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Biomedical subjects

Ichiro Hirao

Publications and source records attributed to Ichiro Hirao.

At least 19 recordsLinked to original sources
PubMed2006

Unnatural base pair systems for DNA/RNA-based biotechnology.

The development of unnatural, extra base pairs could expand genetic information, by enabling the site-specific incorporation of functional components into nucleic acids and proteins. Researchers have been trying to create unnatural base pairs by drawing upon non-standard hydrogen-bonding topologies, shape complementarity, and/or hydrophobic interfaces, to confer sufficient selectivity and efficiency to work with the natural A-T(U) and G-C base pairs in replication, transcription and translation. The development of artificial genetic systems has been broadly advanced by combining the extra base pairs with modified natural components, including the base pairs, substrates and polymerase enzymes.

Base Pairing↗
PubMed2006

An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA.

Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.

Base Pairing↗
PubMed2006

An unnatural base pair system for in vitro replication and transcription.

The development of unnatural base pairs that function in replication, transcription, and translation could expand the genetic alphabet and enable the site-specific incorporation of functional components into nucleic acids and proteins. We present an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and pyrrole-2-carbaldehyde (denoted by Pa). In replication, the Ds-Pa pair exhibits high selectivity in combination with the usual and modified triphosphate substrates and exonuclease-proficient DNA polymerases. In transcription, the Ds-Pa pair mediates the site-specific incorporation of the substrates of both Ds and Pa into RNA by T7 RNA polymerase. This unnatural base pair system could facilitate the specific incorporation of functional components into RNA molecules at desired positions using DNA templates containing the unnatural base pair, which can be amplified by PCR.

Base Pairing↗
PubMed2005

Site-specific fluorescent labeling of RNA molecules by specific transcription using unnatural base pairs.

Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine (v) and y function in transcription, and the substrates of y and 5-modified y bases can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Ribonucleoside 5'-triphosphates of 5-fluorophore-linked y bases were chemically synthesized from the nucleoside of y. These fluorescent substrates were site-specifically incorporated into RNA by transcription mediated by the s-y and v-y pairs. By using this fluorescent labeling method, specific positions of Raf-binding and theophylline-binding RNA aptamers were fluorescently labeled, and the specific binding to their target molecules was detected by their fluorescent intensities. This site-specific labeling method using an unnatural base pair system will be useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.

Aptamers, Nucleotide↗
PubMed2005

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs.

Direct site-specific biotinylation of RNA molecules was achieved by specific transcription mediated by unnatural base pairs. Unnatural base pairs between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y), or 2-amino-6-(2-thiazolyl)purine (denoted as v) and y specifically function in T7 transcription. Using these unnatural base pairs, the substrate of biotinylated-y (Bio-yTP) was selectively incorporated into RNA, opposite s or v in the DNA templates, by T7 RNA polymerase. This method was applied to the immobilization of an RNA aptamer on sensor chips, and the aptamer accurately recognized its target protein. This direct site-specific biotinylation will provide a tool for RNA-based biotechnologies.

Base Pairing↗
PubMed2005

An efficient unnatural base pair for a base-pair-expanded transcription system.

For the site-specific incorporation of artificial components into RNA by transcription, an efficient, unnatural base pair between 2-amino-6-(2-thiazolyl)purine (denoted as v) and 2-oxo(1H)pyridine (denoted as y) was developed. The substrates of y and 5-substituted y were site-specifically incorporated into RNA by T7 RNA polymerase opposite v in templates. The efficiency and fidelity of the v-y pairing in transcription were as high as those of the natural A-T(U) and G-C pairings. Furthermore, RNAs containing two adjacent y bases were also transcribed from DNA templates containing two v bases. This specific transcription allows the large-scale preparation of artificial RNAs and can be combined with other systems to simultaneously incorporate several different components into a transcript.

Base Pairing↗
PubMed2005

Interaction analysis between tmRNA and SmpB from Thermus thermophilus.

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.

Bacterial Proteins↗
PubMed2005

Fluorescent properties of an unnatural nucleobase, 2-amino-6-(2-thienyl)purine, in DNA and RNA fragments.

Nucleoside derivatives of 2-amino-6-(2-thienyl)purine (s) are fluorescent and can be site-specifically incorporated into RNA by transcription mediated by unnatural base pairs between s and its complementary bases. To utilize the fluorescent s base as a probe, we examined the fluorescent properties of s in DNA and RNA fragments. The nucleoside of s exhibited a fluorescence emission centered at 432 nm, characterized by two major excitation maxima (299 and 352 nm), and its quantum yield was 0.41 at pH 7.0. The fluorescence intensity of s in DNA fragments (12-mer) differed depending on circumstances such as the base components, stacking, and pairings. The s base was introduced into a tRNA at specific positions by specific transcription using the unnatural base pairs. The fluorescence intensity of s at each position reflected the local structural features of the tRNA molecules.

Base Pairing↗
PubMed2005

Site-specific incorporation of fluorescent probes into RNA by specific transcription using unnatural base pairs.

To analyze the local conformational changes of RNA molecules, we developed a site-specific fluorescent labeling method for RNA fragments by T7 transcription, using unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y) and between 2-amino-6-(2-thiazolyl)purine (v) and y. Ribonucleoside 5'-triphosphates of 5-fluorescence-linked y derivatives can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Using this specific transcription, the substrate of a fluorescein-linked y was introduced into a theophylline-binding RNA aptamer. The replacement of U6 by the fluorescein-linked y maintained both the binding ability and selectivity of the aptamer to theophylline. Furthermore, the fluorescence intensity was increased upon theophylline binding, but was not changed by the addition of caffeine.

