Interactions of heteroaromatic compounds with nucleic acids. 2. Influence of substituents on the base and sequence specificity of intercalating ligands.

This paper presents the results of a systematic study on the effects of substituents on the base and sequence specificity of tricyclic heteroaromatic compounds interacting with DNA by intercalation. All the compounds tested are derived from proflavine and acridine orange analogs with different heteroatoms in the middle ring. Their base and sequence specificities were determined by differential dialysis of the ligand against DNA samples of differing G-C content. The main results indicate that (a) the introduction of a phenyl substituent into one of the two available positions of the middle ring increases or decreases the G-C specificity of the ligand depending on the position where the substitution takes place; (b) compounds of the substitution type of neutral red (2-methyl-3-amino-7-dimethyl-amino-phenazine) show unexpectedly high G-C specificities and (c) DNA ligands of pronounced sequence specificity for adjacent G-C pairs can be constructed by combining the structural elements of neutral red with an additional phenyl residue in the same molecule. The further study of compounds related to the phenylated neutral red revealed that the G-C specificity can be improved or destroyed by additional substituents. The comparison of the G-C specificity and the DNA-affinity data of the compounds studied leads to the suggestion that the specificity arises mainly from electronic factors which are strongly controlled through steric constraints on possible ocmplex geometries. As a basis for the discussion a possible structure for the DNA complex of the phenylated neutral red is considered in which the extra phenyl ring at N-5 of the phenazinium system, protrudes into the large groove of the DNA helix while the tricyclic part of the ligand is inserted between the DNA base-pairs.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

RNA-Ascorbate Interaction

Abstract

Ascorbic acid and divalent iron salts have been widely used to investigate the effects of reactive oxygen species in different biological targets such as nucleic acids, proteins and lipids. This study was designed to examine the interaction of yeast RNA with vitamin C in aqueous solution at physiological pH with drug/RNA(P)(P=phosphate) molar ratios of r= 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy were used to determine the ascorbate binding mode, binding constant, sequence selectivity and RNA secondary structure in aqueous solution.

Spectroscopic evidence showed that at low drug concentration (r=1/80 and 1/40), no major ascorbate-RNA interaction occurs, while at higher drug concentrations (r>1/40), a major drug-RNA complexation was observed through both G-C and A-U base pairs and the backbone phosphate groups with k=31.80 M^?1. Evidence for this comes from large perturbations of the G-C vibrations at 1698 and 1488 cm^?1 and the A-U bands at 1654 and 1608 cm^?1 as well as the phosphate antisymmetric stretch at 1244 cm^?1. At r>1/10, minor structural changes occur for the ribose-phosphate backbone geometry with RNA remaining in the A- family structure. The drug distributions around double helix were about 55% with G-C, 33% A-U and 12% with PO₂ groups. A comparison between ascorbate-RNA and ascorbate-DNA complexes showed minor differences. The ascorbate binding (H-bonding) is via anion CO and OH groups.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

Poly(L-valine). Conformational dependence on the degree of polymerization: infrared studies.

The infrared spectra of poly(L -valine)'s with varying degrees of polymerization have been investigated, as well as copolymers of L -alanine and L -valine. The spectra of nujol mulls of various molecular-weight poly(L -valine)'s, isolated directly from the polymerization media, as well as spectra of these same samples after treatment with strong acid, are recorded. In the 700–250-cm^?1 region, bands at 543 and 414 cm^?1 are found to increase with increasing degree of polymerization in the nujol mulls, but are missing in the acid-treated samples. These bands are assigned to the L -valine residues with an β-helixlike local conformation. It is inferred that the polymerization proceeds initially in the β form, and after a critical degree of polymerization the chains adopt an appreciable amount of an α-helixlike local conformation.     

Complexes of silver ion with natural and synthetic polynucleotides

A study of silver-ion binding by nucleic acids and synthetic ribo and deoxyribopolynucleotides, has been carried out by means of potentiometric titration, thermal transition, and difference spectra. It is clearly demonstrated that a strong complex between Ag⁺ and nitrogen atoms of bases is made reversibly. Binding constants and site numbers are determined for each type of polynucleotide. Base reactivity varies strongly with chain length, and a cooperative phenomenon is found in each case. Two successive complexes with DNA are seen in all the three techniques, and they have the same characteristics as complexes with respectively poly-dGC and poly dAT. In the first complex, Ag⁺ is linked to four bases, provided two of them are a G-C pair. Calculated and experimental values of site numbers agree very well for DNA of different G-C content. Thermal stabilization occurs simultaneously, and the increase of melting temperature corresponds to calculated changes of stacking energy between base pairs. In the second complex a new ordered structure insensitive to temperature is formed, with simultaneous release of protons. The stoichiometry can be related to base sequence. Complexing with silver increases the resistance of TMV RNA to both temperature and ribonuclease; a tentative explanation is given in the latter case.     

Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the A in equilibrium B transition of DNA in fibers and gels by laser Raman spectroscopy.

