Affinity of reverse transcriptase for some polynucleotide inhibitors.

The relative affinity of avian myeloblastosis virus reverse transcriptase for (U)n and a series of (U)n analogs has been measured directly in solution under conditions previously used to demonstrate the inhibitory properties of these polynucleotides. The affinities were measured by electron spin resonance through a quantitative competition approach where the concentration of each polynucleotide required to compete with the macromolecular spin probe (ls4U,U)n for reverse transcriptase was observed. Using this approach the following relative affinities were determined: K(dUfl)n = 1.6K(dUz)n = 13K(dT)n = 20K(U)320-640 = 57K(dU)n = 167K(U)80 greater than 167K(Um)n These results show that the affinity of (U)n for reverse transcriptase is affected by modifying the (U)n matrix and by the molecular weight of (U)n. In addition, the effect of some factors such as Mg+2 and salt on the polynucleotide affinity for the enzyme was measured. The results show that the same binding, i.e., the fraction of saturation F as a function of the nanomoles of reverse transcriptase added, was observed in the presence or absence of Mg+2, whereas increasing the KCl concentration from 0.04 to 0.5M completely dissociates the polynucleotide X enzyme complex.

Interaction of polymerases with 2'-deoxyuridine-5'-triphosphate spin-labeled at the 5-position.

2'-Deoxyuridine-5'-triphosphate spin-labeled at the 5-position with N-[1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl]-O- was found to be an inhibitor of some DNA and RNA polymerases including avian myeloblastosis virus reverse transcriptase. Furthermore, the spin-labeled nucleotide was found to be incorporated internally into polydeoxythymidylic acid via reverse transcriptase to an extent of 1.0 spin-labeled base per 10(3) bases. The incorporation, monitored by electron spin resonance, is analogous to some other nucleotide inhibitors of polymerases, and the results indicate that it may be feasible to obtain sequence specific, spin-labeled DNA, enzymatically.

Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode.

This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins. The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids. It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used. The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid.