Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester.

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0-8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39-85% yield. This method has potential uses in biotechnology and chemical biology.

Single cell analysis using a glass microchamber for studying movement fluctuations of Navicula pavillardii and Seminavis robusta diatom cells.

We propose a new technique for quantitative trajectory analysis of gliding phenomenon of Navicula pavillardii (NP) and Seminavis robusta (SR) diatom cells by single cell observation using a glass microchamber in this short technical note. Two-dimensional trajectory analysis of cell movements was used to determine the angular velocity, velocity, and migration distances of the diatom movement. Based on the trajectory analysis, we found that asymmetrically shaped SR had a larger angular velocity with large fluctuations compared to symmetrically shaped NP, although the velocity of SR was less than that of NP. It suggests that lateral frictional force in a culture medium is an important factor for diatom movements. Our results revealed that the single cell observation using a glass microchamber is effective on quantitative analysis of angular velocity of diatom gliding.