Biomedical subjects
Publications and source records attributed to Makoto Komiyama.
By introducing an intercalator through D-threoninol to the 10-23 DNAzyme at the junction between its catalytic loop and the binding arm, the RNA cleavage activity was greatly improved.
In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.
For the detection of deletion polymorphisms, two pyrene moieties are tethered to an oligodeoxyribonucleotide (ODN) on both sides of the intervening base; one- and two-base deletions can be selectively detected by the strength of the excimer emission.
Azobenzene was additionally introduced into side chain of T7 promoter for the photocontrol of transcription reaction by T7 RNA polymerase (T7 RNAP). When a single azobenzene molecule was introduced into the T7 promoter either at the loop-binding region of the RNAP (-7 to -11 position) or at the unwinding region (-1 to -4 position), transcription was suppressed in the trans-form but proceeded faster in the cis-form. The amount of transcripts after UV irradiation with respect to that in the dark was 1.5-2.0-fold. Kinetic analysis of the transcription reaction revealed that the photoregulatory mechanism was different in these positions. The photoisomerization of an azobenzene at the loop-binding region primarily affected Km. On the other hand, the isomerization of an azobenzene at the unwinding region mainly affected kcat. Still more clear-cut photoregulation was achieved when two azobenzenes were introduced into both loop-binding and unwinding regions, respectively: transcription proceeded 7.6-fold faster after UV irradiation than that in the dark. This synergistic effect was observed only when two azobenzenes were introduced into these two different regions, respectively, and introduction of them into the same loop-binding region drastically lowered the transcription activity. The cooperation of two azobenzenes at loop-binding and unwinding regions would contribute to the clear-cut photoregulation of transcription.
An ordered dye cluster of Methyl Reds was formed in double-stranded DNA by hybridizing two complementary DNA-dye conjugates, each involving a Methyl Red moiety on a threoninol linker and a 1,3-propanediol spacer arranged alternately in the middle of the DNA sequence. In the duplex, Methyl Reds from each strand were axially stacked antiparallel to each other, as determined from NMR analysis. This clustering of Methyl Reds induced distinct changes in both UV/Vis and CD spectra. Single-stranded DNA-Methyl Red conjugates on D-threoninol linkers and (1,3-propanediol) spacers exhibited broad absorption spectra with lambda(max) at around 480 nm, and almost no CD was observed at around the absorption maximum of Methyl Red. However, as Methyl Reds were clustered by hybridization, lambda(max) shifted towards shorter wavelengths with respect to its monomeric transition. This hypsochromic shift increased as the number of Methyl Red molecules increased. Furthermore, a positive couplet was also strongly induced here. These dye clusters are H-aggregates, in which molecular excitons are coupled. The positive couplet demonstrates that the clusters on D-threoninol form a right-handed helix. In contrast, the induced CD became much weaker with Methyl Red on L-threoninol, which intrinsically prefers counterclockwise winding. Thus, mutual orientation of the stacked dye molecules was controlled by the chirality of the linker.
An artificial restriction DNA cutter (ARCUT), recently developed by the authors, was used to construct a fusion protein. The gene of WW-domain-containing oxidoreductase (WWOX) was cut by ARCUT just before its stop codon, and ligated to fuse the gene of enhanced green fluorescent protein (EGFP). The reading frames of two genes were adjusted to coincide each other. Throughout the manipulation, no PCR was employed. The fluorescent fusion protein was successfully expressed in mammalian cells, and showed entirely different subcellular localization from EGFP itself. Apparently, the DNA was kept completely intact during the manipulation. The man-made tool ARCUT has promising features for future biotechnology and molecular biology.
The hydrolysis of cyclic adenosine 3',5'-monophosphate and 2'-deoxythymidylyl(3'-5')2'-deoxythymidine by Ce(NH4)2(NO3)6 was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce2(IV) (OH)4]4+ , showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce2(IV) (OH)4]4+ cluster are bridged by two OH residues. Upon the complex formation with H2 PO4- (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results.
We have synthesized azobenzene-tethered DNAs and have successfully photo-regulated various DNA functions. In the present study, we synthesized azobenzenes substituted with methyl group for still more effective photo-regulation of DNA hybridization. In trans-form, mono substituted azobenzene at ortho position stabilized the DNA duplex more efficiently than the other mono-substituted ones. In contrast, melting temperature (T(m)) for 2-methylazobenzene was lower in cis-form. As a result, change of T(m) (DeltaT(m)) induced by trans-cis isomerization became larger than that of unmodified azobenzene. Furthermore, di-substituted azobenzene at both ortho positions exhibited even larger DeltaT(m). Quite interestingly, thermal cis-to-trans isomerization of this azobenzene was about 10-fold slower than that of unmodified one. Thus, introduction of methyl group at 2, 6 positions raised both photo-regulatory activity and thermal stability of cis-form.
Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.
The activity of RNA cleaving DNA enzyme 10-23 is greatly improved by covalently introducing an intercalator, such as azobenzene, anthraquinone, 2-stilbazole, and pyrene. The intercalator is attached via an amide bond to D-threoninol, which inserts into the backbone of DNA with typical phosphoramidite chemistry. The increase of cleavage activity is observed only when an intercalator is tethered at the junction point between the catalytic loop and the binding arm at 3' side. Structures of the introduced intercalator itself as well as the linker for tethering it greatly influence the cleavage activity. In the case of anthraquinone, the most efficient activator we investigated, about eight folds of activity increase are obtained in comparison with the native DNA enzyme.
Recently, we developed artificial restriction DNA cutter (ARCUT) composed of pseudo-complementary peptide nucleic acid (pcPNA) and Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). Here we promoted the site-selective hydrolysis by attaching phosphate groups to the pcPNAs. The promotion by the phosphates increased with decreasing length of the gap-like site. Furthermore, the scission was successful even when phosphate groups were introduced to 0 base-gap system.
Genomic DNAs of lambda phage (48.5 kbp) and E. coli (4.6 Mbp) were successfully cleaved at the target site by artificial restriction DNA cutter (ARCUT), which we recently developed by combining Ce(IV)/EDTA and PNA (peptide nucleic acid). PNA satisfactorily invaded the target site in such huge DNAs and activate there for Ce(IV)/EDTA-induced scission.
By using two PNA (PNA = peptide nucleic acid) additives, one-base gap structure was formed in RNA. Upon treatment of this gap-forming RNA substrate with Lu(III) ion, the phosphodiester linkage at 5'-side of the unpaired nucleotide was mainly cleaved. Promising feature of PNA as a sequence recognizing moiety of man-made RNA cutters was evidenced.
Nano devices fabricated from DNA and nanoparticles found many important applications in both biotechnology and nanotechnology. Conventionally, DNA was linked to gold surfaces through a linker. In this study, DNAs were directly attached to gold surfaces through S-Au bonds by modifying 5'-end of DNA with a mercapto group. The pH of the reaction mixture played an important role in DNA-gold conjugate fabrication. The DNA-Au conjugates were successfully obtained in pure forms and, with the use of this new type conjugate, Au-DNA-Au nanostructure was prepared. The Au-DNA-Au nanostructure was characterized by AFM.
By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196-198, TAT at 199-201, and ACC at 502-504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation.
Nucleotide insertion/deletion polymorphisms (indels) in ApoE gene were precisely genotyped using artificial ribonucleases and MALDI-TOF MS. The RNA fragments for MS analysis were prepared by treating RNA specimens with our artificial ribonucleases, which consist of LuCl(3) (molecular scissors) and oligonucleotides bearing two acridine groups (RNA-activator for site-selective scission). RNA scission by Lu(III) ion always occurred at the phosphodiester linkages in front of the two acridines, even when the RNA specimens involved consecutive cytidine sequences of different lengths. Thus, even complicated mixtures of these indel specimens were completely genotyped by using only one acridine-bearing oligonucleotide and by subjecting the reaction mixture to single MS measurement. Moreover, single nucleotide polymorphism (SNP) in the consecutive sequences could be genotyped simultaneously with the indels.
Iminodiacetate-DNA conjugates and acridine-DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)-iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine-DNA conjugate.
With the use of two oligonucleotides bearing ethylenediamine-N,N,N'-triacetate groups as additives, gap sites were formed at predetermined sites in substrate DNA. Upon treating these systems with a Ce(4+)/EDTA complex at pH 7.0 and 37 degrees C, the phosphodiester linkages at the gap site were selectively hydrolyzed. The DNA scission was greatly promoted by the introduction of ethylenediaminetriacetate groups, and the scission efficiency increased as the number of these groups increased. Even a one-base gap was successfully hydrolyzed when three ethylenediaminetriacetate groups were placed consecutively at both edges of the gap, although the scission was minimal in the absence of these groups. The site-selective scission could be also achieved at higher temperatures without any significant loss of site-selectivity.