ABSTRACT
Tropic acid was synthesized in a good yield and with high enantioselectivity (81% ee) under non-biphasic conditions via the novel hydrolytic dynamic kinetic resolution of racemic 3-phenyl-2-oxetanone (tropic acid ß-lactone) in the presence of a chiral quaternary ammonium phase-transfer catalyst and strongly basic anion exchange resin as the hydroxide ion donor.
ABSTRACT
Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.
Subject(s)
G-Quadruplexes , 5' Untranslated Regions , Animals , Guanine/chemistry , Humans , Mammals/genetics , Protein Biosynthesis , RNA, Messenger/metabolismABSTRACT
Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.
Subject(s)
Aptamers, Nucleotide , Optical Imaging/methods , Proteins/analysis , Actins/analysis , Aptamers, Nucleotide/chemistry , Fluorescent Dyes , HeLa Cells , Humans , Molecular Imaging/methods , RNA/chemistry , Time-Lapse ImagingABSTRACT
Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous ß-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.
Subject(s)
Cytoplasmic Granules/metabolism , Fluorescent Dyes/chemistry , Optical Imaging , RNA, Messenger/metabolism , Actins/chemistry , Actins/metabolism , Aptamers, Nucleotide/chemistry , Cortactin/chemistry , Cortactin/metabolism , Cytoplasmic Granules/chemistry , HeLa Cells , Humans , Molecular Structure , RNA, Messenger/chemistryABSTRACT
Live-cell imaging of mRNA dynamics is increasingly important to understanding spatially restricted gene expression. We recently developed a convenient and versatile method that uses a gene-specific RNA aptamer and a fluorescent probe to enable spatiotemporal imaging of endogenous mRNAs in living cells. The method was validated by live-cell imaging of the endogenous mRNA of ß-actin. The new RNA-imaging technology might be useful for live-cell imaging of any RNA molecules.
Subject(s)
Aptamers, Nucleotide/chemistry , Fluorescent Dyes/chemistry , Imaging, Three-Dimensional/methods , RNA/metabolism , Base Sequence , Cloning, Molecular , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Reverse TranscriptionABSTRACT
The highly selective mono-C-allylation of oligoglycinates such as a diethylenetriaminepentaacetate, an iminodiacetate, and an ethylenediaminetetraacetate via insertion of a vacuum operation between the N-allylation and C-migration steps is reported. It is contrastive that one-pot N-allylation-C-allylation procedure gave a mixture including multiallylated products. In the reaction with N-ylides, gem-C-diallylation and α,α'-C-diallylation of oligoglycinates are strongly inhibited even with the use of an excess of allyl bromide and base. A mechanism to explain this control of the frequency of C-allylation on oligoglycinates via N-ylides is also proposed.
