Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiplexed Enzyme Activity-Based Probe Display via Hybridization.

ABSTRACT: Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼10⁶ primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼10³ gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

SUBMITTER: Cavett V

PROVIDER: S-EPMC7655629 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Multiplexed miRNA northern blots via hybridization chain reaction.

Project description:Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ?1.5 days independent of the number of target RNAs.

| S-EPMC5009741 | biostudies-literature

Fluorescent Mechanism-Based Probe for Aerobic Flavin-Dependent Enzyme Activity.

Project description:Diversity in non-ribosomal peptide and polyketide secondary metabolism is facilitated by interactions between biosynthetic domains with discrete monomer loading and their cognate tailoring enzymes, such as oxidation or halogenation enzymes. The cooperation between peptidyl carrier proteins and flavin-dependent enzymes offers a specialized strategy for monomer selectivity for oxidization of small molecules from within a complex cellular milieu. In an effort to study this process, we have developed fluorescent probes to selectively label aerobic flavin-dependent enzymes. Here we report the preparation and implementation of these tools to label oxidase, monooxygenase, and halogenase flavin-dependent enzymes.

| S-EPMC5656434 | biostudies-literature

Semiautomated multiplexed quantum dot-based in situ hybridization and spectral deconvolution.

Project description:Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.

| S-EPMC2248801 | biostudies-literature

A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.

Project description:The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.

| S-EPMC5953424 | biostudies-literature

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity.

Project description:Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

| S-EPMC7366380 | biostudies-literature

Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe.

Project description:Ubiquitin activity-based probes have proven invaluable in elucidating structural mechanisms in the ubiquitin system by stabilizing transient macromolecular complexes of deubiquitinases, ubiquitin-activating enzymes, and the assemblies of ubiquitin-conjugating enzymes with ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine, allows for the preparative reconstitution and structural analysis of the interactions between ubiquitin and certain HECT ligases. We present a crystal structure of the ubiquitin-linked HECT domain of HUWE1 that defines a catalytically critical conformation of the C-terminal tail of the ligase for the transfer of ubiquitin to an acceptor protein. Moreover, we observe that ubiquitin-propargylamine displays selectivity among HECT domains, thus corroborating the notion that activity-based probes may provide entry points for the development of specific, active site-directed inhibitors and reporters of HECT ligase activities.

| S-EPMC8453484 | biostudies-literature

Correction to "Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity".

Project description: Not available

| S-EPMC7644119 | biostudies-literature

Multiplexed NMR: an automated CapNMR dual-sample probe.

Project description:A new generation of microscale, nuclear magnetic resonance (CapNMR) probe technology employs two independent detection elements to accommodate two samples simultaneously. Each detection element in the dual-sample CapNMR probe (DSP) delivers the same spectral resolution and S/N as in a CapNMR probe configured to accommodate one sample at a time. A high degree of electrical isolation allows the DSP to be used in a variety of data acquisition modes. Both samples are shimmed simultaneously to achieve high spectral resolution for simultaneous data acquisition, or alternatively, a flowcell-specific shim set is readily called via spectrometer subroutines to enable acquisition from one sample while the other is being loaded. An automation system accommodates loading of two samples via dual injection ports on an autosampler and two completely independent flowpaths leading to dedicated flowcells in the DSP probe.

| S-EPMC2932813 | biostudies-literature

Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples.

Project description:We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we "count" the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity-based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester-hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.

| S-EPMC7065886 | biostudies-literature

Single-round, multiplexed antibody mimetic design through mRNA display.

Project description:In a single round: By combining the high-efficiency enrichment through the continuous-flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, the generation of antibody mimetics with a single round of mRNA display is made possible. This approach eliminates iterative selection cycles and provides a path to fully automated ligand generation (see picture).

| S-EPMC3832244 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data