Biomedical subjects
Publications and source records attributed to Hiroyuki Asanuma.
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.
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.
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.
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.
Efficient photoregulation of transcription by T7 RNA polymerase (RNAP) has been achieved with modified T7-promoter involving two azobenzene moieties at RNAP recognition region and TATA region simultaneously. Transcription proceeded 7.5-fold faster after UV irradiation than that under dark condition. Kinetic parameters (kcat, Km) for the transcription revealed that an azobenzene at each position played a different role and thus synergistic effect of photo-regulation was achieved with the promoter involving two azobenzenes.
Multiple azobenzenes were introduced into the oligonucleotide on D-threoninol for the clear-cut photo-regulation of the formation and dissociation of DNA duplex. When azobenzenes took trans form, introduction of multiple azobenzenes (azobenzenes:nucleobases = 1:2) did not interfere with duplex formation at all compared with their native duplex. In contrast, melting temperature (Tm) uniformly decreased with the number of azobenzenes when they took cis-form. As a result, clear-cut photo-regulation of the duplex formation was attained under physiological conditions. Furthermore, nearest-neighbor parameters for these azobenzene-tethered oligonucleotides were obtained. It was found that Tms estimated from these parameters well coincided with those of measured ones.
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RNA digestion by RNase H, which is responsible for the antisense effect, was efficiently photoregulated by use of the duplex of azobenzene-tethered sense DNA and native antisense DNA. In the dark, RNA digestion was suppressed because antisense DNA was strongly hybridized with azobenzene-tethered sense DNA, and accordingly RNA was isolated. On UV irradiation, antisense DNA was released from the azobenzene-tethered DNA due to the trans-to-cis isomerization and hybridized with RNA, which was digested by RNase H.
Based on a previous study of photo-regulation of transcription by T7 RNA polymerase, the regulatory effect of azobenzene on in vitro green fluorescent protein (GFP) production was studied by introduction of a D-threoninol-tethered p-azobenzene into the non template strand of the T7 promoter in a GFP template. By measurement of the resulting GFP fluorescence intensity after the in vitro transcription/translation coupled reaction, it is found that photo-regulation of GFP gene expression can be achieved in a position-dependent manner by trans-cis isomerization of azobenzene upon UV irradiation.
We have already reported that transcription reaction by phage T7 or SP6 RNA polymerase (RNAP) can be reversibly photo-regulated with azobenzene-tethered promoter. Transcription reaction proceeded faster by UV irradiation than visible light irradiation. In the present study, binding of T7 RNAP to its azobenzene-tethered promoter was directly monitored by use of affinity biosensor. When azobenzene-tethered T7 promoter was immobilized on the sensor surface, response based on the binding of RNAP increased by UV light irradiation rather than by visible light irradiation. Thus, photo-regulation of transcription reaction by azobenzene-tethered promoter was attributed to the change of binding property of RNAP to the promoter by trans-cis isomerization of azobenzene.
Two diastereomers of a photoresponsive oligodeoxyribonucleotide tethering a trans-azobenzene, based on the chirality of the central carbon of a diol linker, were separated by reversed-phase HPLC. On the basis of 2D NMR analysis, absolute configurations of the diastereomers alpha and beta (tentatively designated from differences in their retention time) were determined as R- and S-forms, respectively. For both diastereomers, their NMR-determined duplex structure showed that trans-azobenzene intercalates between base pairs, because distinct NOEs were observed between the protons of azobenzene and those of the adjacent base pairs, such as with the imino protons and methyl protons of thymine. The melting temperatures of both duplexes were higher than that of the corresponding native duplex, which contained no azobenzene residue, due to the intercalated trans-azobenzene stabilizing the duplex by a stacking interaction. Between these two diastereomers, differences in T(m) were also found: the melting temperature of the R-form duplex (alpha-isomer) was higher than that of the S-form (beta-isomer). On the basis of the NMR-determined structure, this difference was attributed to the fact that the S-form (beta isomer) causes more stress forming the duplex than does the R-form (alpha isomer) due to disturbances of the right-hand helix.
The Naphthyl Red moiety, conjugated to DNA, shows distinct chromism by hybridization with its complementary DNA. Single-stranded DNA involving the Naphthyl Red moiety exhibits an orange color and has lambda(max) at 466 nm at pH 7.0. The absorption maximum is shifted towards 545 nm by the presence of its complementary DNA, and the color of the solution changes from orange to magenta accordingly.
Methyl Red H aggregate of predetermined size is successfully synthesized from the DNA conjugate involving multiple Methyl Red moieties in sequence. In the single stranded state, hypsochromicity monotonically increases with the number of incorporated dyes: the peak maximum of the conjugate involving six Methyl Reds appears at 415 nm, and the shift is as great as 69 nm (3435 cm(-)(1)) with respect to the monomeric transition. This large hypsochromicity accompanied by the narrowing of the band clearly demonstrates that H aggregate is formed in the single strand. H aggregation is further promoted at higher ionic strength. Upon addition of complementary DNA below the T(m), however, this H band disappears and a new peak appears at 448 nm, indicating that aggregated structure is changed by the duplex formation. This spectral change is completely reversible so that the H band at 415 nm appears again above T(m). Thus, aggregated structure can be reversibly controlled by the formation and dissociation of the DNA duplex.
Multiple azobenzene moieties were tethered to DNA on D-threoninol linker, and formation and dissociation of DNA duplex could be reversibly photo-regulated either by irradiating UV or visible light. With this photo-responsive DNA, RNase H reaction was also successfully photo-regulated.
Naphthyl Red moiety, conjugated to DNA, shows distinct chromism by hybridization with its complementary DNA. Single-stranded DNA involving Naphthyl Red moiety exhibits orange color and has lambda max at 466 nm at pH 7.0. Absorption maximum shifts towards 545 nm by the presence of its complementary DNA, and accordingly color of the solution changes from orange to magenta.
Effective photo-regulation of transcription reaction by SP6 RNA polymerase (RNAP) was achieved with photo-responsive SP6 promoter tethering two azobenzenes. With one azobenzene in either TATA or RNAP binding region of the SP6 promoter, photo-regulation activity was very small. But when two azobenzenes were introduced into both regions, efficient photo-regulation of transcription was attained: transcription proceeded 3.5 fold faster under UV irradiation than under dark.