Oriented fibers drawn from aqueous gels of calf-thymus DNA were maintained at constant relative humidites of 75 and 92% to yield canonical A-DNA and B-DNA structures, respectively. Raman spectra of the two forms of DNA were recorded over the spectral range 300–4000 cm−1. The authenticated DNA fibers were deuterated in hygrostatic cells containing D2O at appropriate relative humidities, and the corresponding spectra of deuterated DNAs were also obtained. The spectra reveal all of the Raman scattering frequencies and intensities characteristic of A- and B-DNA structures in both nondeuterated and deuterated froms, as well as the frequencies and intensities of adsorbed solvent molecules from which the hydration content of DNA fibers can be calculated. Numerous conformation-sensitive vibrational modes of DNA bases and phosphate groups have been identified throughout the 300–1700-cm−1 interval. Evidence has also been obtained for conformation sensitivity of deoxyribosyl CH stretching modes in the 2800–3000-cm−1 region. Raman lines of both the backbone and the bases are proposed as convenient indicators of A- and B-DNA structures. The results are extended to Z-DNA models investigated previously. Some implications of these findings for the determination of DNA or RNA structure from Raman spectra of nucleoproteins and viruses are considered.

Characterization of DNA structures by laser Raman spectroscopy.

Applications of Raman spectroscopy to investigate the molecular constituents of nucleic acids were initiated in the late 1960s and soon thereafter progressed to studies of synthetic and native nucleic acids and complex biological assemblies containing either DNA or RNA. Raman applications to nucleic acids have continued to increase in number and diversity up to the present time. This paper attempts to provide an overview of this large body of work, with emphasis on studies carried out during the past decade and focusing on problems of biological interest and significance. The specific Raman methodologies included in this review of nucleic acid applications are (i) conventional Raman spectroscopy (i.e. off-resonance Raman excitation), (ii) ultraviolet resonance Raman (UVRR) spectroscopy, and (iii) polarized Raman microspectroscopy. For each methodology, the experimentally obtained nucleic acid spectrum consists of a number of discrete vibrational bands, most of which can be assigned confidently to a base, sugar or phosphate constituent of the macromolecule and many of which can be employed as sensitive indicators or fingerprints of either local structure, global conformation, intermolecular interaction or molecular dynamics. The applications selected for review include numerous examples from the authors' laboratories. The topics addressed include the influences of base composition, base sequence, superhelical stress drug ligation on nucleic acid structure polymorphism, the thermodynamic parameters governing nucleic acid premelting melting phenomena, the molecular mechanisms determinants of protein/nucleic acid recognition the structures dynamics of nucleic acids in virus assemblies. Copyright © 2005 John Wiley & Sons, Ltd.

Raman, polarized Raman and ultraviolet resonance Raman spectroscopy of nucleic acids and their complexes

Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.

The frequencies and intensities of the laser Raman spectra of poly-L-lysine (PLL) have been observed in the following studies: (1) the thermally induced α-to-β transition which occurs with increasing temperature at high pH; (2) the ionized form to α transition at 10°C by increasing pH; and (3) the ionized form to α transition by ionic strength at low pH.



The frequency-dependent bands which have been observed are the amide I (in H2O), amide I′ (in D2O), amide III, and C–C stretch. It has been found possible to assign an unique set of frequencies and intensities to each conformation of PLL of α, β, and ionized form. In this way the nature of the conformations intermediate in the transitions can be determined. The frequencies of the amide III and amide III′ are very weak in the α-helix and somewhat higher than usual in the β form. Hence it appears the amide III and amide III′ bands may differ from one type of polypeptide to another with the same backbone conformation.

Laser Raman studies of conformational variations of poly‐L‐lysine

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT) poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC)poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

Raman spectra are presented on ordered and presumably helical structures of DNA and RNA as well as the poly A·poly U helical complex, polydAT, and the helical aggregates of 5′-GMP and 3′-GMP The changes in the frequency and the intensity of the Raman bands as these structures undergo order-disorder transitions have been measured In general the changes we have found can be placed into three categories: (1) A reduction in the intensities of certain ring vibrations of the polynucleotide bases is observed when stacking or ordering occurs (Raman hypochromism) Since the ring vibrational frequencies are different for each type of base, we have been able to obtain some estimate of average amount of order of each type of base in partially ordered helical systems (2) A very large increase in the intensity of a sharp, strongly polarized band at about 815 cm−1 is observed when polyriboA and polyriboU are formed into a helical complex Although this band is not present in the separated chains at high temperature, a broad diffuse band at about 800 cm−1 is present The 815 cm−1 band undoubtedly arises from the vibrations of the phosphate-sugar portions of the molecule and provides a sensitive handle to the back-bone conformation of the polymer This band also appears upon ordering of RNA, formation of the helical aggregate of 5′-riboGMP, and to some extent in the selfstacking of the polyribonucleotides polyA, polyU in the presence of Mg++, PolyC, and polyG No such intense, polarized band is found, however, in ordered DNA, polydAT, or the 3′-riboGMP aggregate, although there is a conformationally independent band at about 795 cm−1 in DNA and polydAT (3) Numerous frequency changes occur during Conformational changes In particular the 1600–1700 cm−1 region in D2O shows significant conformationally dependent changes in the CO stretching region analogous to the changes in this region which have been observed in these substances in the infrared Thus, Raman scattering appears to provide a technique for simultaneously observing the effects of base stacking, backbone conformation and carbonyl hydrogen bonding in nucleic acids in moderately dilute (10–25 mg/ml) aqueous solutions

Exoch W. Small

Papers

Conformational dependence of the Raman scattering intensities from polynucleotides. III. Order-disorder changes in helical structures