Synthesis of bicyclo-DNA nucleosides with additional functionalization in the carbocyclic ring

Two novel bicyclo nucleoside isomers carrying the base thymine in the furanose ring and an ester substituent in the carbocyclic ring were synthesized from a common bicyclic sugar precursor via a cyclopropanation/fragmentation pathway in nine steps. The relative configuration of the ester substituent in both isomers as well as the anomeric configuration in one nucleoside was determined by 1H-NMR difference NOE spectroscopy

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological functio

The chemical stability of abasic RNA compared to abasic DNA

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological rol

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine•purine-pyrimidine triple-helical binding motif

The nucleoside analogs 7-(2′-deoxy-α-D-ribofuranosyl)hypoxanthine (β7H, 1), 7-(2′-deoxy-β-D-ribofuranosyl)hypoxanthine (β7H, 2) and 7-(2′-O-methyl-β-Dribofuranosyl)hypoxanthine (β7HOMe, 3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine•purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7.0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyldeoxycytidine, X = β7H, β7HOMe) bind to a DNA target duplex forming a H•G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triplehelix formation is specific to G-C base pairs and up to 40 µM third strand concentration, no stable triplex exhibiting H•A-T, H•T-A or H•C-G base arrangements could be found (target duplex concentration ∼0.1 nM). Multiply substituted sequences containing β7H residues either in an isolated [d(TTTTTβ7HTβ7HTβ7HTβ7HTβ7HT)] or in a contiguous [d(TTTβ7Hβ7Hβ7Hβ7HTTTTβ7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing β7H with those containing MeC or N7-2′-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two case

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues Tm and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in Tm when paired to complementary DNA and leads to substantial increases in Tm when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therap

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivit

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

ABSTRACT The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for T m and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV-and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct T m 's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T m closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function

Nuclear antisense effects in cyclophilin A pre‐mRNA splicing by oligonucleotides: a comparison of tricyclo‐DNA with LNA

The nuclear antisense properties of a series of tricyclo (tc)‐DNA oligonucleotide 9-15mers, targeted against the 3′ and 5′ splice sites of exon 4 of cyclophilin A (CyPA) pre‐mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA‐oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence‐ and dose‐dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc‐oligonucleotides was found to be superior to that of the LNA‐oligonucleotides in all cases by a factor of at least 4-5. A tc‐oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2 µM concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction in CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc‐15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc‐ and LNA‐ oligonucleotides. The obtained results confirm the power of tc‐DNA for nuclear antisense applications. Moreover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV‐