with chemical modifications in the phosphate, sugar or base moieties (Section 4.4). In some cases, the result-
ing hetero-duplexes have proved to have higher association constants than the natural DNARNA duplexes
and the oligonucleotide analogues exhibit increased resistance to phosphate diesterase action (Table 2.6).
The subtle differences in conformation between an RNADNA hybrid duplex and either DNADNA or
RNARNA duplexes have significance for enzyme action and also for anti-sense therapy. The therapeutic
objective of antisense oligodeoxynucleotides very much depends on their ability to create a duplex with the
target RNA and thus make it a substrate for ribonuclease H (Table 2.6). Because RNase H cleaves DNARNA
hybrids but does not cleave the corresponding RNARNA duplexes, it can be induced to degrade an endo-
geneous mRNA species through hybridisation with a synthetic antisense oligodeoxyribonucleotide.
X-ray structures of crystals of duplexes having DNA and RNA residues in both strands showed them to
have pure A-form geometry (see above). Duplexes between RNA and DNA also adopt A-form geometry
in the solid state and in two cases it has been shown that self-complementary DNA decamers with a single
incorporated ribonucleotide are in the A-form in the crystal although the all-DNA sequences prefer the
B-form in the crystal and in solution. It is possible that crystal lattice forces and crystallisation kinetics play
a role in the preference of the A-form geometry observed for all crystal structures of DNARNA duplexes.
By contrast, the hybrid duplexes d(GTCACATG)r(CAUGUGAC) and d(GTGAACCTT)r(AAGUU-
CAC) have been analysed by 2D NOE NMR in solution and shown to have neither pure A-form nor pure
B-form structure.^57 The sugars of the RNA strands have the regular C3-endoconformation but those in
the DNA strand have a novel, intermediate C4-endoconformation. Glycosylic torsion angles in the DNA
chain are typical of B-form (near –120°) but those in the RNA chain are typically A-form values (near
–140°). Overall the global structure is that of an A-form helix in which the base pairs have the small rise
and positive inclination typical of an A-form duplex (Figure 2.19a,d). However, the width of the minor
groove appears to be intermediate between A- and B-form duplexes and such structures have been mod-
elled into the active site of RNase H. The results suggest that additional interactions of the protein with the
DNA strand are possible only for this intermediate hybrid duplex conformation but not for an RNARNA
duplex. So, it seems possible that these subtle changes in nucleotide conformation may explain the selectivity
of RNase H for hybrid DNARNA duplexes.^58 Indeed, crystal structures for complexes between a bacterial
60 Chapter 2
Table 2.6 Properties of antisense oligonucleotides and 1st and 2nd generation analogues
Oligonucleotide type Duplex stabilitya Nuclease resistanceb RNase H activationc
Oligodeoxyribonucleotide (PO 2 )Para —Yes
Oligodeoxyribonucleotide phosphorothioate — Yes
Oligodeoxyribonucleotide methylphosphonate — No
Oligodeoxyribonucleotide phosphoramidate No
Oligoribonucleotide (PO 2 ) —No
Oligo (2-O-Me)ribonucleotide (PO 2 ) No
Oligo (2-O-(2-methoxyethyl)ribonucleotided(PO 2 ) No
Oligo (2-O-(3-aminopropyl)ribonucleotidee(PO 2 ) No
Oligo (2-O-(N,N-dimethylaminooxyethyl)f(PO 2 ) No
Oligo (2,4-methylene ...)g(PO 2 ) No
Oligo (2-fluoroarabinonucleotide)h(PO 2 ) Yes
Peptide nucleic acidsi No
Oligodeoxy(5-propyne-cytidine) (PO 2 ) —Yes
aCompared to DNA–RNA stability under physiological conditions.
bCompared to DNA (phosphate diesterase digestion).
cActivation of RNase H by the duplex formed between the oligonucleotide and RNA.
d 2 -O-MOE.
e 2 -O-AP.
f 2 -O-DMAEOE.
gLocked nucleic acid (LNA).
hFANA.
iPNA.