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Biomedical subjects

Masayuki Endo

Publications and source records attributed to Masayuki Endo.

28 records · Page 2Linked to original sources
PubMed2004

Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface.

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group.

Amino Acid Substitution↗
PubMed2004

Stepwise photocleavage of two C-O bonds of 1,8-bis[(4-benzoylphenoxy)-methyl]naphthalene with three-step excitation using three-color, three-laser flash photolysis.

Stepwise photocleavage of two naphthylmethyl-oxygen bonds of 1,8-bis[(4-benzoylphenoxy)methyl]naphthalene (1,8-(BPO-CH2)2Np, 1) was observed during three-color, three-laser flash photolysis at room temperature. The mechanism from 1 to the final product, acenaphthene (2), was clearly elucidated. The first (308 nm, 5 mJ pulse-1) XeCl laser excited 1 to the lowest triplet excited state 1(T1), in which the excited energy was localized in the naphthalene moiety, but the C-O bond cleavage did not occur. The second (430 nm, 7 mJ pulse-1) OPO laser excited 1(T1) to the higher triplet excited states 1(Tn) in which the excited energy is delocalized in the naphthalene moiety and C-O bonds, and one C-O bond cleavage occurred. The third (355 nm, 10 mJ pulse-1) YAG laser excited the carbon-centered radical in the ground state 1-(BPO-CH2)NpCH2*(D0) to its excited states 1-(BPO-CH2)NpCH2*(Dn), from which the second C-O bond cleavage occurred to give 2 as the final product. This is a successful example of stepwise cleavage of two equivalent C-O bonds in a molecule using three-color three-laser photolysis method.

Journal Article↗
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

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↗
PubMed2003

Control of a double helix DNA assembly by use of cross-linked oligonucleotides.

Disulfide cross-linked oligonucleotides for connecting two DNA double helixes have been designed, synthesized, and characterized. Employing these cross-linked oligonucleotides, two double helixes can be arranged side by side, and the orientations can be controlled both in parallel and antiparallel ways by addition of a specific complementary DNA strand.

Base Sequence↗
PubMed2003

Benzophenones in the higher triplet excited states.

Transient phenomena of benzophenone (BP) in the higher triplet excited state (Tn) have been investigated by the two-colour two-laser excitation method. Triplet energy transfer from BP(Tn) to quenchers (Q) occurred within the duration of a laser pulse (5 ns) to give Q(T1) with higher triplet energy than that of BP(T1). The quantum yield of the triplet energy-transfer quenching of BP(Tn) by CCl4 was found to be 0.0023 +/- 0.0002 from the bleaching of the transient absorption of BP(T1) and the absorbed photon number. It appears that internal conversion from BP(Tn) to BP(T1) is the predominant process. The lifetimes (tauTn) of BP(Tn) and several substituted benzophenones (BPs) in the higher triplet excited state [BPs(Tn)] were estimated from the dependence of the Q concentration on the efficiency of the triplet energy-transfer quenching of BP(Tn) by Q, and found to be 110-450 ps, depending on the nature of the substituents on the BPs. The effect of the substituents on tauTn may be explained by the energy gap between the Tn and T1 states, because the main deactivation pathway for BPs(Tn) is the internal conversion process. In contrast, the substituent effect on the lifetimes of BPs(T1) cannot be explained by the energy gap law. The transient behaviour of Q(T1) depends on the properties of the quencher. Sequential triplet energy transfer from Q(T1) to BP occurred for p-dichlorobenzene and tert-butylbenzene as quenchers, while Q(T1) reacted partly with Q to form triplet excimers (3Q2*) for benzene, chlorobenzene, and o-dichlorobenzene as quenchers. When CCl4 was used as the quencher, the homolytic cleavage of a C-Cl bond of CCl4(T1) occurred to give Cl* and Cl3C* radicals.

Journal Article↗
PubMedDate not supplied

A hydrophilic azobenzene-bearing amino acid for photochemical control of a restriction enzyme BamHI.

A novel hydrophilic and negatively charged azobenzene-bearing amino acid, 4'-carboxyphenylazophenylalanine (azoAla 1), has been designed and synthesized for investigation of the photochemical regulation of the enzyme activity. The properties of photoisomerization and thermal stability of the cis-isomer were similar to those of a commonly used phenylazophenylalanine (azoAla 2). For photochemical control of the enzyme, these two azobenzene-bearing amino acids were incorporated into the specific position at the dimer interface of a restriction enzyme BamHI. These trans-azobenzene derivatives in the BamHI suppressed the enzymatic activity, and the following photoirradiation at 366 nm induced the recovery of its activity. Although the activities of both azoAla-BamHI mutants were same level after a long time irradiation, the recovery of the activity of azoAla 1-BamHI was faster than that of azoAla 2-BamHI with a short time irradiation. This result suggests that the negatively charged carboxylate group introduced into an azobenzene moiety affects the behavior of azoAla in the protein scaffold during the trans-cis photoisomerization.

Azo Compounds↗