Circular dichroism of polynucleotides: A simple theory

A particularly simple theory of circular dichroism is described and applied to the polynucleotides. It works surprisingly well and predicts the conservative spectrum of DNA and the more intense nonconservative spectrum of RNA. The anomalously low intensity of the CD of the nucleic acids is shown to be a natural consequence of the cancellation of the rotational strengths of the many CD bands. The theory's many successes and predictions as well as its weaknesses are discussed.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

Circular dichroism of polynucleotides: dimers as a function of conformation

Working within the restrictions of a model, we have calculated the circular dichroism of the dinucleoside phosphates ApA, CpC, and CpA for various conformations. Comparing the calculated curves with those measured in aqueous solution we find agreement for (1) ApA as a right-handed helix with both bases either as in B-form DNA, or else rotated 180° around the glycosidic bond, (2) CpC as the right-handed conformation with both bases as in DNA, (3) ApC as either the right-handed conformation with both bases as in DNA, or else as a left-handed helix with both bases rotated 180°, and (4) CpA as either a left-handed helix with both bases in a left-handed DNA, or else in the right-handed conformation with both bases rotated 180°. In addition, we have investigated circular dichroism as a measure of unstacking. We find that opening the bases to a 90° total angle (base planes perpendicular) reduces the intensity of the calculated bands to 20% of their original value. Further, we find that allowing the sliding of one base past the other does not lead to a temperature dependence consistent with experiment.     

Circular dichroism of polynucleotides: A general method applied to dimers

A method is described which relates the circular dichroism of a polymer to its conformation. The method takes into account near and accidental degeneracies and eliminates the artificial distinction between degenerate and nondegenerate systems. Comparison of this method with perturbation theory indicates that the errors inherent in nondegenerate perturbation theory tend to cancel when a circular dichroism spectrum is calculated. The method is applied to dinucleoside phosphates.     

Circular dichroism of helices formed by purine monomers with pyrimidine polynucleotides

The CD spectra of a number of helical complexes formed by purine monomers and complementary pyrimidine polyribonucleotides have been observed over the range 200–400 nm. Each of these spectra is quite similar to that of the corresponding polymer–polymer helix. The spectra are evidently determined by the geometry of the asymmetric array of bases, largely unperturbed by the ribose–phosphate backbone. The helix structure (A-form), on the other hand, is determined by the backbone of the pyrimidine homopolymer. Data on the monomer–polymer complexes support the conclusion that the CD spectra of ribohomopolymer helices depend primarily on interastrand interactions of the same transition within a given base and are relatively unaffected by transitions of the complementary base.     

Circular dichroism of superhelical DNA

The circular dichroism (CD) spectra of a number of superhelical DNA's have been measured. The introduction of negative superhelical turns causes an increase in magnitude of the positive band around 280 mμ, while the trough around 250mμ is little affected. For two samples of λb2b5c DNA (20 Mdalton) containing different number of negative superhelical turns, the magnitude of the positive band relative to that of the nicked control increases with increasing number of superhelical turns. In 2M NaCl, the small (1.45 Mdalton) superhelical DNA from E. coli 15 shows an unusually large difference in CD compared with that of the same DNA with a few single-chain scissions per molecule. This large difference is not observed in a medium containing p. 0.11M NaCl. These results indicate that the double helix in a superhelical DNA is perturbed somewhat due to the bending and torsional forces in such a molecule. The magnitude of such structural alteration seems to depend on the number of superhelical turns per unit length, the size of the DNA molecule, as well as the ionic medium.     

Circular dichroism of phytochrome intermediates

Phototransformation P_t to P_fr was investigated with 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa L. cv. Pirol) using circular dichroism spectroscopy at -110°C to +30°C. Using absorption spectra of the intermediates formed at the respective temperatures, circular dichroism spectra (300–800 nm) of pure intermediates were calculated.
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of P_r. This would be compatible with a Z→E isomerization taking place at this reaction step. The subsequent intermediates (meta-R_a and meta-R_c) as well as P_fr showed only small circular dichroism. Their absorption spectra were drastically shifted, but had similar spectral shapes. The results are discussed in terms of conformational changes of the phytochrome chromophore presumably taking place at the early steps of phototransformation P_r to P_fr.