Both Raman spectra and X-ray diffraction patterns have been obtained from oriented fibers of sodium deoxyribonucleic acid (Na-DNA) as a function of salt content and relative humidity. We have confirmed the previously reported X-ray results that, for oriented fibers, the A-form always exists between 75 and 92% relative humidity and that the conformation will change to the B-form at 92% relative humidity only if an excess (3–5%) of added salt is present. Oriented fibers containing low amounts of added salt remain in the A-type conformation at 92% relative humidity and higher. An exact correlation has been found between the familiar A- and B-type X-ray diffraction patterns of DNA fibers and the Raman spectra previously reported without X-ray verification from this laboratory for the A- and B-forms. In particular, a band at 807 cm^?1 was always present when a fiber showed the A-type diffraction pattern, and this band shifts to 790 cm^?1 in the B-form. Using the Raman spectrum to determine the specific conformation of DNA in samples less amenable to X-ray analysis, we have studied the A ? Btransformation in unoriented fibrous masses of DNA and in concentrated, oriented gels. We find that in unoriented fibrous masses, the A ? B transition always occurs at 92% relative humidity even at very low salt concentration (0–4%). However, in oriented DNA gels at low salt, the A-form can persist as a metastable state to concentration as low as 20% DNA. The origin of the bands at 807 and 790 cm^?1 and the possible biological implications of these findings are discussed.     

An FT-IR Study of DNA and RNA Conformational Transitions at Low Temperatures

Abstract

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO₂ and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant.

Marker infrared bands for the B conformer have been found to be the strong band at 825 cm^?1 (sugar conformer mode) and a band with medium intensity at 690 cm^?1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm^?1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm^?1 and at 665-600 cm^?1.     

A critical review of the nucleosidic vibration modes appearing in the 800-500-cm-1 spectral region, based on new harmonic dynamics calculations

On the basis of a harmonic dynamics calculation, it is shown that in the 800–500-cm^?1 spectral region of DNA vibrational spectra, the characteristic Raman peaks and ir bands do not arise from the same nucleosidic motions. The Raman spectra involve mainly the ring-breathing modes of nucleic bases while the ir spectra reveal essentially their out-of-plane vibrations. Moreover, the calculated results show the splitting of the guanine- and adenine-residue breathing modes upon their coupling with the sugar-pucker motions. This fact is in agreement with the poly[d(G-C)] and poly[d(A-T)] Raman spectra.     

Infrared spectra of transfer ribonucleic acids. 3. Fragments of formyl-methionine tRNA from Escherichia coli

Four fragments (named K, L, M, and N) of Escherichia coli formylmethionine transfer RNA have been prepared by a partial digestion with ribonuclease T¹ followed by a chromatographic separation with a DEAE–Sphadex (A-25) column and then a DEAE–cellulose column. The fragment K is the anticodon fragment with 19 nucleotides (previously reported). L is a fragment with 57 nucleotides involving the 3′-terminal (CCA). M is a fragment with 51 nucleotides which is equal to L except that M lacks 6 nucleotides at the 3′-terminal. N is a fragment with 20 nucleotides which involved the 5′-terminal and corresponds to the complementary half to L. The infrared absorption spectrum has been observed of each of these fragments and two equimolar mixtures L + N and M + N in D₂O solutions at several temperatures. The results indicate that at 37°C, K has about 4 hydrogen-bonded base-pairs, L about 11, and M about 13. On the other hand, fragment N is found to have only three weak G-C pairs. For both L + N and M + N, 15–17 strong base pairs are found. The observations give direct support to the clover-leaf structure and at the same time provide information on the stability of each of the four arms in the structure.     

Trypanosoma cruzi: Flow Cytometric Analysis of Developmental Stage Differences in DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2–12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4–8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5–13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.     

Properties of poultry litter humic acid fractions and their metal-complexes

Summary Molecularly homogenous fractions of humic acid extracted from poultry litter were characterized by elemental and functional group analysis, molecular weight determination, U.V. and infrared spectroscopy. The divalent and trivalent metal complexes prepared from different fractions of humic acid were characterized by infrared spectroscopy. The molecular weight of molecularly homogenous fractions of poultry litter humic acid ranged from 2545 to 40219. High amounts of functional groups in low molecular weight of humic acid fraction has been indicated by infrared spectra and by chemical analysis. The presence of chromophores C=C and C=O and auxochromes C−OH, C−NH were indicated by infrared and U.V. spectra of these humic acid fractions. Stable complex formation of Fe³⁺, Cu²⁺, and Zn²⁺ with −OH, −NH₂ and −COOH ligands of humic acid fractions involved electrovalent and coordinate-covalent bonds. Intensity of absorption bands of molecularly homogenous fractions of humic acid in I.R. spectra is differing depending upon the functional groups content of humic acid fractions. Journal paper No. 5. Department of Soil Science, R.A.U., T.C.A., Pusa-Dholi Campus, Dholi-843121, Muzaffarpur, Bihar, India.     

