Dataset Information

Expanding the Chemogenetic Toolbox by Circular Permutation.

ABSTRACT: To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB-FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field.

SUBMITTER: Lee YT

PROVIDER: S-EPMC7710497 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Expanding the Chemogenetic Toolbox by Circular Permutation.

Lee Yi-Tsang YT He Lian L Zhou Yubin Y

Journal of molecular biology 20200408 10

To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB-FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID sy ...[more]

PMID: 32277990

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Project description:Genome-wide association studies (GWAS) aim to detect single nucleotide polymorphisms (SNP) associated with trait variation. However, due to the large number of tests, standard analysis techniques impose highly stringent significance thresholds, leaving potentially associated SNPs undetected, and much of the trait genetic variation unexplained. Pathway- and network-based methodologies applied to GWAS aim to detect associations missed by standard single-marker approaches. The complex and non-random architecture of the genome makes it a challenge to derive an appropriate testing framework for such methodologies. We developed a rapid and simple permutation approach that uses GWAS SNP association results to establish the significance of pathway associations while accounting for the linkage disequilibrium structure of SNPs and the clustering of functionally related elements in the genome. All SNPs used in the GWAS are placed in a "circular genome" according to their location. Then the complete set of SNP association P values are permuted by rotation with respect to the genomic locations of the SNPs. Once these "simulated" P values are assigned, the joint gene P values are calculated using Fisher's combination test, and the association of pathways is tested using the hypergeometric test. The circular genomic permutation approach was applied to a human genome-wide association dataset. The data consists of 719 individuals from the ORCADES study genotyped for ~300,000 SNPs and measured for 51 traits ranging from physical to biochemical measurements. KEGG pathways (n = 225) were used as the sets of pathways to be tested. Our results demonstrate that the circular genomic permutations provide robust association P values. The non-permuted hypergeometric analysis generates ~1400 pathway-trait combination results with an association P value more significant than P ≤ 0.05, whereas applying circular genomic permutation reduces the number of significant results to a more credible 40% of that value. The circular permutation software ("genomicper") is available as an R package at http://cran.r-project.org/.

Dataset Information

Expanding the Chemogenetic Toolbox by Circular Permutation.

Publications

Expanding the Chemogenetic Toolbox by Circular Permutation.

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