Warum Pentose- und nicht Hexose-Nucleinsäuren??. Teil V. (Purin-Purin)-Basenpaarung in der homo-DNS-Reihe: Guanin, Isoguanin, 2,6-Diaminopurin und Xanthin

Why Pentose- and Not Hexose-Nucleic Acids? Purine-Purine Pairing in homo-DNA: Guanine,Isoguanine, 2,6-Diaminopurine, and Xanthine This paper concludes the series of reports in this journal [1–4] on the chemistry of homo-DNA, the constitutionally simplifie dmodel system of hexopyranosyl-(6′ → 4′)-oligonucleotide systems stidued in our laboratory as potentially natural-nucleic-acid alternatives in the context of a chemical aetiology of nucleic-acid structure. The report describes the synthesis and pairing properties of homo-DNA oligonucleotides which contain as nucleobases exclusively purines, and gives, together with part III of the series [3], a survey of what we know today about purine-purine pairingin homo-DNA. In addition, the paper discusses those aspects of the chemistry of homo-DNA which, we think, influence the way how some of the structural features of DNA (and RNA) are to be interpreted on a qualitative level. Purine-purine pairing occurs in the homo-DNA domain in great variety. Most prominent is a novel tridentate Watson-Crick pair between guanine and isoguanine, as well as one between 2,6-diaminopurine and xanthinone, both giving rise to very stable duplexes containing the all-purine strands in antiparallel orientation. For the guanine-isoguanine pair, constitutional assignment is based on temperature-dependent UV and CD spectroscopy of various guanine- and isoguanine-containg duplexes in comparison with duplexes known to be paired in the reverse guanine is replaced by 7-carbauguanine. Isoguanine and 2,6-diaminopurine also have the capability of self-pariring in the reverse-Hoogsteen mode, as previously observed for adenine and guanine [3]. In this type of pairing, the interchangeably. Fig. 36 provides an overall survey of the relative strength of pairing in all possible purine-purine combinations. Watson-Crick pairing of isoguanine with guanine demands the former to participate in its 3H-tautomeric form; hitherto this specific tautomer had not been considered in the pairing chemistry of isoguanine. Whereas (cumulative) purine-purine pairing in DNA (reverse-Hoogsten or Hoogsteen) seems to occur in triplexes and tetrapalexes only, its occurrence in duplexes in a characteristic feature of homo-DNA chemistry. The occurrence of purine-purine Watson-Crick base pairs is probably a consequence of homo-DNA's quasi-linear ladder structure [1][4]. In a double helix, the distance between the two sugar C-atoms, on which a base pair is anchored, is expected to be constrained by the dimensions of the helix; in a linear duplex, however, there would be no restrictions with regard to base-pair length. Homo-DNA's ladder-like model also allows one to recognize one of the reasons why nucleic-acid duplexes prefer to pair in antiparallel, rather than parallel strand orientation: in homo-DNA duplexes, (averaged) backbone and base pair axes are strongly inclined toward one another [4]; the stronger this inclination, the higher the preference for antiparallel strand orientation is expected to be (Fig. 16). In retrospect, homo-DNA turns out to be one of the first artificial oligonucleotide systems (cf. Footnote 65) to demonstrate in a comprehensive way that informational base pairing involving purines and pyrimidines is not a capability unique to ribofuranosyl systems. Stability and helical shape of pairing complexes are not necessary conditions of one another; it is the potential for extensive conformational cooperativity of hte backbone structure with respect to the constellational demands of base pairing and base stacking that determines whether or nor a given type of base-carrying backbone structure is an informational pairing system. From the viewpoint of the chemical aetiology of nucleic-acid structure, which inspired our investigations on hexopyranosyl-(6′ → 4′)-oligonucleotide systems in the first place, the work on homo-DNA is only an extensive model study, because homo-DNA is not to be considered a potential natural-nucleic-acid altenratie. In retrospect, it seems fortunate that the model study was carried out, because without it we could hardly have comprehended the pairing behavior of the proper nucleic-acid alternatives which we have studied later and which will be discussed in Part VI of this series. The English footnotes to Fig. 1–49 provide an extension of this summary.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Antisense derivatives of U7 and other small nuclear RNAs as tools to modify pre-mRNA splicing patterns

