Polymerization of alpha-hydroxy acids by ribosomes

Over 30 years ago, Fahnestock and Rich reported intriguing data showing the capability of the ribosome to polymerize phenyllactic acid. Although the polymerization was initiated and terminated randomly on polyuridic acids, the given data convincingly suggested that the generated polymer was composed of an approximately 7:3 mixture of phenyllactic acid and phenylalanine. Despite the fact that Fahnestock's conclusion was very likely correct, there have been no reports to follow up the ribosome-catalyzed polymerization of alpha-hydroxy acids until very recently. At the end of 2007, we reported messenger RNA (mRNA)-directed polyester synthesis by using the new emerging method of genetic-code reprogramming in which alpha-hydroxy acids with various kinds of side-chains are assigned to arbitrarily chosen codons. In this work, we have achieved the ribosomal synthesis of polyesters with the sequence composition and length in a fully controlled manner according to the sequence of mRNA. This Concept article describes the background of the method development and its application to the synthesis of polyesters.     

Investigation of fluorine containing plasma activation for room-temperature bonding of Si-based materials

Wafer direct bonding of Si based materials, such as silicon, silicon oxide and silicon nitride, is a generic technique enabling realization of innovative structures in semiconductor industry. In this paper, a fluorine containing plasma activated bonding method is developed to achieve sufficient bonding at room temperature in air ambient with no heating process. The whole process is facile and cost effective without requiring high-vacuum system. It does work well for bonding of Si-based materials except for Si₃N₄/Si₃N₄ bonded pairs. The bonding strengths of specimens prepared by fluorine containing oxygen plasma are significantly improved at room temperature compared with those by oxygen plasma. The improved bonding strength is possibly attributed to the formation of fluorinated oxide layers on wafer surfaces after the plasma treatment.     

Flexizymes: their evolutionary history and the origin of catalytic function

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.     

Exploring the Minimal RNA Substrate of Flexizymes

Flexizymes are tRNA acylation ribozymes that have been successfully used to facilitate genetic code reprogramming. They are capable of charging acid substrates onto various tRNAs and tRNA analogues. However, their minimal RNA substrate has not been investigated. Here we have designed fluorescently labeled short RNAs corresponding to the four, three, and two bases (4bRNA, 3bRNA, 2bRNA) at the tRNA 3′-end and explored the minimal RNA substrate of flexizymes, dFx and eFx. 3bRNA was the observed minimal RNA substrate of the flexizymes, but the efficiency of acylation of this short RNA was two to three times lower than that of 4bRNA. The efficiency of acylation of 4bRNA was comparable with that of the microhelix, a 22-base RNA conventionally used as a tRNA analogue for analyzing acylation efficiency. We also compared the efficiencies of acylation of the microhelix and 4bRNA with various acid substrates. Thanks to the short length of 4bRNA, its acyl-4bRNA products exhibited larger mobility shifts in gel electrophoresis than those exhibited by acyl-microhelix products with every substrate tested. This indicated that 4bRNA was an ideal RNA substrate for analyzing the efficiency of acylation by flexizymes.     

Saprophytic pleuritis with Candida parapsilosis

A 73-year-old man was admitted to our hospital with pneumonia in the right S6 induced by Streptococcus milleri and with left pleural thickening. He had histories of diabetes mellitus for 30 years and pulmonary tuberculosis 35 years ago. The pneumonia resolved completely after administration of ceftazidime and clindamycin for 10 days, but the pleural thickening remained and computed tomography revealed that it was an encapsulated effusion without calcification. An aspirate was turbid yellow with a high concentration of lipids, and consisted of dominant crystals and scattered cells, 80% of which were yeasts and 20% of which were macrophages phagocytizing them. Only Candida parapsilosis developed in culture. The same silent pleural shadow was identified on chest X-ray films obtained over the previous 7 years. The persistent pleuritis was diagnosed as saprophytic infection with C. parapsilosis.     

NMDA-mediated facilitation in the echo-delay tuned areas of the auditory cortex of the mustached bat

We recorded the responses of single delay-tuned neurons in the dorsal fringe (DF) area and the FM-FM area of the auditory cortex of the mustached bat using multi-barreled carbon-fiber electrodes. An iontophoretic application of N-methyl-D-aspartate (NMDA) or kainate (KA) to a DF neuron evoked a burst of discharges from the neuron. The burst of discharges evoked by NMDA was always smaller than that evoked by KA. Simultaneous application of D-2-Amino-5-phosphonovalerate (APV) with NMDA and KA abolished the NMDA-evoked but not the KA-evoked discharges. APV did not evoke any significant changes in the auditory responses of 43 out of the 47 delay-tuned neurons studied in the DF area, and in all 20 neurons studied in the FM-FM area. In the remaining four DF neurons, however, APV either increased the initial discharges of their auditory response or decreased the late discharges of their response. These results indicate that in the majority of neurons in the DF and FM-FM areas NMDA receptors do not play a significant role in the processing of target-distance information, and that their facilitative auditory responses are basically created by synaptic interactions occurring in the subcortical auditory nuclei.     

A new preparative method for cyclohexenyl alkenyl ketones

New cyclohexenyl alkenyl ketones are prepared by the reaction of various methyl cyclohexenyl carboxylates and vinylmagnesium chloride; for example, 1-(4′-methyl-3′-cyclohexene-1′-yl)-1-oxo-4-pentene is obtained from methyl 4-methyl-3-cyclohexene-1-carboxylate and vinylmagnesium chloride.