Inhibition of protein synthesis through RNA-based tandem G-quadruplex formation.

We demonstrate that an RNA template containing eight GGG repeat sequences exhibits a unique tandem G-quadruplex structure in which two individual G-quadruplexes are aligned in close proximity. Because of their unexpected stability, tandem G-quadruplexes formed in the coding region of mRNA strands effectively inhibited in vitro protein synthesis.

Novel NanoLuc-type substrates with various C-6 substitutions.

NanoLuc (NLuc)-furimazine bioluminescence system offers several advantages over established systems, including improved stability, smaller size, and >150-fold enhancement in bioluminescence. Herein, we designed and synthesized a series of bioluminescent substrates with varying at the C-6 position of furimazine for NLuc-furimazine bioluminescence system. Among all derivatives, compounds A6 and A11 provided excellent bioluminescence characteristics compared with furimazine in vitro and in vivo. We believe that these new NLuc substrates can broaden the application of NLuc bioluminescence techniques, especially in vivo bioluminescent imaging.

Synthesis and evaluation of the luciferase-oligodeoxynucleotide for the sequence-selective detection of nucleic acids.

A new method for the synthesis of ODN-luciferase conjugate was investigated as a signal-amplifying sensor of the target nucleic acids. The conjugation of the luciferase was successfully achieved between the cysteine residue and the 2-amino-6-vinylpurine nucleoside of the ODN probe without significant inactivation of luciferase. The ODN-luciferase conjugate modified with PEG retained the luciferase activity and selectivity during the hybridization with the target ODN.

Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.

Simultaneous in vitro protein synthesis using solid-phase DNA template.

In vitro protein synthesis is rapidly becoming an accepted tool in functional genomic analysis. We have demonstrated the in vitro synthesis of firefly luciferase on solid-phase template DNA, bound to wells in 96-well plates, using simultaneous transcription and translation in a wheat-germ extract system. The bound DNA template was stable and did not release during transcription. Coupled translation resulted in ca. 1.2 ng/microL luciferase synthesized, which is ca. one-fifth of that synthesized using conventional solution-phase coupled transcription and translation. Reuse of the DNA template was influenced by the complexity of the wheat-germ extract, which resulted in fouling of the transcription surface and reduction of protein synthesis after extended use. The approach developed in this study may enable the development of high-throughput, microscale protein synthesis platforms for use in functional genomic analysis.

Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene.

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .