Electron spin resonance for detecting polyadenylate tracts in RNA's.

Electron spin resonance is used to detect RNA's that contain polyadenylate tracts. The method depends on the ability of RNA's that contain polyadenylate sequences to associate with poly(2'-deoxy-2'-fluoro)uridylic acid, which has been spin-labeled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl. The formation of the hybridization product can be detected by monitoring the decrease in mobility of the spin probe.

Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.

Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy.

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by electron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.

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.

Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease.

Deoxyuridine analogs spin labeled in position 5 have been enzymatically incorporated sequence specifically into an oligodeoxyribonucleotide to form a spin-labeled 26-mer. The 26-mer contains the EcoRI-binding site and two labels which are located symmetrically close to the binding site. The labels are separated from one another far beyond the Heisenberg spin-exchange distance. The local base motion as determined by ESR spectroscopy is of the order of 4 ns in the oligonucleotide duplex. This is the same value as reported earlier for local T motions in polynucleotide duplexes, thereby providing direct experimental evidence that the ESR line shape of spin levels covalently attached to nucleic acids depends primarily on the local dynamics of the nucleic acid building blocks.

Variability in the nucleic acid binding site size and the amount of single-stranded DNA-binding protein in Escherichia coli.

The Escherichia coli single-stranded DNA binding protein (SSB), essential for DNA replication, recombination and repair, can undergo a thermally induced irreversible conformational change which does not eliminate its biological activity, but changes the number of nucleotides it covers (binding site size) when binding to a single-stranded nucleic acid lattice. The binding site size of native and conformationally changed SSB was also found to be a function of the molecular mass of the polynucleotide, an observation which is unusual for single-stranded DNA binding proteins and will greatly affect the affinity relationship of this protein for nucleic acids. A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB tetramers to be larger than initial estimates by a factor of as much as six. All these data suggest that the biological role of SSB and its mechanism of action is by far more complex than originally assumed.

Spin probes as mechanistic inhibitors and active site probes of thymidylate synthetase.

C-4- and C-5-substituted analogues of dUMP were examined as inhibitors of thymidylate synthetase and as topographical probes of its active site by electron spin resonance (ESR). The C-5-substituted spin-labeled analogues pDUAP (2) and a pDUTT (3) as well as the unlabeled AAdUMP (1) were competitive inhibitors with Ki's of 9.2, 89, and 7.9 microM, respectively. The C-4-spin-labeled pls4dU (4) displayed no inhibition activity. Scatchard plots as determined by ESR gave similar association constants for 2 (Kassoc = 1.9 X 10(5) M-1) and for 3 (Kassoc = 2.4 X 10(5) M-1). Both of these values are similar to the Kassoc of FdUMP indicating that the bulky substituent in position 5 does not interfere with the formation of the binary complex. The enzyme-C-5-spin-labeled nucleotide complexes indicate the presence of similarly immobilized spin labels by ESR, whereas no binding and immobilization were noticed with the C-4-spin-labeled nucleotide. A model for the active-site geometry of the enzyme was derived which suggests that the C-5 substituents point toward the opening of the binding cavity whose depth is at least 12 A. Also, the approximate 10-fold increased inhibitory activity of 2 as compared to that of 3 may be attributed to the significant electron withdrawing properties of the C-5 substituent in 2. Finally, the set of probes used for the binding and inhibition of thymidylate synthetase gives direct experimental evidence that an electron-withdrawing C-5 substituent primarily affects the formation of the ternary complex and will not substantially influence the stability of the binary complex.

5-O-Alkylated derivatives of 5-hydroxy-2'-deoxyuridine as potential antiviral agents. Anti-herpes activity of 5-propynyloxy-2'-deoxyuridine.

Alkylation of 5-hydroxyuridine or 5-hydroxy-2'-deoxyuridine with various activated alkylating agents in the presence of 1 equiv of NaOH gave a series of new nucleoside analogues which were evaluated for antiviral activity against vaccinia virus, herpes simplex-1 virus, and vesicular stomatitis virus in both primary rabbit kidney cells and human skin fibroblasts. One of these analogues, 5-propynyloxy-2'-deoxyuridine, was a potent inhibitor of herpes simplex virus. Structure-activity considerations suggest that the anti-herpes activity is dependent on the integrity of the acetylene group since substitution of phenyl, p-nitrophenyl, vinyl, carboxamido, or carboxyl for the triple bond led to diminished antiviral activity.

