Photo-Cross-Linking between Br U and Pyrene Residues in an RNA/DNA Hybrid.

In this study, we investigated the photoreaction of Br U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of Br U changed dramatically from hydrogen abstraction to cross-linking by changing the conformation of the duplex from the B-form to the A-form. Among three A-form structures, the largest amount of cross-linked products was observed when Br U was incorporated into the RNA strand and the pyrene was conjugated to the 5' end of the DNA. These results indicate that the contact manner of pyrene was different between A- and B-form duplexes. This is a rare example of the use of the reactivity of bromouracil to analyze the contact between a small molecule with a weak binding affinity and a nucleic acid.

Photoreaction of DNA Containing 5-Halouracil and its Products.

5-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage. In DNA strands, the uracil radical generated from 5-halouracil causes DNA damage via a hydrogen-abstraction reaction. We analyzed the photoreaction of 5-halouracil in various DNA structures and revealed that the reaction is DNA structure-dependent. In this review, we summarize the results of the analysis of the reactivity of 5-halouracil in various DNA local structures. Among the 5-halouracil molecules, 5-bromouracil has been used as a probe in the analysis of photoinduced electron transfer through DNA. The analysis of groove-binder/DNA and protein/DNA complexes using a 5-bromouracil-based electron transfer system is also described.

Effects of Physical Damage in the Intermediate Phase on the Progression of Amyloid ß Fibrillization.

Understanding the mechanism responsible for the progression of amyloid deposition is important for developing methods to suppress this process in the treatment of Alzheimer's disease. The effects of physical damage during the transition phase of amyloid ß fibril formation are unclear. In this study, we used high-speed atomic force microscopy to investigate the effects of damage to the intermediates of amyloid ß in real time. Physical damage to intermediates did not suppress, but instead promoted fibrillization. This progression was accompanied by morphological changes from globular oligomers to protofibrils. These results suggest that the properties of the intermediates, such as structural fragility and stability, are highly related to the rate of fibrillization.

A Photoregulated DNA-Based Rotary System and Direct Observation of Its Rotational Movement.

Various DNA-based nanodevices have been developed on the nanometer scale using light as regulation input. However, the programmed controllability is still a major challenge for these artificial nanodevices. Herein, we demonstrate a rotary DNA nanostructure in which the rotations are controlled by light. A bar-shaped DNA rotor, fabricated as a stiff double-crossover molecule, was placed on the top of a rectangular DNA tile. The photoresponsive oligonucleotides modified with azobenzenes were employed as switching motifs to release/trap the rotor at specific angular position on DNA tile by switching photoirradiations between ultraviolet and visible light. As a result, two reconfigurable states (perpendicular and parallel) of rotor were obtained, in which the angular changes were characterized by AFM and fluorescence quenching assays. Moreover, the reversible rotary motions during the photoirradiation were directly visualized on the DNA tile surface in a nanometer-scale precision using a second-scale scanning of the high-speed AFM.

Linking two DNA duplexes with a rigid linker for DNA nanotechnology.

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.

Controlling electron rebound within four-base π-stacks in Z-DNA by changing the sugar moiety from deoxy- to ribonucleotide.

Charge transfer through DNA is of great interest because of the potential of DNA to be a building block for nanoelectronic sensors and devices. The photochemical reaction of 5-halouracil has been used for probing charge-transfer processes along DNA. We previously reported on unique charge transfer following photochemical reaction of 5-bromouracil within four-base π-stacks in Z-DNA. In this study, we incorporated a guanosine instead of a deoxyguanosine into Z-DNA, and found that electron transfer occurs in a different mechanism through four-base π-stacks.

Photoreactivities of 5-bromouracil-containing RNAs.

5-Bromouracil ((Br)U) was incorporated into three types of synthetic RNA and the products of the photoirradiated (Br)U-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA(Br)UGC)(2) and r(CGAA(Br)UUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2'-keto adenosine ((keto)A) product at the 5'-neighboring nucleotide, such as r(GC(keto)AUGC) and r(CGA(keto)AUUGC), respectively. The photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in Z-form RNA produced the 2'-keto guanosine ((keto)G) product r(CGC(keto)GUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2' position to provide a 2'-keto RNA product.

The distance between donor and acceptor affects the proportion of C1' and C2' oxidation products of DNA in a BrU-containing excess electron transfer system.

We have investigated the products of (Br)U in excess electron transfer and have demonstrated that in DNA the proportion of products changes with the distance between the donor and acceptor. On the basis of a labeling experiment using H(2)(18)O, we have shown that hole migration from Py(•+) formed after charge separation is involved in the reaction.

Photoreaction at 5'-(G/C)AA(Br)UT-3' sequence in duplex DNA: efficient generation of uracil-5-yl radical by charge transfer.

