Donald M. Gray

[3] Absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes

Absorption and CD measurements of complementary oligomers and mixtures are described. The concentrations of oligomers may be estimated from absorption measurements and nearest-neighbor calculations of molar extinction coefficients. Interactions between complementary strands in mixtures can lead to obvious differences between measured CD spectra and the average of the spectra of the individual strands. CD spectra also allow an assessment of whether the individual strands are in self-complexes, which could compete with duplex or triplex formation. Isodichroic and isoabsorptive points provide important indicators of the stoichiometry of the strands in base-paired complexes. CD spectra provide an important means of characterizing differences in the conformations of DNA, RNA, and hybrid duplexes or triplexes having analogous sequences.

Circular dichroism spectroscopy of DNA.

Publisher Summary Circular dichroism (CD) measurements are used to study the conformations of nucleic acids in solution. The reliance on CD spectroscopy to study DNA conformations has stemmed from the sensitivity and ease of CD measurements, the nondestructive nature of such measurements, the fact that conformations can be studied in solution, and the requirement for relatively small amounts of material. Although detailed structural information, such as from X-ray crystallography or NMR spectroscopy, is not available from CD spectra, the CD spectrum of DNA in solution can provide a reliable determination of its overall conformational state when compared with the CD spectra of reference samples. Moreover, CD spectroscopy is applicable to a wide range of samples, including those that are difficult to crystallize or to obtain at high concentrations. The chief practical application of CD spectroscopy to the study of DNA structures has been by making empirical comparisons with the CD spectra of known structures. The chapter provides examples from laboratory of CD spectral changes in the near ultraviolet range that illustrate the CD characteristics of structural transitions in natural and synthetic DNAs.

Derivation of nearest‐neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors

The constraints on combinations of nearest neighbors in nucleic acid sequences and the numbers of independent sequences needed to describe nearest-neighbor properties of oligomers and polymers are derived and summarized. It has been pointed out in previous work [D. M. Gray and I. Tinoco, Jr. (1970) Biopolymers, Vol. 9, pp. 223-244; R. F. Goldstein and A. S. Benight (1992) Biopolymers, Vol. 32, pp. 1679-1693] that these constraints restrict the information available from measurements of properties of sequence combinations. The emphasis in this paper is on the properties of oligomer sequences that vary in length, where each nucleotide or base pair at the end of the sequence makes a significant contribution to the measured property by interacting with its boundary of fixed sequence or solvent. In such cases it is not be possible to determine values of properties of individual nearest neighbors, except for the like neighbors [e.g., d(A-A), d(G-G), d(T-T), and d(C-C) nucleotide neighbors in single-stranded DNA or d(A-A)/d(T-T) and d(G-G)/d(C-C) base pair neighbors in double-stranded DNA], solely from measurements of properties of different sequences. Even values for properties of the like neighbors cannot be determined from such oligomeric sequences if the sequences are all of the same length. Nearest-neighbor properties of oligomer sequences that vary in length can be summarized in terms of the values for independent sets of sequences that are nearest neighbors and monomers all with boundaries of the fixed sequence or solvent. Straightforward combinations of the values for the independent sequences will give the values of the property for any dependent sequence, without explicit knowledge of the individual nearest-neighbor values. These considerations have important consequences for the derivation of widely used thermodynamic parameters, as discussed in the following paper.

Independence of photoproduct formation on DNA conformation.

Abstract— In an ethanolic solution native T7 DNA can undergo conformational transitions from the B conformation (0% ethanol) to the C‐like (60% w/w ethanol) and the A (80% w/w ethanol) conformations. We have investigated the formation of three classes of thymine‐derived photoproducts in T7 DNA irradiated (280 nm) in the B, C‐like, and A conformations, which were monitored by circular dichroism measurements. We find that the predominant class of thymine‐derived photoproducts in any conformational state is cyclobutyl dipyrimidines. While the ‘spore product,’ 5‐thyminyl‐5,6‐dihydrothymine, which belongs to another class of photoproductsf does form in native DNA in the A conformation, its yield in denatured DNA at 80% ethanol is the same as that in native DNA. The yield of pyrimidine adduct, a third photoproduct class, is a maximum at 50–60% ethanol. This effect of ethanol is probably not due to the ethanol‐induced C‐like conformation, however, since pyrimidine adduct formation is not enhanced when T7 DNA is irradiated in the C conformation in 6 M CsCl or in intact phage. We conclude from these and other data in the literature that the degree of hydration rather than the conformational state is the critical factor in determining which of the photoproducts will form in native DNA.

