Soon Jin Oh

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.

Catalytic Hydrolysis of Phosphate Diesters by Lanthanide(III) Cryptate (2.2.1) Complexes.

Lanthanide(III) Cryptate (2.2.1) chlorides (Ln(2.2.1)Cl(3); Ln = La (1a), Ce(1b), and Eu(1c); (2.2.1) = 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tricosane) are effective for the catalytic hydrolysis of bis(4-nitrophenyl) phosphate. Kinetic studies reveal that the europium(III) complex (1c) catalyzes the hydrolysis to produce 6 equiv of 4-nitrophenol with a significant rate (k(1) = 1.5 x 10(-)(4) s(-)(1) at 0.40 mM) at pH 8.5 and 50 degrees C. The catalytic activity of the complexes is increased with decreasing the ionic size, i.e, La < Ce < Eu. While the use of hydrogen peroxide further increase the activity of 1b (k(1) = 1.6 x 10(-)(3) s(-)(1) at 0.40 mM), the presence of molecular oxygen does not affect the activity at all. Crystal of 1a.CH(3)OH([La(2.2.1)(Cl)(2)](Cl)(CH(3)OH)) belongs to the space group Pnma with a = 17.072(3) A, b = 19.037(3) A, c = 14.725(2) A, V = 4786(1) A(3), Z = 8, D(x)() = 1.691 g cm(-)(3), m = 21.7 cm(-)(1). The encryptated metal ion is nine-coordinated, and all the heteroatoms of the cryptate (2.2.1) ligand coordinate the metal center to form a bowl-shaped structure. Two coordinating chloride anions are located on the open face with a cis geometry. The existence of coordinated water to the europium(III) complex 1c in the aqueous solution was confirmed by time-resolved Eu(III) luminescence spectroscopy. From the decay constants in H(2)O and D(2)O, the numbers of coordinated water molecules (q) are found to be 3.02 at pH of 5.0. The above kinetic and spectroscopic observation are supportive of mechanisms in which the metal complexes act as a center for binding and activation as well as a source of nucleophilic metal hydroxides.

Structure and phosphodiesterase activity of Bis-Tris coordinated lanthanide(III) complexes

A commonly used buffer, 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol (Bis-Tris) coordinates lanthanide(III) ion strongly in water to form molecular species that are highly active for the hydrolysis of a phosphate diester, bis(4-nitrophenyl) phosphate.

Catalytic hydrolysis of phosphate monoesters by lanthanide(III) cryptate (2.2.1) complexes

Lanthanide(III) Cryptate (2.2.1) chlorides (Ln(2.2.1)Cl(3); Ln = La (1a), Ce(1b), and Eu(1c); (2.2.1) = 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tricosane) are effective for the catalytic hydrolysis of bis(4-nitrophenyl) phosphate. Kinetic studies reveal that the europium(III) complex (1c) catalyzes the hydrolysis to produce 6 equiv of 4-nitrophenol with a significant rate (k(1) = 1.5 x 10(-)(4) s(-)(1) at 0.40 mM) at pH 8.5 and 50 degrees C. The catalytic activity of the complexes is increased with decreasing the ionic size, i.e, La < Ce < Eu. While the use of hydrogen peroxide further increase the activity of 1b (k(1) = 1.6 x 10(-)(3) s(-)(1) at 0.40 mM), the presence of molecular oxygen does not affect the activity at all. Crystal of 1a.CH(3)OH([La(2.2.1)(Cl)(2)](Cl)(CH(3)OH)) belongs to the space group Pnma with a = 17.072(3) Å, b = 19.037(3) Å, c = 14.725(2) Å, V = 4786(1) Å(3), Z = 8, D(x)() = 1.691 g cm(-)(3), &mgr; = 21.7 cm(-)(1). The encryptated metal ion is nine-coordinated, and all the heteroatoms of the cryptate (2.2.1) ligand coordinate the metal center to form a bowl-shaped structure. Two coordinating chloride anions are located on the open face with a cis geometry. The existence of coordinated water to the europium(III) complex 1c in the aqueous solution was confirmed by time-resolved Eu(III) luminescence spectroscopy. From the decay constants in H(2)O and D(2)O, the numbers of coordinated water molecules (q) are found to be 3.02 at pH of 5.0. The above kinetic and spectroscopic observation are supportive of mechanisms in which the metal complexes act as a center for binding and activation as well as a source of nucleophilic metal hydroxides.

Mesospaced surface for DNA micro-array and other applications

We have studied ways to control density as well as spacing among functional groups. In particular, we observed that use of aziridine for the surface hyperbranching polymerization yielded extremely high surface density of primary amine that is useful for the immobilization of molecules of biological relevance such as oligo DNA. Also, an employment of dendrons of appropriate molecular architecture provided mesospacing among the reactive functional groups. The spacings was expected to guarantee the freedom of the biological macromolecules so that their properties are close to that in solution in spite of the confinement in the two dimensional world. We demonstrated that this was the case for oligonucleotide microarrays.

DISSOCIATION KINETICS OF EUROPIUM(III) CRYPTATE COMPLEXES IN AQUEOUS BUFFERS

Dissociation of the [EuL 1 ] 3+ complex (L 1 = 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tricosane) in aqueous buffer solutions of pH 7.0–9.0 was studied by monitoring the absorbance change of its charge-transfer (c.t.) band. While the dissociation rate is linearly dependent on complex concentration, the rate constant (k d ) is dependent on both the concentration and the type of buffer employed. In tris(hydroxymethyl)aminomethane (Tris) the measured rate constant is composed of a concentration-independent term and another term based on the square of the concentration of the basic form of Tris, i.e. k d = k 0 k 2 [NH 2 C(CH 2 OH) 3 ] 2 , which indicates that a general base mechanism is dominant at high buffer concentrations. The dissociation of the analogous complex of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane is more rapid. The unique c.t. band of the europium complexes was utilized to elucidate their thermodynamic behaviour in aqueous buffers. No absorbance was observed even at relatively high concentrations of the europium ion and the cryptands (0.100 mol dm -3 ). This enables an upper limit of the formation constants of 0.50 dm 3 mol -1 to be set in aqueous buffer.

Stepwise Growth of Oligodeoxynucleotides on Solid Hydroxylated Substrates: Characterization of the Growth by UV-Vis Spectroscopy and Ellipsometry

Abstract A stepwise growth of oligonucleotides on silicon wafer and fused silica is described. Spectroscopic analysis such as ellipsometry and UV-Vis spectroscopy, and contact angle measurement after each synthetic step have confirmed that a 15-mer oligonucleotide was successfully grown on the hexaethylene glycol linker bound to a silicon wafer or fused silica via a glycidoxypropylsilane.

CATALYTIC HYDROLYSIS OF PHOSPHATE TRIESTERS BY LANTHANIDE(III) CRYPTATE (2.2.1) COMPLEXES

Lanthanide(III) cryptate (2.2.1) chlorides [Ln(2.2.1)Cl3; Ln = La, Ce, Eu] are effective in catalysing the hydrolysis of 4-nitrophenyl diphenyl phosphate. Kinetic studies reveal that the europium(III)(2.2.1) chloride catalyses the hydrolysis efficiently (k= 1.8 × 10–2 s–1 at 1.6 mmol dm–3, turnover number = 12 at pH 9.00 and 25 °C). The reactivity of the cerium(III) complex is enhanced by the presence of molecular oxygen. Analysis of the products of the hydrolysis shows that the selectivity toward yielding 4-nitrophenol against phenol is unusually high (ratio = 17 : 1).