The photoreactivities of 5-halouracil-containing DNA have widely been used for analysis of protein-DNA interactions and have recently been used for probing charge-transfer processes along DNA. Despite such practical usefulness, the detailed mechanisms of the photochemistry of 5-halouracil-containing DNA are not well understood. We recently discovered that photoirradiation of BrU-substituted DNA efficiently produced 2'-deoxyribonolactone at 5'-(G/C)AABrUBrU-3' and 5'-(G/C)ABrUBrU-3' sequences in duplex DNA. Using synthetic oligonucleotides, we found that similar photoreactivities were maintained at the 5'-(G/C)AABrUT-3' sequence, providing ribonolactone as a major product with concomitant release of adenine base. In this paper, the photoreactivities of various oligonucleotides possessing the 5'-BrUT-3' sequence were examined to elucidate the essential factors of this photoreaction. HPLC product analysis indicated that the yield of 2'-deoxyribonolactone largely depends on the ionization potential of the purine derivatives located 5'-upstream of 5'-BrUT-3', as well as the electron-donating ability of their pairing cytosine derivatives. Oligonucleotides that possess G in the complementary strand provided the ribonolactone with almost the same efficiency. These results clearly suggest that the photoinduced charge transfer from the G-5' upstream of 5'-BrUT-3' sequence, in the same strand and the complementary strand, initiates the reaction. To examine the role of intervening A/T base pair(s) between the G/C and the 5'-BrUT-3' sequence, the photoreactivities of a series of oligonucleotides with different numbers of intervening A/T base pairs were examined. The results revealed that the hotspot sequence consists of the electron-donating G/C base pair, the 5'-BrUT-3' sequence as an acceptor, and an appropriate number of A/T base pairs as a bridge for the charge-transfer process.

Photochemical determination of different DNA structures.

The various conformations of DNA are thought to have important biological roles. Investigation of the local DNA conformational changes associated with biological events is therefore essential to an understanding of the functions of DNA. We have reported the photoreactivities of 5-halouracil in the five characteristic local DNA structures: the A, B and Z forms, protein-induced DNA kinks and the G-quadruplex form. These studies demonstrate the detailed relationships between the local DNA structures and the photochemical products of photoinduced hydrogen abstraction by the resulting uracil-5-yl radicals, and show that this photochemical method can be used to detect DNA structures. Here, we describe in detail procedures that have been developed in our laboratory for probing DNA conformations by product analysis of photoirradiated 5-halouracil-containing DNA. The protocol includes the preparation of 5-halouracil-containing DNA and the characterization of the photoproducts, and it can be completed in 2 weeks.

Detection of photoinduced electron transfer from Sso7d to DNA.

Sso7d is a chromosomal protein of hyperthermophilic Archaea. The crystal structure of Sso7d-d(GTAAT(Br)UAC)(2) has been clarified at high resolution, showing that the protein binds in the minor groove of DNA, causing a sharp kink of about 60 degrees . We demonstrated that electron transfer occurs efficiently from Sso7d to DNA by using inosine (I)-containing 5'-d(ITAAT(Br)UAC)-3'. Product analysis showed that Trp24 of Sso7d, located at the surface of the DNA, is consumed to produce N'-formylkynurenine during photoirradiation, indicating that Trp24 acts as an electron source. To explore the possibility of electron transfer between Sso7d and other DNA substrates, we examined the photochemical repair of the thymine dimer 5'-d(GTAAT<>TAC)-3' by Sso7d. Sso7d effectively repaired 5'-d(GTAAT<>TAC)-3' to 5'-d(GTAATTAC)-3' under irradiation conditions.

Photoreactivation of DNA by an archaeal nucleoprotein Sso7d.

Sso7d is a chromosomal protein of hyperthermophilic Archaea. The crystal structure of Sso7d-d(GTAAT(I)UAC)(2) has been clarified at high resolution, showing that the protein binds in the minor groove of DNA, causing a sharp kink of approximately 60 degrees. Recently, we found that photoirradiation of Sso7d and 5-iodouracil-((I)U)-containing 5'-d(GTAAT(I)UAC)-3' efficiently induced the abstraction of hydrogen from the methyl group of T(5) at the kink. In the present study, we found that the photoreactivity of 5-bromouracil ((Br)U)-containing 5'-d(GTAAT(Br)UAC)-3' was enhanced in the presence of Sso7d. Using hypoxanthine (I)-containing 5'-d(ITAAT(Br)UAC)-3', we demonstrated that electron transfer occurs efficiently from Sso7d to DNA. Product analysis showed that Trp-24 of Sso7d, located at the surface of the DNA, is consumed to produce N'-formylkynurenine during photoirradiation, indicating that Trp-24 acts as an electron source. To explore the possibility of electron transfer between Sso7d and other DNA substrates, we examined the photochemical repair of the thymine dimer 5'-d(GTAAT<>TAC)-3' by Sso7d. Sso7d effectively repaired 5'-d(GTAAT<>TAC)-3' to 5'-d(GTAATTAC)-3' under irradiation conditions. During this reaction, Trp-24 was not oxidized significantly, indicating that the anion radical of the repaired TT sequence is oxidized by the cation radical of Trp-24, and that a so-called "circular electron transfer" mechanism is operating in this system.

