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Here we present the synthesis, the biophysical properties, and the RNase H profile of 6’-difluorinated [4.3.0]bicyclo-DNA (6’-diF-bc4,3-DNA). The difluorinated thymidine phosphoramidite building block was synthesized starting from an already known gem-difluorinated tricyclic glycal. This tricyclic siloxydifluorocyclopropane was converted into the [4.3.0]bicyclic nucleoside via cyclopropane ring-opening through the addition of an electrophilic iodine during the nucleosidation step followed by reduction. The gem-difluorinated bicyclic nucleoside was then converted into the corresponding phosphoramidite building block which was incorporated into oligonucleotides. Thermal denaturation experiments of these oligonucleotides hybridized to complementary DNA or RNA disclosed a significant destabilization of both duplex types (ΔTm/mod = −1.6 to −5.5 °C). However, in the DNA/RNA hybrid the amount of destabilization could be reduced by multiple insertions of the modified unit. In addition, CD spectroscopy of the oligonucleotides hybridized to RNA showed a similar structure than the natural DNA/RNA duplex. Furthermore, since the structural investigation on the nucleoside level by X-ray crystallography and ab initio calculations pointed to a furanose conformation in the southern region, a RNase H cleavage assay was conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand.

Here we report on the synthesis, biophysical properties and molecular modeling of oligonucleotides containing unsaturated 6’-fluoro[4.3.0]bicyclo nucleotides (6’F-bc4,3-DNA). Two 6’F-bc4,3 phosphoramidite building blocks (T and C) were synthesized starting from a previously described [3.3.0]bicyclic sugar. The conversion of this sugar to a gem-difluorinated tricyclic intermediate via difluorocarbene addition followed either by a NIS-mediated or Vorbrüggen nucleosidation yielded in both cases the β-tricyclic nucleoside as major anomer. Subsequent desilylation and cyclopropane ring opening of these tricyclic intermediates afforded the unsaturated 6’F-bc4,3 nucleosides. The successful incorporation of the corresponding phosphoramidite building blocks into oligonucleotides was achieved with tert-butyl hydroperoxide as oxidation agent. Thermal melting experiments of the modified duplexes disclosed a destabilizing effect versus DNA and RNA complements, but with a lesser degree of destabilization versus complementary DNA (ΔTm/mod = −1.5 to −3.7 °C). Molecular dynamics simulation on the nucleoside and oligonucleotide level revealed the preference of the C1’-exo/C2’-endo alignment of the furanose ring. Moreover, the simulation of duplexes with complementary RNA disclosed a DNA/RNA-type duplex structure suggesting that this modification might be a substrate for RNase H.

Spinal muscular atrophy (SMA) is a recessive disease caused by mutations in the SMN1 gene, which encodes the protein survival motor neuron (SMN), whose absence dramatically affects the survival of motor neurons. In humans, the severity of the disease is lessened by the presence of a gene copy, SMN2. SMN2 differs from SMN1 by a C-to-T transition in exon 7, which modifies pre-mRNA splicing and prevents successful SMN synthesis. Splice-switching approaches using antisense oligonucleotides (AONs) have already been shown to correct this SMN2 gene transition, providing a therapeutic avenue for SMA. However, AON administration to the CNS presents additional hurdles. In this study, we show that systemic delivery of tricyclo-DNA (tcDNA) AONs in a type III SMA mouse augments retention of exon 7 in SMN2 mRNA both in peripheral organs and the CNS. Mild type III SMA mice were selected as opposed to the severe type I model in order to test tcDNA efficacy and their ability to enter the CNS after maturation of the blood brain barrier (BBB). Furthermore, subcutaneous treatment significantly improved the necrosis phenotype and respiratory function. In summary, our data support that tcDNA oligomers effectively cross the blood-brain barrier and offer a promising systemic alternative for treating SMA.

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

The structure of homo-DNA duplex [[dd(CGAATTCG)].sub.2] (dd =2',3'-dideoxy-[beta]-D-glucopyranose sugars) is described. It exposes a weakly twisted right-handed duplex with a strong leaning between the hexose-phosphate backbones and base-pair axes, and highly unbalanced values for helical rise and twist at individual base steps.

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.

We report on the pairing properties of oligodeoxynucleotides containing chemically functionalized biphenyl residues instead of natural nucleobases. The synthesis of the corresponding C-nucleosides with one or two nitro- or methoxy groups, resp. in the distal phenyl ring was accomplished via Suzuki coupling of a protected 4-bromophenyl-C-nucleoside with the correspondingly substituted phenylboronic acids or esters. The interstrand hydrophobic and/or stacking recognition properties of the aromatic units were probed by Tm-measurements on duplexes containing single or triple incorporations. We found a strong dependence of duplex stability from the nature of the substituents (donor vs. acceptor). The stability of duplexes with one biphenyl-pair increases in the order of donor–donor < acceptor–donor < acceptor–acceptor pairs. On the other hand in the triply modified duplexes stability increases in the order acceptor–acceptor < donor–acceptor < donor–donor pairs. It thus appears that interactions to the nearest neighbor natural base-pairs are preferred by acceptor modified biphenyls while the interactions between biphenyls are most stable with opposing donor modified biphenyls.

Research has been conducted on the chimaeric DNA/RNA triplex-forming oligonucleotides. The preparation of these oligonucleotides and the triplex formation's structural, kinetic and thermodynamic properties have been investigated via spectroscopic methods and the details are discussed.

This minireview highlights three aspects of our recent work in the area of sugar modified oligonucleotide analogues. It provides an overview over recent results on the conformationally constrained analogue tricyclo-DNA with special emphasis of its antisense properties, it summarizes results on triple-helix forming oligodeoxynucleotides containing pyrrolidino-nucleosides with respect to DNA recognition via the dual recognition mode, and it highlights the advantageous application of the orthogonal oligonucleotidic pairing system homo-DNA in molecular beacons for DNA diagnostics.

Research was conducted to examine the synthesis, duplex and triplex forming characteristics of the DNA analogue tricyclo-DNA. A further objective was the thermodynamic investigation of duplex formation of a range of tricyclo-DNA sequences having the nucleobases adenine and thymine. Results suggest that the tricyclo-DNA duplexes are thermodynamically more stable than the corresponding bicyclo-DNA duplexes. They also demonstrated unusual supramolecular association properties and potentially useful biological properties.

In our search for novel oligonucleotide analogues for use in therapy and DNA-based diagnostics we explored the concept of conformational preorganization as a means to improve duplex stability with complementary DNA and RNA. This research effort has led to a number of novel DNA analogues and has contributed to a deeper understanding of the interrelation between the structure of nucleosides and the properties of corresponding oligonucleotides. Some of these analogues show interesting biological properties. This article is a short review summarizing the properties of bicyclo-DNA and tricyclo-DNA, developed in our laboratory.

This article contains a short account of the chemical and biophysical properties of the oligonucleotide analogues bicyclo-DNA, tricyclo-DNA, [3.2.1]-DNA, [3.2.1]amide-DNA and the PNA analogue OPA, recently developed in our laboratory. Emphasis is given to various aspects of conformational restriction as a designer tool for enhancing the performance of oligonucleotide analogues and for exploring the supramolecular chemistry of DNA. A short summary of current problems in the field of DNA triple-helix chemistry as well as contributions from our laboratory in this area concludes this communication.