Biomedical subjects

I Hirao

Publications and source records attributed to I Hirao.

At least 19 recordsLinked to original sources

PubMed2001

Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides.

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.

Base Pairing↗

PubMed2001

Unnatural base pairs for specific transcription.

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

Base Pairing↗

PubMed2001

Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase.

Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity. Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg. We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS. The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.

Amino Acid Sequence↗

PubMed2001

Efforts toward creating unnatural base pairs for an expanded genetic code.

A series of unnatural base pairs was designed and examined for the expansion of the genetic alphabet and for a better understanding of the mechanism of nucleic acid biosyntheses. To improve the shape complementarity of the previously developed unnatural base pairs, 2-amino-6-(N,N-dimethylamino)purine (x)--pyridon-2-one (y) and 2-amino-6-(2-thienyl)purine (s)--y, the pyrimidine analogue, y, was replaced by a five-member ring, 4-imidazolin-2-one (z), and the s-z pairing in replication was examined. Unnatural bases based on the five-member ring were also applied to the development of non-hydrogen-bonded base pairs.

Base Pairing↗

PubMed2001

X-ray analyses of d(GCGAAAGC) and d(GXGAAAGCT), where X = 2'-deoxy-5-iodocytidine.

DNA fragments containing a sequence d(GCGAAAGC) are known to be highly thermostable. To investigate the structural basis for such a specific property, crystallographic studies of the DNA octamer and a nanomer d(GXGAAAGCT) (X = 2'-deoxy-5-iodocytidine) have been performed. The present higher resolution X-ray analyses have shown that both DNA oligomers are stabilized respectively to form a zipper-type duplex homodimer. PAGE of these oligomers, however, indicates that they are monomeric even when their crystals were dissolved at room temperature.

Base Sequence↗

PubMed2001

X-ray analysis of d(GCGAACGC); intra-duplex and inter-duplex hand-in-pocket motifs.

The structure of DNA octamer with the sequence d(GCGAACGC) has been determined by the X-ray crystallography to investigate the specific DNA structural motifs that are useful for designing various functional DNA molecules. Two kinds of interaction motifs for the bulged out adenosine residues have been found. One is an intra-duplex and the other is an inter-duplex hand-in-pocket motif. These interactions consist of the novel A:G pairs that have never been found so far in any crystal and solution studies on DNA molecules.

Base Sequence↗

PubMed2001

Photo-regulation of RNA polymerase reaction by use of modified DNA carrying an azobenzene.

Transcription reaction by T7-RNA polymerase was photo-regulated on the basis of two strategies as depicted in Scheme 1. It was found that incorporation reaction of azobenzene-tethered uridine triphosphate proceeded only when azobenzene took trans-form (Scheme 1(A)). On the other hand, the transcription was more efficient when the azobenzene moiety, tethered to the non-template strand of the promoter DNA, was in its cis-form under UV irradiation (Scheme 1(B)). Thus, azobenzene-tethered DNAs are promising for the photo-regulation of gene-expression.

Azo Compounds↗

PubMed2000

RNA aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin.

Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.

Animals↗

PubMed2000

Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

The unnatural base, 2-amino-6-(2-thienyl)purine (designated as s), instead of 2-amino-6-(N,N-dimethylamino)purine (designated as x), was designed in order to improve the specificity and efficiency of the base pairing with pyridin-2-one (designated as y). DNA fragments containing s were chemically synthesized, and the thermal stability and the enzymatic reactions involving the s-y pairing were examined. Thermal denaturation experiments showed that the DNA duplex (12-mer) containing the s-y pair was more stable than that containing the x-y pair. The incorporation of dyTP was also more advantageous to the s-y pairing than the x-y pairing in single-nucleotide insertion experiments using the Klenow fragment of Escherichia coli DNA polymerase I.

Base Pairing↗

PubMed2000

Synthesis of 6-(2-thienyl)purine nucleoside derivatives toward the expansion of the genetic code.

Unnatural bases specifically pairing with pyridin-2-one, 2-amino-6-(2-thienyl) purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace 2-amino-6-(N,N-dimethylamino)purine. It was expected that these novel purine analogues, as compared with 2-amino-6-(N,N-dimethylamino)purine, might reduce the interference in the stacking interactions with the neighboring bases in a duplex and improve the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite these unnatural bases. The syntheses of these nucleoside derivatives and the DNA fragments were examined.

