Marina G. Ivanovskaya

Detection of Glycosylase, Endonuclease and Methyltransferase Enzyme Activities Using Immobilized Oligonucleotides

A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme‐dependent label release (in case of glycosylase and endonuclease), or non‐release (in case of methyltransferase) into solution from end‐labeled DNA immobilized on solid support (CPG or Tenta Gel S‐NH2). The assay has been validated for monitoring activity of repair enzyme uracil‐DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine‐5)‐DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase‐immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary. IUBMB Life, 56: 139‐143, 2004

Modified DNA fragments specifically and irreversibly bind transcription factor NF-κB in lysates of human tumor cells

Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-κB has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used 32P-labeled 17/19 bp nucleotide DNA duplex containing an NF-κB recognition site (κB-site) in which one of internucleotide phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 32P-labeled DNA duplex with NF-κB in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3′-end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-α promoted partial translocation of the DNA reagent into the nucleus.

Genetic Analysis of Fluorescent Labeled Amplified DNA Using Series of Oligonucleotide Probes Covalently Bound to Membranes

Recently proposed reverse hybridization method is very convenient for simultaneous detection of a set nucleotide sequences in a DNA fragment of interest (1,2). The main idea of this method is immobilization of an oligonucleotide probes set on a solid support and their hybridization with the labeled DNA fragment. Here we report on an improved non-radioactive reverse hybridization method and provide an example of its application for testing point mutations responsible for β-thalassemia.