Neighboring base sequence effect on DNA damage

Guanine is the most strongly oxidized base in DNA; generation of a guanine radical cation as an intermediate in an oxidation reaction leads to migration through a resulting cationic hole in the DNA π-stack until it is trapped by irreversible reaction with water or other free radicals. In the case of normal sequences, the primary position of Guanine oxidations by one-electron oxidants such as carbonate radical anions, BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene), and riboflavin are 5′-G in GG doublets and the central G in a GGG triplet. According to results, the properties of guanine oxidation on abasic site containing sequences are independent from the position of AP(apurinic/apyrimidinic) site in the presence of carbonate radical anions under a short irradiation time, although this radical is exposed to solvent by the existence of an abasic site. The lack of abasic site effect on guanine oxidative damage by the carbonate radical may be due to a sequence-independent property of the initial electron transfer rate in the hole injection step, or may relate to an electron transfer mechanism with large reorganization energy dependency. Consequently, the carbonate radical anions may easily migrate to another single G in the charge re-distribution step. Meanwhile, there is a strong dependency on the presence of an AP(apurinic/apyrimidinic) site in the cleavage patterns of guanine oxidations by physically large oxidizing agents, such as BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene) and riboflavin. These radicals show strong AP(apurinic/apyrimidinic) site dependency and clear G-site selectivity. Communicated by Ramaswamy H. Sarma</p

Interaction of G‑Quadruplex with RecA Protein Studied in Bulk Phase and at the Single-Molecule Level

As in the human genome there are numerous repeat DNA sequences to adopt into non-B DNA structures such as hairpin, triplex, Z-DNA, G-quadruplex, and so on, an understanding of the interaction between DNA repair proteins and a non-B DNA forming sequence is very important. In this regard, the interaction between RecA protein and human telomeric 5′-TAGGG-(TTAGGG)₃-TT-3′ sequence and the G-quadruplex formed from this sequence has been investigated in bulk phase and at the single-molecule level. The RecA@ssDNA filament, which is formed by the interaction between RecA protein and a G-rich sequence, was dissociated by the addition of K⁺ ions, and the dissociated G-rich sequence was quickly folded to a G-quadruplex structure, indicating that the G-quadruplex structure is more favorable than the RecA@ssDNA filament in the presence of K⁺ ions. In addition, we demonstrate that the conformation of the G-quadruplex, which is heterogeneous in the absence of RecA, converged to the specific G-quadruplex with one double-chain-reversal loop upon association of RecA protein

Chiral Selective Stacking of a Cationic Porphyrin along Z‑Form Poly[d(A-T)₂]

In this study, the binding mode of porphyrin-free single-stranded poly­[d­(AT)] and <i>trans</i>-BMPyP was observed in the Z-form <i>trans</i>-BMPyP–poly­[d­(A-T)₂] complex induced by extensive stacking depending on the temperature and concentration through circular dichroism (CD). The Z-form <i>trans</i>-BMPyP–poly­[d­(A-T)₂] complex (<i>R</i> = 0.30) retained the Z-form DNA structure at a low temperature (20 °C) by the <i>trans</i>-BMPyP molecules. When the temperature was increased to 60 °C, the DNA was almost unfolded as a single-stranded poly­[d­(AT)], but the extensive stacking binding mode of <i>trans</i>-BMPyP was maintained and the shape of the porphyrin Soret band was symmetrically changed in comparison with the shape of the Z-form DNA. However, when the temperature was raised to 80 °C, the extensive stacking binding mode of <i>trans</i>-BMPyP was also unfolded almost completely. The binding mode of the <i>trans</i>-BMPyP-single-stranded poly­[d­(AT)] complex was very similar to the already known binding mode of porphyrins and a double-stranded DNA. The binding mode was dependent on the concentration ratio ([porphyrin]/[DNA]): a monomeric binding mode at a concentration ratio of 0.04, a moderate groove binding mode at a concentration ratio between 0.08 and 0.16, and extensive stacking at a concentration ratio between 0.20 and 0.30. The same result was obtained when the temperature of the Z-form DNA (<i>R</i> = 0.30) was increased to 60 °C. However, those binding modes were not found in <i>cis</i>-BMPyP, which was because, in the extensive stacking of <i>trans</i>-BMPyP along the DNA skeleton, the distance between the two positive methylpyridine ions at the trans site and thymine, one of the DNA bases, is decreased, creating a much more hydrophobic environment. In addition, the poly AT sequences found from the CD spectra for the binding of <i>trans</i>-BMPyP–poly­[d­(A-T)₂] and <i>trans</i>-BMPyP–poly­[d­(AT)] (<i>R</i> = 0.30) showed that both of them underwent effective extensive stacking and that the chirality of extensive stacking was dependent on the form of DNA

Effect of periphery cationic substituents of porphyrin on the B-Z transition of poly[d(A-T)₂], poly[d(G-C)₂] and their binding modes