An FTIR Spectroscopic Study of Calf-thymus DNA Complexation with Al(III) and Ga(III) Cations

Abstract

The interaction of calf-thymus DNA with trivalent Al and Ga cations, in aqueous solution at pH =6–7 with cation/DNA(P) (P=phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2 was characterized by Fourier Transform infrared (FTIR) difference spectroscopy.

Spectroscopic results show the formation of several types of cation-DNA complexes. At low metal ion concentration (r=l/80, 1/40), both cations bind mainly to the backbone PO₂ group and the guanine N-7 site of the G-C base pairs (chelation). Evidence for cation chelate formation comes from major shifting and intensity increase of the phosphate antisymmetric stretch at 1222 cm-¹ and the mainly guanine band at 1717 cm¹. The perturbations of A-T base pairs occur at high cation concentration with major helix destabilization. Evidence for cation binding to A-T bases comes from major spectral changes of the bands at 1663 and 1609 cm-¹ related mainly to the thymine and adenine in-plane vibrations. A major reduction of the B-DNA structure occurs in favor of A-DNA upon trivalent cation coordination.     

Raman spectroscopic studies of the DNA cro binding site conformation, free and bound to cro protein.

Raman spectra of the DNA binding site for cro repressor protein were obtained in the presence and absence of bound cro protein. The 17 base pair fragment is a consensus sequence of the six cro binding sites in phage lambda, except that the second base to the right of the center of pseudosymmetry is altered. Analysis of the spectrum of the free DNA indicates that the molecule exists in a B-like conformation with deviations from the usual B form occurring mainly in the bands assigned to A-T vibrations. The spectrum of the bound DNA was obtained by subtracting the spectrum of free cro from the spectrum of the complex which was estimated to be 90% bound. The DNA undergoes significant structural changes upon binding to the protein; most notable of these changes is a destacking of the G-C bases reflected by increases in the 1240, 1262, and 1320 cm-1 bands. A decrease in the 1361 cm-1 band that occurs has also been assigned to a destacking in guanine bases. The appearance of a 705 cm-1 band and the decrease and downshift of the 670 cm-1 band are consistent with the appearance of A-like character in the A-T region of the binding site when the protein binds; however, the spectra indicate that the entire binding site remains in a distorted B-like conformation. We use the 705 cm-1 band to estimate A-like character because the 800-850 cm-1 region is obscured by interference from strong protein bands. Other shifts in both intensity and position cannot be assigned to characteristic changes in conformation and therefore must be attributed to the protein influencing the structure in a novel way.     

Trypanosoma cruzi: flow cytometric analysis of developmental stage differences in DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2-12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4-8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5-13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.     

Vibrational spectroscopy of seaweed galactans

Information from classical infrared spectroscopy studies has been of significance for characterizing seaweed galactans. The development of Fourier transform infrared spectroscopy and of Fourier transform laser Raman spectroscopy has produced great advances in the application of vibrational spectroscopy to the structural study of polysaccharides. Computational facilities in the spectrometers allow the arithmetic manipulations of the spectra. The second-derivative mode in the FT IR spectrocopy provided more information by increasing the number and resolution of the bands in the spectra as compared to the parent ones. A review of literature data on vibrational spectroscopy of sulfated polysaccharides and new results are presented. Agar-type polymers showed two diagnostic bands in the second-derivative mode in the region 800–700 cm^–1. Carrageenans exhibited a number of bands in the region 1600–1000 cm^–1. Fourier transform laser Raman spectroscopy in the solid state gave well-defined characteristic spectra of agar and carrageenans. Both techniques can be applied to small samples in the solid state and allow differentiation in a few minutes between agar and carrageenan-type seaweed galactans. The second-derivative mode of the FT IR spectra can be applied to distinguish agar-producing from carrageenan-producing seaweeds. The spectra on KBr pellets of dried, ground agarophyte and carrageenophyte seaweed samples showed the same bands as the corresponding polysaccharides.     

Interaction of anthracyclines with DNA and chromosomes

Daunomycin and adriamycin were previously found to produce Q-like banding patterns on chromosomes. The interaction of several anthracyclines with both natural and synthetic DNAs and chromosomes has been investigated in more detail. Daunomycin fluorescence is almost completely quenched by natural DNAs with varying base composition from 31 to 72% G-C and by the alternating polymer poly-d(G-C)·poly-d(G-C). In contrast, daunomycin fluorescence is quenched by only 50% when the dye interacts with synthetic A-T polymers. Thus, differential quenching of daunomycin fluorescence can account for the production of bright bands at contiguous A-T sequences along the chromosome. Slight differences in fluorescence quenching between the repeating and homopolymeric A-T duplex DNAs were observed which can be attributed to differences in affinity of daunomycin for these DNAs. The aminosugar moiety of daunomycin, daunosamine, increases the binding of daunomycin to DNA and also enhances chromosome banding. — Nogalamycin, which displays no differential quenching with the different DNAs in solution, also fails to produce bands on chromosomes. — These findings suggest that non-random nucleotide sequence arrangements along the chromosome are a basic determinant for dye interaction to produce the observed banding patterns. Specific banding procedures may determine the accessibility of these sites within the chromosomal DNA.