Stacking interactions between neighboring heterocyclic bases are the main factor determining the conformation of single-stranded oligo- and polynucleotides.

Since monomer units in oligo- and polynucleotides are linked by covalent phosphodiester bonds, the stacking interactions of bases are more pronounced than in nucleoside and nucleotide associates and, secondly, occurs at any concentrations. The latter is the reason why stacked conformations can be observed in dilute solutions where intermolecular interactions (i.e., association of individual oligo- and polynucleotides) are minimal.

In this case, use is made of two fundamental optical properties of these compounds, directly related to the existence of stacking conformations, namely, hypochromism and Cotton effect in the UV region of the spectrum. Let us now consider these phenomena at greater length.

Being essentially aromatic chromophores, purine and pyrimidine bases absorb light intensely at 180 to 300 nm. Their UV absorption spectra usually feature two bands with maxima near 200 and 260 nm (Fig. 7-16).

The absorption spectra of heterocyclic bases are affected by all factors responsible for changes in their electron density distribution (introduction of substituents, protonation of bases, changeover from one tautomeric form to another, etc.).

It is important that the UV spectrum of a base changes as it comes closer together to another. In other words, the UV absorbance spectra of oligonucleotides (beginning with dinucleoside phosphates) differ from those of their constituent mononucleotides.

The absorption of oligo- and polynucleotides near 260 nm is less intense than that of all constituent monomer units taken together. This phenomenon has become known as the hypochromic effect. When the ordered conformation of oligo- and polynucleotides is upset or they are hydrolysed to nucleosides or nucleotides, increasing intensity of optical absorption known as the hyperchromic effect is observed.

The theory of hypochromism was elaborated in the early sixties by Tinoco. As detailed analysis of this theory goes beyond the scope of this chapter, we shall only dwell on the qualitative conclusions following from it and being of direct interest to us.

In its classical version the theory regards two closely spaced molecules interacting with light as two oscillators. The oscillators vibrate in the direction of transition moment vectors. The theory makes it possible to predict how the optical absorption of the system will vary as a function of the distance and angle between these vectors. In particular, if the oscillators are arranged one above the other in parallel planes, hypochromic effects will be observed in the long-wave part of the spectrum at certain values of the angle between their projections on the same plane (i.e., in the case of oligo- and polynucleotides in the region of the absorption band with a maximum near 260 nm).

The magnitude of the hypochromic effect is usually expressed as percentage hypochromism h calculated from the formula:

As was emphasized above in connection with association of nucleosides and nucleotides (7.2), their stacking interactions are strongly dependent on the heterocyclic base species. Hence, the magnitude of the hypochromic effect for oligo- and polynucleotides must be determined by their nucleotide composition. Table 7-3 shows the percentage hypochromism of some dinucleoside monophosphates (at 25⁰ C, ionic strength 0.1, pH 7).

Just as expected, the values of h for dinucleoside phosphates containing purine bases are usually higher.

As the polynucleotide chain increases in length, the values of h gradually go up and reach a maximum at a chain length of 7 to 10 nucleotides.

Table 7-3. Hypochromicity of selected dinucleoside monophosphates.

The ORD and CD spectra of oligo- and polynucleotides in the UV region (200-320 nm) also differ from those of their constituent monomer units, the differences in the magnitudes and positions of the maxima of the Cotton effects being usually greater, as compared to those in optical absorption of the same compounds (see Fig. 7-17). It should be pointed out that in the course of time the CD method almost completely displaced the ORD method by virtue of its higher sensitivity. However, we have included here some results obtained by the ORD method because it was rather important in studying the three-dimensional structure of nucleic acids and for quite some time was the principal tool for observing their conformational changes.

A remarkable property of the CD and ORD spectra is their sensitivity to nucleotide sequence. This important feature can be illustrated by CD spectra of two pairs of isomeric dinucleoside phosphates (see Fig. 7-18).

ORD and CD spectra have led to certain conclusions as regards the structure of oligo- and polynucleotides in aqueous solutions. Here are some of them.

(1) It has been confirmed that the stacking of purine oligonucleotides is more pronounced than that of pyrimidine ones; in this case, uracils and their derivatives interact with one another and with other heterocyclic bases to a much lesser degree than cytosine derivatives. This can be easily seen, for example, from comparison of ORD spectra of two isomeric trinucleotides - UpApGp and ApUpGp (Fig. 7-19). In the trinucleotide ApUpGp, the strongly interacting purine bases are separated by a uridine, and the Cotton effects of this compound are much less pronounced than in the case of UpApGp.

Fig. 7-18. CD spectra of isomeric dinucleoside phosphates (adapted from M. Warshaw and C. R. Cantor, Biopolymers 9, 1079, 1971).

(2) The ORD and CD spectra of oligoribo- and polyribonucleotides differ widely in amplitude and shape from those of oligodeoxyribo- and polydeoxyribonucleotides with the same nucleotide sequence, which is indicative of marked conformational dissimilarity. At the same time, the spectra of 2'-O-substituted oligoribonucleotides are closely similar to those of unsubstituted oligoribonucleotides. Thus, the most likely reason for the difference in conformation between single-stranded polyribo- and deoxyribonucleotides is the different conformation of the sugar in monomer units rather than involvement of the 2'-OH group of riboderivatives in intra-molecular hydrogen bonding.

(3) The ordered conformation of oligo- and polynucleotides, observable by ORD and CD methods, is determined chiefly by the interaction between adjacent bases in the polynucleotide chain. Long-distance interactions (even between monomers separated by a single nucleotide) are extremely weak. The lack of cooperativity during formation of an ordered structure of single-stranded oligo- and polynucleotides as well as the fact that the conformation of any two adjacent monomer units is the same both in diand polynucleotides make it possible to calculate the CD and ORD spectra of the latter from those of the constituent dinucleoside phosphates.

(4) As the temperature increases, the ordered structure of single-stranded oligo- and polynucleotides is gradually destroyed (which is a direct indication of the non-cooperative nature of the intermolecular interactions in them). Since hydrophobic interactions are involved in stabilization of this structure, it is also disrupted when organic solvents are added to aqueous solutions of oligo- and polynucleotides.

The transition of oligo- and polynucleotides from ordered to disordered state can be easily followed by changes in their CD and ORD spectra (when the stacking conformations break down completely, the spectra of oligo- and

polynucleotides become almost indistinguishable from the sum of spectra of the constituent monomer units). From changes in the spectra one can calculate the thermodynamic parameters of this transition, which are important characteristics of single-stranded oligo- and polynucleotides.

Detailed molecular models of di- and longer oligonucleotides can be proposed only if their fine structure is known well enough, which, in particular, becomes possible by resorting to NMR spectroscopy and X-ray structural analysis.

At present, X-ray diffraction patterns for many dinucleoside phosphates are available with a resolution of about 0.1 nm (1 Å). Examined in the first place were compounds capable of forming, during crystallization, intermolecular complexes with complementary base pairing. This has provided direct information on the precise geometry of Watson-Crick pairs. As regards stacking interactions, a rather interesting pattern has been revealed here as well. It has turned out that in dinucleoside phosphates, which contain both a purine base and a pyrimidine one, much more marked overlapping of the bases is observed in the case of isomers with a PupPy sequence. This can be illustrated using GpC as an example (Fig. 7-20).

As can be seen from the structure in Figure 7-20, if the positions of the bases in this dinucleoside phosphate are interchanged without affecting the sugar-phosphate backbone, the bases will overlap to much less extent.