813 ◽  
Author(s):  
G. Padroni ◽  
N. N. Patwardhan ◽  
M. Schapira ◽  
A. E. Hargrove
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
Rna Binding ◽  
Rna Recognition ◽  
Systematic Analysis

This study underscores privileged interactions for RNA binding small molecules, an emerging focus in drug discovery.

scholarly journals STEM-20. A CANCER STEM CELL-SELECTIVE APOPTOSIS-INDUCING SMALL MOLECULE FOR THE TREATMENT OF GBM

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii200-ii200
Author(s):  
Stephen Skirboll ◽  
Natasha Lucki ◽  
Genaro Villa ◽  
Naja Vergani ◽  
Michael Bollong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Small Molecules ◽  
Small Molecule ◽  
Large Scale ◽  
Critical Role ◽  
Tumor Formation ◽  
Cell Type ◽  
Caspase 1 ◽  
Activation Assay

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

scholarly journals Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ahmed Al Qaffas ◽  
Salvatore Camiolo ◽  
Mai Vo ◽  
Alexis Aguiar ◽  
Amine Ourahmane ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Sequence Data ◽  
Laboratory Strain ◽  
Serial Passage ◽  
Wild Type Virus ◽  
Protein Coding ◽  
Genetic Changes ◽  
Long Read

AbstractThe advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

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