Aptamers, Nucleotide↗
PubMed2005

Non-hydrogen-bonded base pairs for specific transcription.

Specific transcription mediated by unnatural base pairs could create novel RNA molecules with increased functionality and expand the genetic code. Here, we report an unnatural base pair between pyrrole-2-carbaldehyde (Pa) and 2-amino-6-(2-thienyl)purine (s) or 6-(2-thienyl)purine (s') for the site-specific incorporation of s or s' into RNA by T7 RNA polymerase, using DNA templates containing Pa. Despite the absence of significant hydrogen bonding interactions between the unnatural bases, the efficiency and fidelity of the s-Pa pairing in transcription were as high as those of the natural base pairings. As shown in replication, this indicates the importance of shape complementarity between pairing bases in transcription. Since the s base is fluorescent, this transcription mediated by the s-Pa pair provides a useful tool for site-specific fluorescence probing of RNA molecules.

Base Pairing↗
PubMed2004

A two-unnatural-base-pair system toward the expansion of the genetic code.

Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription. The combination of the s-y and s-z pairs provides a powerful tool to prepare both y-containing mRNA and s-containing tRNA for efficient coupled transcription-translation systems, in which the genetic code is expanded by the codon-anticodon interactions mediated by the s-y pair. In addition, the nucleoside of s is strongly fluorescent, and thus the s-z pair enables the site-specific fluorescent labeling of RNA molecules. These unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.

Base Pairing↗
PubMed2004

Unnatural base pairs between 2- and 6-substituted purines and 2-oxo(1H)pyridine for expansion of the genetic alphabet.

An unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y) shows high selectivity in transcription and translation. Toward the further development of unnatural base pairs that also have exclusive selectivity in replication, we examined the roles of the 2-amino and 6-thienyl groups of s using base pairs between y and purine-analogs, 6-thienylpurine and 2-amino-6-furanylpurine, as well as s. The results obtained from the thermal stability and DNA polymerase single-nucleotide insertion experiments suggest that the 2-amino group of s contributes toward the shape complementarity of the pairing with y, rather than the hydrogen bonding with the 2-keto group of y. In addition, the bulkiness of positions 2 and 6 of the unnatural purines cooperatively determines the selectivity of the noncanonical pairing with y or the natural pyrimidines in replication. This information is useful not only for the development of unnatural, orthogonal base pairs, but also for understanding the mechanisms of base pair formation in replication.

Base Pairing↗
PubMed2004

Unnatural base pairs mediate the site-specific incorporation of an unnatural hydrophobic component into RNA transcripts.

Site-specific incorporation of a hydrophobic nucleotide analog into RNA, by T7 transcription mediated by unnatural base pairs, was developed. The nucleotide analog, 5-phenylethynyl-3-(beta-D-ribofuranosyl)pyridin-2-one 5-triphosphate (denoted by Ph-yTP), was chemically synthesized and then site-specifically incorporated by T7 RNA polymerase into RNA opposite the pairing partner, 2-amino-6-(2-thienyl)purine (denoted by s) in DNA templates. The introduction of Ph-y into a theophylline-binding RNA aptamer, in which a uridine in the internal loop was replaced by Ph-y, raised the thermal stability of the aptamer. Thus, this unnatural nucleotide analog would be useful for stabilizing RNA tertiary structures and complexes between RNA and other molecules.

Base Pairing↗
PubMed2004

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA.

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.

Base Sequence↗
PubMed2004

Structures of d(GCGAAGC) and d(GCGAAAGC) (tetragonal form): a switching of partners of the sheared G.A pairs to form a functional G.AxA.G crossing.

The DNA fragments d(GCGAAGC) and d(GCGAAAGC) are known to exhibit several extraordinary properties. Their crystal structures have been determined at 1.6 and 1.65 A resolution, respectively. Two heptamers aligned in an antiparallel fashion associate to form a duplex having molecular twofold symmetry. In the crystallographic asymmetric unit, there are three structurally identical duplexes. At both ends of each duplex, two Watson-Crick G.C pairs constitute the stem regions. In the central part, two sheared G.A pairs are crossed and stacked on each other, so that the stacked two guanine bases of the G.AxA.G crossing expose their Watson-Crick and major-groove sites into solvent, suggesting a functional role. The adenine moieties of the A(5) residues are inside the duplex, wedged between the A(4) and G(6) residues, but there are no partners for interactions. To close the open space on the counter strand, the duplex is strongly bent. In the asymmetric unit of the d(GCGAAAGC) crystal (tetragonal form), there is only one octamer chain. However, the two chains related by the crystallographic twofold symmetry associate to form an antiparallel duplex, similar to the base-intercalated duplex found in the hexagonal crystal form of the octamer. It is interesting to note that the significant difference between the present bulge-in structure of d(GCGAAGC) and the base-intercalated duplex of d(GCGAAAGC) can be ascribed to a switching of partners of the sheared G.A pairs.

Base Pairing↗
PubMed2004

Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs.

A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (Iy) capable of site-specific incorporation into transcripts was developed. The site-specific Iy incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between Iy and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, Iy was incorporated into an anti(Raf-1) RNA aptamer, which binds to human Raf-1 and inhibits the interaction between Raf-1 and Ras. Protein-dependent photo-dimerization of the aptamer was observed when Iy was located at specific positions in the aptamer, showing that the site-specific incorporation of the photo-sensitive component into RNA achieves highly specific crosslinking. This specific transcription mediated by the unnatural base pair would be a powerful tool for generating high-affinity RNA ligands and for analyzing RNA-RNA and RNA-protein interactions, as well as for constructing RNA-based nanostructures.

Bacteriophage T7↗
PubMed2004

A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer.

A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5'-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication.

Algorithms↗