Derivation of nearest-neighbor properties from data on nucleic acid oligomers. II. Thermodynamic parameters of DNA · RNA hybrids and DNA duplexes

Using nearest-neighbor models consisting of independent short sequence combinations of nearest neighbors (ISS models), values of thermodynamic parameters for sets of independent sequences are derived from published oligomer data for DNA.RNA hybrids [N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, and M. Sasaki (1995) Biochemistry, Vol. 34, pp. 11211-11216] and dsDNA duplexes [J. SantaLucia, Jr., H. T. Allawi, and P. A. Seneviratne (1996) Biochemistry, Vol. 35, pp. 3555-3562]. The results are compared with those from models that assign values of thermodynamic parameters to individual nearest neighbors (INN models). Differences in the use of ISS and INN models are also illustrated in an appendix, which shows examples of analyses for values of a fictitious nearest-neighbor property. INN models that include an initiation parameter contain an implicit assumption that combinations of end neighbors have the same value of a property. It is found that combinations of end neighbors (e.g., base pairs neighboring solvent) in oligomers can have significant and different apparent values of thermodynamic properties, so that the assumption inherent in INN models is not always correct. Even though ISS models do not allow the assignment of values to individual nearest neighbors, except for the like neighbors [such as d(AA)/r(UU), etc., for hybrids and d(AA)/d(TT) and d(GG)/d(CC) for DNA duplexes], they do provide physically meaningful values for the like neighbors, for sequence combinations, and for specified combinations of end neighbors.

Dehydrated circular DNA: electron microscopy of ethanol-condensed molecules.

Abstract The ethanol-induced condensation of linear DNA from phage φ29, and circular DNA from phage PM2 and plasmid R6K, on air-dried electron microscope specimen grids has been analyzed by measuring size and shape of the resulting particles. Upon such dehydration, single DNA molecules are condensed into tight particles of three different classes of superstructure, appearing shorter and thicker with increasing ethanol concentration, as described earlier with coliphage T7 DNA (Lang, 1973), except that circularity of DNA impairs the formation of condensates of second and third order to a degree that depends on the molecular weight. It is suggested that tight supercoils are formed by induction of numerous kinks in the double helix between short, straight DNA segments. This would be consistent with DNA kinks proposed by Crick & Klug (1975).

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the fd DNA--gene 5 protein complex in solution. A neutron small-angle scattering study.

Neutron small-angle scattering has been used to investigate the fd DNA-gene 5 protein complex in solution. Results are as follows. 1. (1) The mass per unit length is found to be 1380 or 1610 daltons/A, depending upon whether one gene 5 protein molecule is assumed to bind to four or five nucleotides, respectively. These values correspond to axial subunit repeats of 7.9 or 7.0 A and to total contour lengths in solution of 1.27 or 0.90 μm. For a helix of pitch 90 A there are between about 11 and 13 proteins per turn. 2. (2) The cross-sectional radius of gyration at infinite contrast of the complex is 34.5 ± 1 A. 3. (3) The structure must be quite open and solvated as indicated by the dry volume per subunit, the mass per unit length, and the radius of gyration. The volume occupied per subunit in a uniform cylinder having the measured radius of gyration and mass per unit length is about four times greater than the measured subunit dry volume in the complex. 4. (4) From the change with contrast of both the measured radius of gyration and the position of a subsidiary maximum we conclude that the DNA could not be on the outer periphery of the helical structure. This is supported by a calculation of the maximum radius of the DNA backbone. With the possible exception of the positioning of the DNA, our results for the complex in solution are in good agreement with a model proposed by McPherson et al. (1979b) for the complex structure. 5. (5) The complex formed by reconstitution in vitro is not substantially different in its solution structure from the in vivo complex isolated from infected cells.

STRUCTURAL PROPERTIES OF FRIEDREICH'S ATAXIA D(GAA) REPEATS

The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAA.TTC)n, a sequence whose expansion is associated with Friedreichs ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272-274 nm over the temperature range of 5-50 degrees C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm) of d(GAA)15 was 40 degrees C. At 5 degrees C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35 degrees C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5 degrees C, unknown but partially base-paired at 37 degrees C, and an approximately random coil above 65 degrees C.

CD Spectral Comparisons of the Acid-Induced Structures of Poly[d(A)]Poly[r(A)], Poly[d(C)], and Poly[r(C)]

CD spectra were used to compare the acid-induced structural transitions of poly[d(A)] and poly[d(C)] with those of poly[r(A)] and poly[r(C)], respectively. The types of base pairing were probably the same in the acid self-complexes of both A-containing polymers and in the acid self-complexes of both C-containing polymers. Similar base pairings were indicated by similarities in the difference CD spectra showing the changes during the first major acid-induced transitions of the polymers. Information from the CD spectra and pKa values of the transitions suggested that the transitions for the RNA polymers involved similar structural changes. The two DNA polymers were markedly different. Single-stranded poly[d(A)] was in the most stacked structure and had the lowest pKa for forming an acid self-complex. Single-stranded poly[d(C)] was in the least stacked structure and had the highest pKa for forming a protonated duplex.