Microfluidic devices were designed to perform on micromoles of biological macromolecules and viruses the search and the optimization of crystallization conditions by counter-diffusion, as well as the on-chip analysis of crystals by X-ray diffraction. Chips composed of microchannels were fabricated in poly-dimethylsiloxane (PDMS), poly-methyl-methacrylate (PMMA) and cyclo-olefin-copolymer (COC) by three distinct methods, namely replica casting, laser ablation and hot embossing. The geometry of the channels was chosen to ensure that crystallization occurs in a convection-free environment. The transparency of the materials is compatible with crystal growth monitoring by optical microscopy. The quality of the protein 3D structures derived from on-chip crystal analysis by X-ray diffraction using a synchrotron radiation was used to identify the most appropriate polymers. Altogether the results demonstrate that for a novel biomolecule, all steps from the initial search of crystallization conditions to X-ray diffraction data collection for 3D structure determination can be performed in a single chip.

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5P-terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a`transzyme' molecule, the autocatalytic activity of which liberates a 5P-OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNA yr , starting with 5P-C I , and operates as well for yeast tRNA esp with 5P-U I . Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k t and K m parameters quasi identical to those of their phosphorylated counterparts.z 1998 Federation of European Biochemical Societies.