Biomolecule-based switching devices that respond inversely to thermal stimuli.

We demonstrate a molecular switch, on the basis of the characteristic properties of DNA and RNA, which indicates a completely inverted response to thermal stimuli using the transition between right- and left-handed helices. We designed a system using aminopurine (Ap), which can convert the pi-stack information of the transition from right-handed to left-handed DNA (B-Z transition) and RNA (A-Z transition) into an output giving a fluorescent signal. These two biomolecular devices together serve as "right-left" or "off-on" switches. When the temperature is changed from low to high, the RNA device changes from the off to on signal; however, the DNA device changes from on to off. The response of these RNA and DNA based devices against thermal stimulus was completely reversible.

Efficient generation of 2'-deoxyuridin-5-yl at 5'-(G/C)AA(X)U(X)U-3' (X = Br, I) sequences in duplex DNA under UV irradiation.

The photoreactivity of 5-halouracil-containing DNA was investigated using 450 base pair DNA fragments under 302 nm irradiation. Heat-dependent cleavage selectively occurs at 5'-(G/C)AAXUXU-3' and 5'-(G/C)AXUXU-3' (X = Br, I) sequences in double-stranded DNA. HPLC product analysis indicated that 2'-deoxyribonolactone residues are effectively generated at these sequences. These observations will be useful in studying the molecular basis of the sequence-dependent DNA-damaging process in UV-irradiated 5-halouracil-containing DNA.

The molecular-thermometer based on B-Z transition of DNA.

A molecular thermometer utilizing the different pi-stacking of B- and Z-DNA was designed. Aminopurine, which is a fluorescent probe for charge-transfer processes, was introduced into B- and Z-DNA and the emission monitored. Z-DNA showed a marked increase in fluorescence compared with B-DNA, with the fluorescence intensity correlating linearly and reproducibly with temperature.

Unique charge transfer properties of the four-base pi-stacks in Z-DNA.

To investigate the photoreactions of BrU in Z-DNA, the photoirradiation of 5'-d(C1G2C3G4BrU5G6C7G8)-3'/5'-d(C9mG10C11A12C13mG14C15G16)-3'(ODN 1-2) was investigated. In accord with previous observations, B-form ODN 1-2 with the 5'-GBrU sequence showed very weak photoreactivity. However, Z-form ODN 1-2 in 2 M NaCl underwent photoreaction to afford 5'-d(CGC)rGd(UGCG)-3' together with the formation of imidazolone (Iz) contained 5'-d(CIzCACmGCG)-3'. The results clearly indicate that structural changes caused by the B-Z transition dramatically increased the photoreactivity of ODN 1-2. Inspection of the molecular structure of Z-DNA suggests that there is unique four-base pi-stacks at the G4-BrU5-C11-mG10 in ODN 1-2. These results suggest that the intriguing possibility that the mG10 in a complementary strand located at the end of the four-base pi-stacks may act as an electron donor. To test the hypothesis of interstrand charge transfer from mG10 to BrU5 within the four-base pi-stacks in Z-DNA, ODN 1-3 samples in which the putative donor G10 residue was replaced with 8-methoxyguanine (moG) were prepared, since moG is known to trap cation radicals to yield Iz moieties in DNA. Photoirradiation of ODN 1-3 efficiently produced 5'-d(CGC)rGd(UGCG)-3' together with formation of 5'-d(CIzCACmGCG)-3'. These results clearly indicate that the interstrand charge transfer from mG10 to BrU5 initiates the photoreaction. In clear contrast, other replacements of G with moG did not enhance the photoreactivity. The present study revealed the presence of unique four-base pi-stacks in Z-DNA and photoirradition of BrU in Z-DNA causes efficient electron transfer from G within this cluster.

Photochemistry of bromouracil containing Z-DNA.

To investigate the mechanism underlying the photoreaction of bromouracil in DNA, a detailed analysis of photoirradiated 5'-d(CGCG(Br)UGCG)-3'/5'-d(C(8MeO)GCAC(m)GCG)-3' was undertaken. We found that rG- and 2-aminoimidazolone-containing octanucleotides are formed efficiently in Z-form DNA. These results suggest that interstrand electron transfer initiates the photoreaction of bromouracil in Z-DNA.