Recent years have witnessed an explosion of interest in the use of DNA-nanoparticle bioconjugates for programmed nanostructures, 1D or 2D nanoparticle arrays, nanoelectronics, and biosensing and biodiagnostics. [1][2][3][4][5][6][7][8][9] DNA was chosen as a polymeric material in these studies owing mainly to the specificity of DNA base-pairing, the predictability of inter-or intramolecular interactions, its physicochemical stability, and mechanical rigidity. In addition, DNA can be manipulated and modified by a wide range of enzymes, including DNA polymerase, ligase, and restriction endonucleases. The powerful, convenient, and specific enzymatic manipulations make DNA a highly desirable building block for the construction of various nanostructures. In the current study, we set out to investigate whether we can perform rolling circle amplification (RCA) between a DNA oligonucleotide tethered to gold nanoparticles (AuNPs) as the primer and a single-stranded circular DNA as a template, catalyzed by a special DNA polymerase known as f29 DNA polymerase (f29DNAP). RCA is a powerful but simple biochemical method that can be used to generate long single-stranded DNA (ssDNA) with a repeating sequence unit. [10][11] In a typical RCA process, a DNA polymerase, such as f29DNAP, which has a strong ability to displace newly synthesized DNA strands, makes continuous nucleotide additions to a growing DNA chain over a short circular single-stranded DNA as the template under isothermal conditions. As a result, long, linear tandemly repetitive single strands of DNA are produced. As the synthesized long DNA molecules contain many repeating sequence motifs, RCA coupled with ensuing hybridization with fluorescent DNA probes has been used as an on-chip signal-amplification tool for sensitive biosensing. [12][13][14][15][16] Recently, it has been shown that long DNA molecules from RCA can be used as scaffolds for assembling nanoparticles to form 1D nanoparticle arrays. [17][18] However, to our knowledge, performing the RCA reaction on gold nanoparticles and using the resultant special DNA-AuNP assemblies to form 3D nanoparticle array have not been demonstrated. Figure 1 schematically illustrates the RCA process on gold nanoparticles and its scaffolding for 3D periodical nanoassemblies. Gold nanoparticles of 15 nm diameter were first prepared by the classical citrate reduction route. Thiolmodified DNA primers (41 nucleotides) were then functionalized onto the nanoparticles following Mirkins approach. [5] The concentration of DNA primers on the gold nanoparticles, referred to hereafter as primer-Au, was determined using UV/Vis spectroscopy by measuring the amount of DNA in solution before and after coupling; each primer-Au contained approximately 230 DNA primer strands. A 63-nucleotidelong circular DNA template was then annealed with the DNA-functionalized gold nanoparticles. The hybridization efficiency, as examined by measuring the radioactivity on the gold nanoparticles and in solution after annealing with a radiolabeled circular DNA ...

We previously demonstrated—through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions—that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539–7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a kobs of ∼1 min−1 at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring ∼80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of ∼106-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA.

An efficient two-step method for the synthesis of 2-bromo-4-nitroimidazole, 6, a key building block for nitroimidazole drugs, has been developed. The synthesis involves dibromination of 4-nitroimidazole 10 followed by selective debromination using in situ reductive deiodination strategy. The reactions are facile, safe, and easy to scale up. The large-scale applicability of this improved method was tested by conducting the reactions on kilogram scale to produce the desired product in high yield and quality.

We previously described a cis-acting RNA-cleaving deoxyribozyme known as pH3DZ1 that exhibits optimal catalytic activity at pH 3.0 (. Am. Chem. Soc. 125, 7539 (2003)). This DNA catalyst was made of a 99-nucleotide (nt) catalytic domain covalently linked to a 23-nt DNA-RNA chimeric substrate containing a single ribonucleotide as the cleavage site. In the present work, we conducted an extensive sequence examination of this deoxyribozyme via nucleotide truncation and reselection experiments, with a goal to minimize its size and identify the nucleotides that are crucial to its catalytic function. A trans-acting deoxyribozyme that can process an external substrate was also successfully designed. Stretches of 30 and 17 nucleotides from the 5′ and 3′ ends of the trans catalyst, respectively, were found to be completely dispensable; in contrast, few nucleotides could be deleted internally without producing a detrimental effect. The reselection experiment led to the discovery of 7 and 5 absolutely conserved nucleotides located at the 5′ and 3′ ends of the minimized catalyst, respectively, separated by a 31-nt element in which 14 highly conserved nucleotides were scattered among 17 variable nucleotides. The shortened deoxyribozyme and the original catalyst showed a similar pH profile with the optimal activity at pH 3; however, the minimized deoxyribozyme still exhibited strong catalytic activity at pH 2.5, while the fulllength catalyst was barely active at this pH. Finally, it was found that this deoxyribozyme generated two cleavage fragments, one with 2′,3′-cyclic phosphate and the other with 5′-OH. a décrit un désoxyribozyme connu sous le nom de code pH3DZ1, qui est capable de cliver l'ARN, qui agit d'une façon cis et dont l'activité catalytique est optimale à un pH de 3,0; ce catalyseur d'ADN a été fait en liant le domaine catalytique 99-nucléotide (nt) à un substrat chimérique 23-nt ADN-ARN ne contenant qu'un seul ribonucléotide comme site de clivage. Dans le travail présenté maintenant, on a fait appel à des expériences de tronquage et de resélection pour effectuer un examen extensif de la séquence de ce désoxyribozyme dans le but de minimiser sa taille et d'identifier les nucléotides qui sont cruciaux à son fonctionnement catalytique. On a aussi déve-loppé avec succès un désoxyribozyme agissant de façon trans qui peut traiter un substrat extérieur. On a identifié des blocs respectivement de 30 et de 17 nucléotides des extrémités 5′ et 3′ du catalyseur trans dont on peut se dispenser complètement; par opposition à cette situation, il n'y a que peu de nucléotides internes qui peuvent être éliminés sans produire des effets néfastes. L'expérience de resélection a permis de découvrir des blocs de 7 et 5 nucléotides absolument nécessaires qui sont situés respectivement aux extrémités 5′ et 3′ du catalyseur minimisé et qui sont séparés par un élément de 31-nt dans lequel 14 des nucléotides à conserver sont éparpillés parmi les 17 nucléotides variables. Le désoxyribozyme raccourci et le catalyseur original présentent tous l...

Recent years have witnessed an explosion of interest in the use of DNA-nanoparticle bioconjugates for programmed nanostructures, 1D or 2D nanoparticle arrays, nanoelectronics, and biosensing and biodiagnostics. [1][2][3][4][5][6][7][8][9] DNA was chosen as a polymeric material in these studies owing mainly to the specificity of DNA base-pairing, the predictability of inter-or intramolecular interactions, its physicochemical stability, and mechanical rigidity. In addition, DNA can be manipulated and modified by a wide range of enzymes, including DNA polymerase, ligase, and restriction endonucleases. The powerful, convenient, and specific enzymatic manipulations make DNA a highly desirable building block for the construction of various nanostructures. In the current study, we set out to investigate whether we can perform rolling circle amplification (RCA) between a DNA oligonucleotide tethered to gold nanoparticles (AuNPs) as the primer and a single-stranded circular DNA as a template, catalyzed by a special DNA polymerase known as f29 DNA polymerase (f29DNAP). RCA is a powerful but simple biochemical method that can be used to generate long single-stranded DNA (ssDNA) with a repeating sequence unit. [10][11] In a typical RCA process, a DNA polymerase, such as f29DNAP, which has a strong ability to displace newly synthesized DNA strands, makes continuous nucleotide additions to a growing DNA chain over a short circular single-stranded DNA as the template under isothermal conditions. As a result, long, linear tandemly repetitive single strands of DNA are produced. As the synthesized long DNA molecules contain many repeating sequence motifs, RCA coupled with ensuing hybridization with fluorescent DNA probes has been used as an on-chip signal-amplification tool for sensitive biosensing. [12][13][14][15][16] Recently, it has been shown that long DNA molecules from RCA can be used as scaffolds for assembling nanoparticles to form 1D nanoparticle arrays. [17][18] However, to our knowledge, performing the RCA reaction on gold nanoparticles and using the resultant special DNA-AuNP assemblies to form 3D nanoparticle array have not been demonstrated. Figure 1 schematically illustrates the RCA process on gold nanoparticles and its scaffolding for 3D periodical nanoassemblies. Gold nanoparticles of 15 nm diameter were first prepared by the classical citrate reduction route. Thiolmodified DNA primers (41 nucleotides) were then functionalized onto the nanoparticles following Mirkins approach. [5] The concentration of DNA primers on the gold nanoparticles, referred to hereafter as primer-Au, was determined using UV/Vis spectroscopy by measuring the amount of DNA in solution before and after coupling; each primer-Au contained approximately 230 DNA primer strands. A 63-nucleotidelong circular DNA template was then annealed with the DNA-functionalized gold nanoparticles. The hybridization efficiency, as examined by measuring the radioactivity on the gold nanoparticles and in solution after annealing with a radiolabeled circular DNA ...

An in vitro selection endeavor previously executed by our laboratory led to the isolation of a set of RNA-cleaving deoxyribozymes that thrive under acidic conditions [Liu, Z., Mei, S. H., Brennan, J. D., and Li, Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One of these sequences, coined pH5DZ1, is a 100-nucleotide (nt) cis-acting enzyme that was found to exhibit high cleavage activity near pH 5. Herein, we seek to deduce the properties and sequence requirements of this enzyme. This deoxyribozyme was found to cleave a 23-nt chimeric DNA-RNA substrate, which contains a single ribonucleotide flanked by fluorophore- and quencher-modified nucleotides on each side of the cleavage junction. Extensive nucleotide deletion experiments indicated that only 42 bases within the original enzyme sequence are required for catalysis. Results from a reselection experiment further revealed that 26 of these nucleotides are absolutely conserved. In addition to sequence analysis and minimization studies, we successfully designed a trans-acting variant of this enzyme. Characterization of the cleavage products produced upon pH5DZ1-mediated RNA cleavage and analyses of possible structures of pH5DZ1 provided us with insights into the catalytic mechanism of pH5DZ1 and characteristics of deoxyribozymes that retain their activity under acidic conditions.

A convenient method involving 32P-labeling of an RNA substrate at the cleavage site and subsequent enzymatic digestion of cleavage products via phosphatases reveals that pH4DZ1--an RNA-cleaving deoxyribozyme with optimal activity at pH 4--forms a 5'-cleavage fragment with 2',3'-cyclic phosphate group and a 3'-cleavage fragment with 5'-OH group.

The biomimetic oxidation of etodolac, an anti-inflammatory drug (1) with iodosylbenzene catalyzed by 5,10,15,20-tetraarylporphyrinatoiron(III) chlorides TAPFe(III)Cl (7a-e) in dichloromethane gives 4-hydroxyetodolac (6) and 4-oxoetodolac (5) regioselectively in moderate yields.Key words etodolac; halogenated and perhalogenated metalloporphyrin; iodosylbenzene; 4-hydroxyetodolac; 4-oxoetodolac Etodolac, 1,3,4,-indole-1-acetic acid (1) is a clinically effective antiinflammatory agent, possesses an exceptional safety profile with respect to the gastrointenstinal tract and renal function.1,2) The major primary oxidative metabolites of 1 in man are 6-hydroxyetodolac (2), 7-hydroxyetodolac (3), and 8-(1Ј-hydroxy)etodolac (4), whereas the major metabolite in rat is 4-oxoetodolac (5). The biomimetic oxidation of selected drugs with monooxygen donors catalyzed by 5,10,15,20-tetraarylporphyrinatoiron(III) chlorides (TAPFe(III)Cl) has been studied to understand the drug metabolism as well as to isolate the drug metabolites and reactive intermediates in sufficient amount without the use of experimental animals.3-6) The catalytic activity of metalloporphyrins can be increased either by introduction of bulkier substituents at ortho position of the phenyl rings or by substitution of hydrogens by halogens of main porphyrin ring of metalloporphyrins (TAPFe(III)Cl). Herein, we report the biomimetic oxidation of etodolac with iodosylbenzene catalyzed by halogenated and perhalogenated iron(III) porphyrins TAPFe(III)Cl (7a-e) 7) in organic solvents to mimic the reactions of natural cytochrome P450 enzyme.Iodosylbenzene (1 mmol, 220 mg) was added to a mixture of 1 (1 mmol, 287 mg) and Cl 8 TPPFe(III)Cl (7b) (0.01 mmol) in dichloromethane (10 ml). The reaction mixture was stirred for 2 h at room temperature. The solvent was removed under reduced pressure and the residue was chromatographed on silica gel. The elution of column with petrol : chloroform The reaction of 1 with iodosylbenzene catalyzed by TPPFe(III)Cl (7a) in dichloromethane in after 2 h gives regioselctively 6 in 11.3% and 5 in 5.0% yield (Chart 1).