Seog K. Kim

A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.

Classification of CD and absorption spectra in the Soret band of H2TMPyP bound to various synthetic polynucleotides

The binding mode of porphyrins, namely meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (H(2)TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H(2)TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)].

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.

Base specific complex formation of norfloxacin with DNA.

We examined the base specificity of the norfloxacin-DNA interaction by measuring the binding constant of norfloxacin to various synthetic polynucleotides, using the Stern-Volmer and the Benesi-Hildebrand methods. The equilibrium constants were largest for poly[d(G-C)2] and poly(dG).poly(dC), suggesting that norfloxacin binds preferentially to the G-C bases of calf thymus DNA. We also found that norfloxacin has a greater affinity for purine than for pyrimidine. The binding mode of norfloxacin to double-stranded polynucleotide was studied using circular and linear dichroism (CD and LD). When the norfloxacin was complexed to poly[d(G-C)2], poly(dG).poly(dC) and DNA, all of the complexes exhibited a similar weak, positive CD band and negative LD in the 300-350-nm region. A closer examination of the LD spectra suggests that the molecular plane of norfloxacin is near perpendicular relative to DNA helix axis that excludes the groove binding mode or surface binding of norfloxacin.

DNA STRUCTURAL FEATURES RESPONSIBLE FOR SEQUENCE-DEPENDENT BINDING GEOMETRIES OF HOECHST 33258

The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.

Binding of meso-Tetrakis(N-methylpyridinium-4-yl)porphyrin to AT Oligomers: Effect of Chain Length and the Location of the Porphyrin Stacking

Circular dichroism (CD) spectra of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) that are associated with various duplex and triplex AT oligomers were investigated in this study. A strong positive CD was apparent for both the TMPyP complexed with duplex d[(A-T)(12)](2), d(A)(12).d(T)(12) and triplex d(A)(12).d[(T)(12)](2) at a low mixing ratio. As the mixing ratio increased, bisignate excitonic CD was produced for TMPyP complexed with duplexes, whereas the positive CD signal remained the same for the TMPyP-d(A)(12).d[(T)(12)](2) complex. This difference in the CD spectrum in the presence of duplex and triplex oligomers indicates that the moderate stacking of TMPyP occurs at the major groove of the duplex and the monomeric binding occurs in (or near) the minor groove. When TMPyP forms a complex with duplex d[(A-T)(6)](2) only excitonic CD was observed, even at a very low mixing ratio. Therefore, at least seven or more basepairs are required for TMPyP to exhibit a monomeric CD spectrum. After close analysis of the CD spectrum, the TMPyP-poly[d(A-T)(2)] complex could be explained by a combination of the CD spectrum of the monomeric, moderately stacked, and extensively stacked TMPyP.

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.

Enantioselective binding of S- and R-ofloxacin to various synthetic polynucleotides.

The binding properties of S- and R-ofloxacin to poly[d(A-T)(2)], poly[d(G-C)(2)] and poly[d(I-C)(2)] were studied by circular dichroism (CD) and various fluorescence techniques. The spectral properties of R-ofloxacin did not change when it was mixed with poly[d(A-T)(2)] and poly[d(I-C)(2)], indicating that R-enantiomer does not interact with these polynucleotides. On the other hand, when S-ofloxacin was mixed with any polynucleotide, or R-enantiomer with poly[d(G-C)(2)], characteristic changes in CD and fluorescence were observed. Therefore, it is clear that enantiomers of ofloxacin selectively recognize B-form DNA. The overall spectral properties of the ofloxacin-polynucleotide complex are similar to those of the norfloxacin-polynucleotide complex [Eur. J. Biochem. 267 (2000) 6018], suggesting that this quinolone also binds in the minor groove of DNA and therefore it may be partially inserted between DNA bases or interact with purine bases.

Conformational analysis of genotoxic benzo[a]pyrene- 7,8-dione-duplex DNA adducts using a molecular dynamics method.

Abstract Benzo[a]pyrene-7,8-quinone (BPQ), a metabolite of the wide spread carcinogen benzo[a] pyrene, has been known to form adducts with DNA bases, namely guanine and adenine. The adducts include stereoisomers of the eight BPQ-G1,2 and products of the formation of the cyclic five-member ring between BPQ and the G base. The stereochemical characteristics, structural properties, and thermodynamic aspects of the formation of the BPQ-G modified duplexes were investigated in this work using molecular modeling and a molecular dynamics (MD) simulation technique. Three conformations for the BPQ- oligonucleotides adduct showed the most favorable free energy (AG). The molecular plane of the BPQ is nearly perpendicular to the DNA base plane for all three stable adducts. In two adducts, BPQ is located in the minor groove with either anti or syn conformations around the glycosidic bond. One of adducts in which BPQ locates into the major groove with the anti conformation around the glycosidic bond was also energetically possible. The resulting detailed structures of the three conceivable BPQ-oligonucleotide adducts are elucidated in this work.

Real-time detection of DNA cleavage induced by [M(2,2'-dipyridylamine)2(NO3)n]x+ (M = Cd, Cu, Ni, Zn, n = 1,2, x = 0,1): Effect of central metal ions

[M(Hdpa)(2)(NO(3))(n)](x+) (M=Zn(II), Cd(II), Cu(II) and Ni(II), n=1,2, and x=0, 1) complexes were synthesized, and their activity as catalysts for DNA cleavage reactions were investigated using electrophoresis and linear dichroism technique (LD). All four metal complexes effectively cleaved pBR322 super-coiled DNA. The electrophoresis analysis showed that the [Zn(Hdpa)(2)(NO(3))](+) and [Cd(Hdpa)(2)(NO(3)) (2)] complexes most effectively cleaved the super-coiled DNA, whereas the [Ni(Hdpa)(2)(NO(3))](+) complex was least effective. The magnitude of LD in the DNA absorption region reflects the flexibility and length of DNA when the conditions for measurement are properly adjusted. The double stranded DNA cleavage increases the flexibility of DNA and reduces the length, reducing the magnitude of LD in DNA absorption region. Utilizing this LD property, the cleavage was detected in real-time by measuring the LD magnitude with respect to time. The decrease in the LD magnitude was described as the sum of two exponentials. The fast component was tentatively assigned to the cleavage of the single strand, reflecting the increase in the flexibility of DNA, and the slow component was assigned to the cut of the double strand which reduced the length of DNA. The average reaction time was the fastest for the Zn(II) complex and the slowest for the Ni(II) complex. The reaction time of the Cd(II) complex was as fast as that of the Zn(II) complex. Both the Zn(II) and Cd(II) belong to group 12, suggesting that [M(Hdpa)(2)(NO(3))(n)](x+) with central metal ions from group 12 most efficiently cleaved double stranded DNA.