Binding of actinomycin D to DNA revealed by DNase I footprinting

We have analyzed the specificity of the actinomycin D-DNA interaction. The 'footprint' method has been used in this investigation. It is shown that: (i) The presence of dinucleotide GC or GG is required for binding of a single drug molecule. (ii) The strong binding sites are encoded by tetranucleotide XGCY; where X not equal to G and Y not equal to C in accordance with RNA elongation hindrance sites [1]. (iii) There is a positive cooperativity in binding of actinomycin D with DNA.     

Investigation of the binding of Escherichia coli RNA polymerase to DNA from bacteriophages T2 and T7 by kinetic formaldehyde method and electron microscopy.

The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy. For electron-microscopic investigations the effect of different conditions of formaldehyde fixation for DNA-RNA-polymerase complexes was studied and optimal fixation conditions were found. The suggested fixation method for DNA-RNA-polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ration of enzyme to DNA etc.). The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence. The electron-microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA. A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found.     

In vitro selection of single-stranded DNA aptamers that bind human pro-urokinase

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase (urinary plasminogen activator) with a Mr of 50,000 which is converted to the active two-chain form by catalytic amounts of plasmin. It is used for thrombolytic therapy of acute myocardial infarction and acute ischemic stroke. We have isolated single-stranded DNA molecules with significantly increased binding affinity for human pro-urokinase by SELEX (systematic evolution of ligands by exponential enrichment) procedure from a pool of 10(15) molecules containing 24 randomized positions which are flanked by defined regions. ssDNA from this library was hybridized with helper "fixture", thus allowing the central random chain to fold into complex three-dimentional shapes. Sequencing data from pro-urokinase aptamers obtained after 12 selection cycles displayed a highly conserved 12-14 base region.