George J. Thomas

A phase diagram for sodium and potassium ion control of polymorphism in telomeric DNA

Switching between antiparallel and parallel quadruplex structures of telomeric DNA under the control of intracellular Na+ and K+ has been implicated in the pairing of chromosomes during meiosis. Using Raman spectroscopy, we have determined the dependence of the interquadruplex equilibrium of the telomeric repeat of Oxytricha nova, upon solution concentrations of Na+ and K+. Both alkali cations facilitate the formation of an antiparallel foldback quadruplex at low concentration, and a parallel extended quadruplex at higher concentration. However, K+ is more effective than Na+ in inducing the parallel association. We propose a phase diagram relating d(T4G4)4 polymorphism to intracellular [Na+]/[K+] ratios. The phase diagram indicates that the interquadruplex equilibrium is highly sensitive to changes in the mole fraction of either cation when the total concentration falls within the interval 65 to 225 mM, a range which encompasses total of the Na+ and K+ concentrations occurring in a typical mammalian cell. These results support a role for the guanine-rich overhang of eukaryotic DNA in promoting chromosome association during meiotic synapsis.

Solution conformations of nucleoside analogues exhibiting antiviral activity against human immunodeficiency virus

Abstract The molecular-conformational basis for HIV-1 antiviral activity of dideoxynucleoside analogues is unknown. A recent proposal by van Roey [1] that furanose sugar puckering in the C2′ -endo family (namely C3′ -exo) may account for the enhanced anti-HIV-1 activity of azidothymidine (AZT), dideoxythymidine (ddT) and dideoxycytidine (ddC) has been tested by conformational analysis of these and related agents, using laser Raman spectroscopy of their solutions and crystal structures. The results show that nucleoside analogues exhibiting anti-HIV-1 activity, including AZT, ddT and ddC, exist in solution with C3′ -endo as the predominating sugar pucker. The C3′ -endo solution conformations differ fundamentally from the C3′ -exo conformations observed in the corresponding crystal structures. Accordingly, the crystal conformation cannot be responsible for enhanced recognition of these agents, either by nucleoside kinase or reverse transcriptase, as a mechanism to explain antiviral activity. The present findings suggest that C3′ -endo sugear pucker, rather than C3′ -exo pucker, or other puckers of the C2′ -endo family, is more probably the required conformation for antivaral activity. The present work also shows that nucleoside phosphorylation does not, in general, change the preferred solution conformation of a nucleoside. Therefore, C3′ -endo sugar pucker is likely to be the preferred conformation for both nucleoside kinase and reverse transcriptase recognition. In this study, the list of thymidine nucleoside conformation markers available from Raman spectra is extended and additional group frequency assignments for C3′ -azido, C3′ -deoxy and related nucleoside derivatives are provided.

Raman Scattering Tensors in Biological Molecules and Their Assemblies

INTRODUCTION The tensor associated with a Raman band plays an important role in determining the band intensity and its structural significance. Each Raman tensor interrelates two electric vectors, that of the exciting radiation (i.e. laser photon) and that of the Raman scattered radiation (i.e. the inelastically scattered photon which results from the exchange of a vibrational quantum between the exciting photon and the molecule). The Raman tensor is obtained formally as the first derivative of the molecular polarizability tensor, the derivative being taken with respect to the vibrational normal coordinate. In other words, the Raman tensor associated with a vibrational Raman band is an indicator of how the polarizability of the molecule oscillates with the molecular normal mode of vibration.

Vibrational analysis of nucleic acids. III. Conformation-dependent Raman markers of the phosphodiester backbone modeled by dimethyl phosphate☆

Abstract The dimethyl phosphate anion (CH3O)2PO−2 is the simplest structural analogue of the phosphodiester backbone group in nucleic acids. Because of its simplicity, dimethyl phosphate is amenable to experimental and theoretical analyses of conformation-dependent vibrations of the diester (Cue5f8Oue5f8Pue5f8Oue5f8C) and dioxy (PO−2) linkages [Guan et al., Biophys. J., 66 (1994) 225; J. Phys. Chem., 99 (1995) 12054]. In the present work, we report the use of a previously developed generalized valence force field to determine the dependence of vibrational stretching frequencies of the dimethyl phosphate anion upon the conformational geometry of the phosphodiester moiety. Starting with the gauche-gauche Cue5f8Oue5f8Pue5f8Oue5f8C conformation (symmetry point group C2), we have computed the frequency dependence of six diagnostic vibrational stretching modes upon stepwise rotation of each carbon linkage with respect to its phosphoester bond (Oue5f8P torsion). The computations show that both antisymmetric and symmetric phosphodiester (Oue5f8Pue5f8O) stretching vibrations are highly sensitive to Cue5f8Oue5f8Pue5f8Oue5f8C conformation, whereas the corresponding phosphodioxy (PO−2) stretching modes are relatively insensitive to conformation changes. The present calculations provide a theoretical basis for previously established empirical correlations which indicate that phosphodiester stretching modes are the definitive indicators of backbone conformation in the vibrational spectra of DNA and RNA.

Raman tensors for the tryptophan side chain in proteins determined by polarized Raman microspectroscopy of oriented N-acetyl-l-tryptophan crystals

Abstract Polarized Raman spectra have been obtained from oriented single crystals of N -acetyl-l-tryptophan by use of a Raman microscope and 488.0 nm argon excitation. The crystal, of orthorhombic space group P2 1 2 1 2 1 , provides the relative Raman intensities I aa , I bb and I cc corresponding to the aa, bb and cc components of the crystal Raman tensor. The polarized Raman spectra of the crystal have been combined with depolarization ratios from solution spectra of randomly oriented N -acetyl-l-tryptophan and l-tryptophan to yield Raman tensors for each of the following vibrational normal modes of the indole moiety: N 1 ue5f8H stretch (≈3416 cm −1 ), WI (≈1617 cm −1 ), W 2 (≈1576 cm −1 ), W 3 (≈1557 cm −1 ), W 4 (≈1487 cm −1 ), W 5 (≈1458 cm −1 ), W 6 (≈1424 cm −1 ), W 7 (≈1357 cm −1 ), W 7′ (≈1332 cm −1 ), W 16 (≈1010 cm −1 ) and W 18 (≈757 cm −1 ). These Raman tensors determined for the tryptophan residue in N -acetyl-l-tryptophan are proposed as being transferable to tryptophan side chains in proteins. A knowledge of Raman tensors for the tryptophan side chain should facilitate the determination of indole ring orientation in biological complexes amenable to investigation by the method of polarized Raman microspectroscopy.

Water as a Bioactivator and Probe of DNA Structure: Investigation by Laser Raman Spectroscopy

The water molecule may be regarded as the quintessential ‘inorganic bioactivator’ of DNA or RNA. Here we consider the use of laser Raman spectroscopy to examine the role of water in stabilizing equilibrium structures of nucleic acids. Both high frequency (3000–4000 cm-1) and low frequency (0–300 cm-1) regions of the DNA vibrational spectrum provide information which can be correlated with adsorbed and bulk water molecules, and with effects of H2O upon DNA conformation. New correlations between the Raman frequencies of DNA and the macromolecular conformation are presented and tabulated with previously established correlations. A second objective of this paper is to demonstrate the power of the Raman method to monitor the dynamics of interaction between solvent water molecules and specific purine sites along the grooves of the DNA double helix. Solvent-mediated isotope exchanges of DNA purines (8C deuterations) may be accurately and quantitatively discriminated by means of Raman optical multichannel analysis to provide a sensitive probe of structural micro-heterogeneity in DNA.