Abstract
The hyper-constrained nucleoside, methylene-bridged hexopyranosyl nucleoside (BHNA) was incorporated into the antisnese oligonucleotides (AON), which show more preference for binding toward the complementary RNA (Tm loss by ca 5°C) than that with the complementary DNA (Tm loss by 10°C), vis-a-vis corresponding native duplex. The origin of reduction of Tm of the duplexes formed by the BHNA incorporated AON and the complementary RNA or DNA was further investigated by thermal denaturation study with the single-mismatched DNA or RNA, CD spectroscopy, RNase H digestion study, as well as by molecular model building. These studies showed that the introduction of BHNA causes only a limited local conformational perturbation in the AON/RNA heteroduplex, whereas it affects the global conformation in the AON-DNA duplex. BHNA incorporated AONs also show improved stability in the human blood serum, which may prove to have some potential therapeutic application.
Highlights
Many different types of pyranosyl modified nucleosides have been so far incorporated in to antisnese oligonucleotides (AON), and their affinity toward complementary RNA or DNA, as well as the biochemical features have been studied by the groups of Herdewijn and Eschenmoser:[2] AONs composed of homo-DNA (Figure 1) is almost linear, and not able to form duplexes with either RNA or DNA19, 20, While HNA21, 22, CNA23 and CeNA24-26 all have improved affinity toward RNA and show improved nuclease resistance compared to the native counterpart
The CD spectra showed the BHNA modified AON/RNA adopt a conformation comparable to that of the native AON/RNA duplex, which means that BHNA can only cause limited local distortion in the AON/RNA duplex, which was confirmed by the molecular model of AON/RNA duplex
(1) The hyper-constrained nucleoside, methylene-bridged hexose (BHNA) was purified and incorporated into antisnese oligonucleotides (AON). (2) The BHNA incorporated AON show preference toward complementary RNA than complementary DNA, both AON/RNA and AON/DNA duplex have lower Tm compared to the native DNA/RNA and DNA/DNA duplex
Summary
Conformationally-constrained oligonucleotides have attracted considerable interest in the last two decades because they enable us to dictate specific conformational character in to the therapeutically interesting oligonucleotides, thereby allowing to steer their biological properties, as required in the antisense, siRNA or triplex strategy.[1,2,3] The pentose-sugar moiety in natural nucleos(t)ide is very flexible compared to those of the hexose-based oligos, and can adopt a dynamic equilibrium of several sugar pseudorotamers.[4, 5] It has been found that the North-type (34o > P> -1o, Φm > 30o) or South-type (194o > P > 137o, Φm > 48o). Oligonucleotides with these types of covalently constrained nucleosides can potentially preorganize the single-stranded oligonucleotide in to a helical form, which, in turn, show much improved affinity toward complementary RNA (38oC/modification, with the exception of C1′ and C2′ constrained systems) as well as to DNA18; they show improved resistance toward enzyme degradation.[13, 17] (3) The AONs produced by the replacement of the pentofuranosyl sugar with the hexopyranosyl sugar[19] give thermodynamically stable duplexes with the target DNA or RNA It is because, first, the sixmembered ring system adopt a rigid chair conformation, requiring a less negative entropy change during the duplex formation; and second, the interstrand phosphate distance in the six-membered pyranosyl-modified system is larger than in the natural nucleic acid duplexes giving a relatively less interstrand charge repulsion compared to the native counterpart.[19] Many different types of pyranosyl modified nucleosides have been so far incorporated in to AONs, and their affinity toward complementary RNA or DNA, as well as the biochemical features have been studied by the groups of Herdewijn and Eschenmoser:[2] AONs composed of homo-DNA (Figure 1) is almost linear, and not able to form duplexes with either RNA or DNA19, 20, While HNA21, 22, CNA23 and CeNA24-26 all have improved affinity toward RNA and show improved nuclease resistance compared to the native counterpart. It has turned out that modification of the AON with the new hyper-modified pyranose-modified molecules, as in 1, greatly improve human blood serum stability in comparison with the native counterpart
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