A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and single-strand binding proteins.

An EPR competition formalism is developed which provides relative affinities of proteins for nucleic acids. Two models for analyzing protein-nucleic acid interactions, one assuming independent binding sites (Model 1) and the other considering site overlap (Model 2), are examined with respect to their validity and limitations. The models are employed to derive affinity ratio relationships which are used to calculate the relative affinities of gene 32, gene 5, and SSB proteins for various nucleic acids. It is determined that although Model 2 must be used when determining absolute binding constants, by taking the ratio of binding constants the site overlap becomes unimportant under conditions of moderate to high cooperativity and relatively small site size. This allows Model 1 to considerably simplify binding analyses. Both models are applied to the single-strand binding proteins of bacteriophage T4 gene 32, bacteriophage fd gene 5, and the Escherichia coli ssb gene, and the results are compared.

Gene 5 protein-DNA complex: modeling binding interactions.

A helical (not toroidal) complex consisting of eight gene 5 protein dimers per turn is proposed for the extension of DNA from dimer to dimer using known bond length constraints, postulated protein-nucleic acid interactions (determined from NMR and chemical modification studies), other physical properties of the complex, and data from electron micrographs. The binding channel has been dictated by these known parameters and the relative ease of geometrically fitting these constituents. This channel is different from that previously reported by other modelers. The channel lies underneath the long arm "claw-like" extension of the monomer, so that it rests inside the outer surface of the protein complex. An explanation is proposed for the two binding modes, n = 4 (the predominate mode) and n = 3, based on the weak binding interaction of Tyrosine 34. Also, the site of the less mobile nucleic acid base as reported from ESR studies (S.-C. Kao, E.V. Bobst, G.T. Pauly and A.M. Bobst, J. Biom. Struc. Dyn. 3,261 (1985)) is postulated as involving the fourth nucleotide, and this particular base is stacked between Tyrosine 34 and Phenylalanine 73'.

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities in poly(deoxythymidylic acid)-gene 5 protein complexes.

A set of differently spin labeled (dT)n is used to evaluate thymidine dynamics and some of the structural features in a (dT)n-gene 5 protein complex. ESR evidence is presented that only one of the four thymidine residues bound in the DNA binding channel shows strong immobilization, whereas the other three display significant mobility of the order of nanoseconds. It is hypothesized that the accessability of such mobile bases could be critical to the recognition of the (dT)n-gene 5 protein complex in auxiliary interactions with other proteins and competitive DNAs.

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes.

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.

Spin-labeled nucleotide mobility in the boundary of the EcoRI endonuclease binding site.

A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 +/- 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S = 0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.

A low-cost microcomputer-based data acquisition and analysis system for an electron spin resonance spectrometer: data handling of dilute spin labeled nucleic acids.

This article describes the construction of an inexpensive and reliable data acquisition system for a Varian E-line Century Series ESR spectrometer utilizing an Apple II Plus microcomputer. All necessary hardware is readily available and used without modification. A BASIC program for routine collection, display, plotting and disk storage of experimental data has been written and subsequently compiled into machine code for high speed operation. The interface offers distinct advantages in spectral resolution as well as instrument control. An example of signal enhancement via computer controlled time averaging is presented for a spin labeled DNA experiment. The technique has recently been applied to studies of relative binding affinities of gene-32 protein for various spin-labeled polynucleotides.

Light-induced electron-transfer reactions between chlorophyll a and hydrogenated pteridine derivatives in solution.

Preliminary studies of the formation of cationic 2 - amino - 6, 7 - dimethyl - 4 - hydroxy - 5, 6, 7, 8 - tetrahydropteridine radicals in alcoholic solution are reported. The results indicate that the same type of radical is formed either by oxidizing agents like hydrogen peroxide and iodine, or in a photoinduced reaction with chlorophyll a. A similarity between the epr signal of the light-induced cationic pteridine radical and the photoinduced epr signal II in photosynthesis is observed.