Base Pairing↗

PubMed1999

Chemical synthesis of novel base pairs and their enzymatic incorporation into DNA.

A novel base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and the counter part, pyridin-2-one (denoted y) were designed. The bulky 6-dimethylamino group of x is expected to eliminate base pairing with all natural bases. The phosphoramidite of x for DNA templates and the 2'-deoxyribonucleoside triphosphate of y (dyTP) for a substrate were synthesized, and the selectivity of the enzymatic incorporation of dyTP opposite x in the templates was examined. dyTP was preferentially incorporated opposite x than canonical dNTPs by Klenow fragment of Escherichia coli DNA polymerase I. While dyTP was also incorporated opposite A and G, the misincorporation was suppressed in the presence of dTTP and dCTP, respectively.

Base Pairing↗

PubMed1998

RNA aptamers that specifically bind to the Ras-binding domain of Raf-1.

RNA aptamers that bind to the Ras-binding domain (RBD) of a proto-oncogene product, Raf-1, were isolated from a pool of random sequences using a glutathione S-transferase-fused RBD (GST-RBD). The RNA molecules bind to the GST-RBD, but not to GST, with dissociation constants of about 300 nM. In contrast, these RNA aptamers do not bind to the Ras-binding domain of the RGL protein, which is also known to be activated by Ras. The aptamers actually compete with Ras for binding to the Raf-1 RBD. The anti-Raf-1 aptamers may be used to specifically inhibit the Ras-Raf interaction in the complicated signaling network in mammalian cells.

Base Sequence↗

PubMed1998

Crystal structures of a series of RNA aptamers complexed to the same protein target.

We have determined the crystal structures, at 2.8 A resolution, of two different RNA aptamers, each bound to MS2 coat protein. One of the aptamers contains a non-Watson-Crick base pair, while the other is missing one of the unpaired adenines that make sequence-specific contacts in the wild-type complex. Despite these differences, the RNA aptamers bind in the same location on the protein as the wild-type translational operator. Comparison of these new structures with other MS2-RNA complexes allows us to refine further the definition of the minimal recognition elements and suggests a possible application of the MS2 system for routine structure determination of small nucleic acid motifs.

Base Pairing↗

PubMed1998

Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution.

The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined. It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem. The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions. In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures.

Asparagine↗

PubMed1997

GNA trinucleotide loop sequences producing extraordinarily stable DNA minihairpins.

d(GCGAAAGC) and d(GCGAAGC) fragments form extraordinarily stable DNA minihairpins containing only two G-C base pairs and a GAAA or GAA loop, respectively, with a Tm of 76 degrees C. These sequences are frequently found in some important regions such as replication origins and promoter regions for transcription. We examined all 64 possible DNA fragments, d(GCNNNGC), in which the triloop region of the d(GCGAAGC) minihairpin was randomized and found that only four fragments, d(GCGNAGC) (N = A, G, C, or T), formed extraordinarily stable minihairpins as shown by their gel mobility and resistance to a single-stranded DNA-specific exonuclease. Structural and thermodynamic analyses suggest that the extraordinary stability is caused by a unique structural property of the trinucleotide sequences corresponding to the GNA loop.

DNA↗

PubMed1997

In vitro selection of aptamers that bind to ribosome-inactivating toxins.

Ribosome-inactivating proteins, such as ricin, pepocin and gypsophilin, catalyze the hydrolysis of a single N-glycosidic bond at a specific position in rRNAs. Aptamers targeting pepocin were selected from a random sequence RNA pool that spanned 30 positions. After 8 rounds, the anti-pepocin aptamers were sequenced and a conserved hairpin motif was identified. Interestingly, the selected motif is quite different from the toxin-binding domains of rRNAs.

Base Sequence↗

PubMed1995

Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity.

A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Re-creating the RNA world.

Results from in vitro selection experiments can be used to construct and test models for the evolution of the RNA world. Surprisingly, the success of selected RNAs at binding ligands and catalyzing reactions may make it difficult to determine precisely the lineage of molecular fossils, molecules that are believed to have survived from the RNA world to the present.

Evolution, Molecular↗