Nucleic Acids

If proteins perform many crucial tasks in living organisms, it is the mac-romolecules DNA and RNA, called 90&-"(&?&(=), which carry the genetic information in a form which can be transmitted from generation to gen-eration. These long linear polymers are made of a large number of linked 90&-"%$(=" units, which themselves consist of a sugar, a phosphate group, and a ,?)" (Figure 2.2). Sugars linked by phosphates form a common backbone, whereas the base may vary among four kinds: adenine (A), cytosine (C), guanine (G), and thymine (T) in DNA, the latter base being replaced by uracil (U) in RNA. A and G belong to the family of the /0*(9"), C, T, and U to that of the /+*(#(=(9"). The genetic information is stored in the base sequence along a nucleic acid chain. The DNA nu-cleotides are often referred to as dA, dC, dG, and dT, where the prefix d indicates that the sugar is deoxyribose to make the distinction from RNA nucleotides in which the sugar is ribose. Another special property of the bases is that they form specific pairs with one another across two nucleic strands which are stabilised by hydrogen bonds: A with T or U, C with G

[131].¹³ This base pairing results in the formation of a =%0,-"'"-(7 if the sequence of two strands is complementary.

DNA and RNA do not only differ in one of the bases. The sugar in DNA is deoxyribose, whereas it is ribose in RNA. Ribose is identical to deoxyribose except for one additional hydroxyl group. This minor chemical variation has two important consequences. Indeed, due to the additional free hydroxyl group which promotes hydrolysis, RNA is chemically less stable than DNA. Secondly, the hydroxyl group induces a different sugar conformation in RNA, which in turn causes the RNA double helix to adopt a conformation different from that of DNA.

a) Nucleic Acid Conformations

Three double-stranded (ds) nucleic acid conformations (Figure 2.3, Table 2.1) are believed to be found in nature: A, B, and Z [132]. The right-handed B form predominates in cells and is the one originally described by Watson and Crick [131]. It extends 3.4 Å per base pair and makes one complete turn about its axis every 10.4 base pairs (this quantity is known as the helical /($&'). Because the glycosidic bonds of a base pair are not diametrically opposite each other, B-DNA contains two kinds of

13 Other minor forms of base pairing, known as Hoogsteen and wobble base pairs, have been identified [127]. Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Uracil (U)

Figure 2.2. Molecular structure of the nucleic acid bases (top) and of the DNA, RNA, and LNA polymers.

grooves, called the #?Q%*.*%%8" (12 Å wide) and the #(9%* .*%%8" (6 Å wide) [126]. The grooves offer many possibilities for hydrogen-bond for-mation with other molecules and are often the target of small molecules [133] or proteins which recognise specific DNA sequences.

Because of its 2’-hydroxyl group, the sugar in RNA adopts another conformation than in DNA. Double-stranded RNA folds into a double-helical form termed the A helix. It is shorter (2.3 Å per base pair) and wider than the B helix, and its base pairs are tilted rather than normal to the helix axis. The pitch of 11 base pairs per turn is however similar to that of the B form. Moreover, the minor groove nearly vanishes in the A helix, whereas the major groove gets very deep. Dehydrated DNA also adopts the A form when the relative humidity is reduced below about 75 %. The driving force for this conformational transition upon dehydra-tion is apparently that the A helix is more economical to hydrate than the B helix: in A-DNA, the oxygen atoms of two adjacent phosphate groups are close enough to get hydrated by a single bridging water molecule, whereas this is not possible in B-DNA where individual hydration of the phosphate groups occurs [134].

The Z-DNA form is adopted by short oligonucleotides which have se-quences of alternating pyrimidines and purines. It is left-handed and the phosphates in the backbone zigzag. High salt concentrations are needed to induce this form and minimise electrostatic repulsion between the phosphate groups which are closer to each other than in A- or B-DNA.

Figure 2.3. Structural models of A-, B-, and Z-DNA (drawn from 2d47.pdb, 2bna.pdb, and 3zna.pdb). Two different grey tones are used to distinguish the two strands.

The first crystal structure of a short DNA oligomer was actually found to be in the Z form [135, 136].

A-, B-, and Z-DNA can easily be differentiated on the basis of their circular dichroism spectrum [137]. Conformational transitions from B to A or Z are believed to be induced by the binding of proteins to DNA [138] and are investigated by various approaches [138-141]. Other DNA conformations (C-, D-, E-, H-, L-, and P-DNA [142-146]) have been iden-tified but are not biologically relevant, according to the current knowl-edge. Furthermore, the existence of DNA triple and quadruple strands has been demonstrated [147-149].

b) Nucleic Acid Base Analogues

In the search for nucleic acid mimics capable of binding strongly to a complementary nucleic acid strand and satisfying the requirements for potential pharmaceutical applications, the group of Wengel introduced an RNA analogue containing 2’-R,4’-E-methylene linked bicyclic ribofurano-syl nucleotides [150-152]. Because the sugar conformation is locked in a conformation which is preferable for the formation of hybrids with a complementary DNA or RNA strand, they called this analogue locked nucleic acid (LNA, Figure 2.2). This feature gives the LNA probes very high binding affinity without compromising their sequence specificity [153, 154]. LNA strands have indeed been shown to perfectly hybridise Table 2.1. Comparison of A-, B-, and Z-DNA [126].

A-DNA B-DNA Z-DNA

Shape Broadest Intermediate Narrowest

Rise per base pair 2.3 Å 3.4 Å 3.8 Å

Helix diameter 25.5 Å 23.7 Å 18.4 Å

Base pairs per turn 11 10.4 12

Screw sense Right-handed Right-handed Left-handed

with DNA, RNA, and LNA strands, the effect of LNA being that the melting temperature of the duplex, that is, the temperature at which hy-drogen bonds are broken and the two strands separate to form single strands, increases in an unprecedented way (several degrees per inserted LNA nucleotide). Nonetheless, slight structural changes in LNA:DNA or LNA:RNA helices have been observed experimentally [155, 156]. The actual conformation (A form, B form, or a mixture of both) depends on the fraction of LNA nucleotides: the greater the amount of LNA mono-mers, the more prominent is the A form. Molecular dynamics simula-tions have shown that the perturbation introduced by a single LNA modi-fication is fairly localised, extending mostly to the next extending base pair [157].

Many other nucleic acid variants have been developed and investi-gated [158], among which the promising expanded DNA double helix (xDNA) [159], but they are not relevant to this work and are therefore not further discussed.