The binding mode of cationic porphyrin (trans-BMPyP) with poly[d(G-C)2] and poly[d(A-T)2] was examined according to the site of the periphery cationic methyl pyridine ion of the cationic porphyrin (o-, m-, p-) as well as the possibility of a B-Z transition depending on the binding modes by measuring the absorption spectrum and circular dichroism (CD). The negative band found in the soret region showed the intercalation mode of m- and p-trans-BMPyP-poly[d(G-C)2] to the DNA base pairs, but no B-Z transition was induced. On the other hand, the distinctive bisignate band found in the soret region of the CD spectrum for m- and p-trans-BMPyP-poly[d(A-T)2] suggests that m- and p-trans-BMPyP have an effective extensive stacking-based binding mode along with the skeleton of poly[d(A-T)2], wherein the B-Z transition was induced through extensive stacking. The difference in binding mode was attributed to the difference in the molecular structure depending on the site of the periphery cationic methyl pyridine ion in the cationic porphyrin. In other words, o-trans-BMPyP is nonplanar because of the steric hindrance of the cationic methyl pyridine ion at the o-site. In contrast, m- and p-trans-BMPyP are planar, but not all porphyrins with a planar structure undergo the B-Z transition. In conclusion, a B-Z transition is induced if the structure of a porphyrin is planar and the binding mode allows the porphyrins to be stacked effectively along the DNA skeleton, not in a binding mode where the porphyrin is intercalated to the DNA. Communicated by Ramaswamy H. Sarma</p

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G3T2G3TGTG3T2G3) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I2 to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction. Communicated by Ramaswamy H. Sarma</p

Photoinduced Reduction of Manganese(III) <i>meso</i>-Tetrakis(1-methylpyridinium-4-yl)porphyrin at AT and GC Base Pairs

The photoreduction of water-soluble cationic manganese­(III) <i>meso</i>-tetrakis­(1-methylpyridium-4-yl)­porphyrin (Mn^III(TMPyP)⁴⁺) bound to a synthetic polynucleotide, either poly­[d­(A-T)₂] or poly­[d­(G-C)₂], was examined by conventional absorption and circular dichroism (CD) spectroscopy, transient absorption, and transient Raman spectroscopy. Upon binding, Mn^III(TMPyP)⁴⁺ produced a positive CD signal for both polynucleotides, suggesting external binding. In the poly­[d­(A-T)₂]–Mn^III(TMPyP)⁴⁺ adduct case, an interaction between the bound porphyrin was suggested. The transient absorption spectral features of Mn^III(TMPyP)⁴⁺ in the presence of poly­[d­(A-T)₂] and poly­[d­(G-C)₂] were similar to those of the photoreduced products, Mn^II(TMPyP)⁴⁺, whereas Mn^III(TMPyP)⁴⁺ in the absence of polynucleotides retained its oxidation state. This indicated that both poly­[d­(A-T)₂] and poly­[d­(G-C)₂] act as electron donors, resulting in photo-oxidized G and A bases. The transient Raman bands (ν₂ and ν₄) that were assigned to porphyrin macrocycles exhibited a large downshift of ∼25 cm^–1, indicating the photoreduction of Mn^III to Mn^II porphyrins when bound to both polynucleotides. The transient Raman bands for pyridine were enhanced significantly, suggesting that the rotation of peripheral groups for binding with polynucleotides is the major change in the geometry expected in the photoreduction process. These photoinduced changes do not appear to be affected by the binding mode of porphyrin

Solvent-to-Polymer Chirality Transfer in Intramolecular Stack Structure

Solvent-to-polymer chirality transfer was examined using conjugated polymer with intramolecular stack structure (IaSS). When achiral poly­(diphenylacetylene)­s (PDPAs) dissolved in limonene, the solvent chirality was successfully transferred to the side phenyl stack structure, leading to intramolecular axial chirality. The phenyl–phenyl IaSS was under thermodynamic control to readily undergo asymmetric changes in chiral limonene, leading to optical activity in the isotropic structure between the main chain and resonant side phenyl rings. The axial chirality was significantly affected by the chain length and substitution position of the side alkyl groups. The longer alkyl chains and bulkier alkyl group prevented direct intermolecular interactions between the side phenyl rings and the chiral limonene molecules. PDPA with sterically congested, highly stable, and regulated IaSS was not favorable for efficient solvent-to-polymer chirality transfer

Photosensitized Oxidative DNA Damage: From Hole Injection to Chemical Product Formation and Strand Cleavage

Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5‘-d(CAT[G1Py]CG2TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py•+ and hydrated electrons; the latter are trapped by O2, thus forming the superoxide anion O2•-. The decay of the Py•+ radical is correlated with the appearance of the G•+/G(−H)• radical on microsecond time scales, and O2•- combines with guanine radicals at G1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz1Py). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in single-stranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G2 occurs only after G1Py, an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, khh 3 s-1 from G1•+−Py to G2 can be estimated from the known rates of the combination reaction of the G(−H)• and O2•- radicals. The formation of Iz products in the unmodified complementary strand compared to the modified strand in the duplex is ∼10 times smaller. The formation of tandem lesions is observed even at low levels of irradiation corresponding to “single-hit” conditions when less than ∼10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed

Bioconjugation of l-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications