Biomedical subjects

Arti Pothukuchy

Publications and source records attributed to Arti Pothukuchy.

4 recordsLinked to original sources

PubMed2006

A potentially insect-implantable trehalose electrooxidizing anode.

The dominant sugar in the body fluids of many insects is not glucose, the sugar of the vertebrates, but trehalose. In a step toward a cell that would operate in insects, we describe here a trehalose electrooxidizing anode. The novel component of the anode is its engineered, trehalose oxidation catalyzing, FAD-glucose-3-dehydrogenase (G3DH). Screening for gene-sources of G3DH pointed to the G3DH of Agrobacterium tumefaciens. Sequencing of the A. tumefaciens genome revealed a 1.7 kb fragment which contained the G3DH coding gene. The fragment was isolated, cloned and expressed in E. coli strain BL-21, to yield the approximately 65 kDa his-tagged flavoenzyme, with a specific activity of approximately 2.5U/mg protein. Electrical wiring of its reaction center to a carbon electrode through a high apparent electron diffusion coefficient (5.8 x 10(-6)cm(2)/s) redox hydrogel with a -0.2V versus Ag/AgCl redox potential resulted in the trehalose electrooxidizing anode. Trehalose was electrooxidized at pH 7.2 already at -0.36 V versus Ag/AgCl. At 0 V versus Ag/AgCl the trehalose electrooxidation current density was 0.1 mA/cm(2).

Animals↗

PubMed2005

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion.

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.

Base Sequence↗

PubMed2004

Detection of approximately 10(3) copies of DNA by an electrochemical enzyme-amplified sandwich assay with ambient O(2) as the substrate.

The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, and Heller (Anal. Chem. 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD). BOD catalyzes the reduction of ambient O(2) to water and obviates the need for adding H(2)O(2). Femtomolar (10(-)(15) M) concentrations of DNA were detected at a 10-microm-diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in approximately 5-microL samples. The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound. Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode. Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of O(2) to water at +0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, approximately 3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.

Animals↗

PubMed2003

Photo-oxidation of duplex DNA with the stable trioxatriangulenium ion.

We show that 5'-Gs in 5'-GG-3' duplex DNA dinucleotide steps are preferentially oxidized by the trioxatriangulenium ion (TOTA( plus sign in circle )) upon photo-activation, an oxidation pattern characteristic of guanine radical cation formation. Some photo-oxidation of the 3'G in 5'GG3' steps and of isolated guanines is also observed but reactions carried out in D(2)O reveal only a minor increase in oxidation damage at these sites, indicating that electron transfer is the primary mechanism of guanine oxidation.

Base Sequence↗