Hairpin formation by single-stranded DNA molecules was exploited in a DNA-based computation in order to explore the feasibility of autonomous molecular computing. An instance of the satisfiability problem, a famous hard combinatorial problem, was solved by using molecular biology techniques. The satisfiability of a given Boolean formula was examined autonomously, on the basis of hairpin formation by the molecules that represent the formula. This computation algorithm can test several clauses in the given formula simultaneously, which could reduce the number of laboratory steps required for computation.

A suppressor tRNA^Tyr and mutant tyrosyl‐tRNA synthetase (TyrRS) pair was developed to incorporate 3‐iodo‐l‐tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA^Tyr gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA^Tyr, which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3‐iodo‐l‐tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA^Tyr, while 3‐iodo‐l‐tyrosine was supplied in the growth medium. 3‐Iodo‐l‐tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3‐iodo‐l‐tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline‐regulated promoter.

Tyrosyl–tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo- l -tyrosine rather than l -tyrosine for the site-specific incorporation of 3-iodo- l -tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo- l -tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the l -tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo- l -tyrosine 10-fold more efficiently than l -tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA ^Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo- l -tyrosine. After the use of the 3-iodotyrosyl–tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of l -tyrosine in the translation. Therefore, the variant enzyme, 3-iodo- l -tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10¹² random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 µM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K_d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.