The importance of alternative splicing for the diversity of the proteome and the large number of genetic diseases that are due to splicing defects call for methods to modulate alternative splicing decisions. Although splicing can be modulated by antisense oligonucleotides, this approach is confronted with problems of efficient delivery and the need for repeated administrations of large amounts of the oligonucleotides. Therefore we have developed methods allowing us to modulate splicing with the help of modified derivatives of the U7 small nuclear RNA involved in histone RNA 3' end processing. Its nuclear accumulation as a stable ribonucleoprotein particle makes U7 snRNA especially useful for this purpose. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 expression cassettes have been successfully introduced into a great number of cell lines, primary cells or tissues with the help of lentiviral and adeno-associated viral vectors. Examples of these therapeutic strategies in the fields of β-thalassemia, Duchenne muscular dytrophy and HIV/AIDS are discussed.

From Ribbons to Networks: Hierarchical Organization of DNA-Grafted Supramolecular Polymers

DNA-grafted supramolecular polymers (SPs) allow the programmed organization of DNA in a highly regular, one-dimensional array. Oligonucleotides are arranged along the edges of pyrene-based helical polymers. Addition of complementary oligonucleotides triggers the assembly of individual nanoribbons resulting in the development of extended supramolecular networks. Network formation is enabled by cooperative coaxial stacking interactions of terminal GC base pairs. The process is accompanied by structural changes in the pyrene polymer core that can be followed spectroscopically. Network formation is reversible, and disassembly into individual ribbons is realized either via thermal denaturation or by addition of a DNA separator strand.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.

Gas-phase fragmentation and conformation of the sugar-modified antisense oligonucleotide tcDNA

Tricyclo-DNA (tcDNA) is a sugar- and backbone-modified analogue of DNA that is currently tested as antisense oligonucleotide for the treatment of Duchenne muscular dystrophy. The name tricyclo-DNA is derived from the modified sugar-moiety: the deoxyribose is extended to a three-membered ring system. This modification is designed to limit the flexibility of the structure, thus giving rise to entropically stabilized hybrid duplexes formed between tcDNA and complementary DNA or RNA oligonucleotides. While the structural modifications increase the biostability of the therapeutic agent, they also render the oligonucleotide inaccessible to enzyme-based sequencing methods. Tandem mass spectrometry constitutes an alternative sequencing technique for partially and fully modified oligonucleotides. For reliable sequencing, the fragmentation mechanism of the structure in question must be understood. Therefore, the presented work evaluates the effect of the modified sugar-moiety on the gas-phase dissociation of single stranded tcDNA. Moreover, our experiments reflect the exceptional gas-phase stability of hybrid duplexes that is most noticeable in the formation of truncated duplex ions upon collision-induced dissociation. The stability of the duplex arises from the modified sugar-moiety, as the rigid structure of the tcDNA single strand minimizes the change of the entropy for the annealing. Moreover, the tc-modification gives rise to extended conformations of the nucleic acids in the gas-phase, which was studied by ion mobility spectrometry-mass spectrometry.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

Supramolecular Polymers in DNA Nanotechnology

Creation of biocompatible functional materials is an important task in supramolecular chemistry. In this contribution, we report on noncovalent synthesis of DNA-grafted supramolecular polymers (SPs). DNA-grafted SPs enable programmed arrangement of oligonucleotides in a regular, tightly packed one-dimensional array. Further interactions of DNA-grafted SPs with complementary DNA strands leads to the formation of networks through highly cooperative G-C blunt-end stacking interactions. The structural changes in the polymeric core enable to monitor spectroscopically the stepwise formation of networks. Such stimuli-responsive supramolecular networks may lead to the development of DNA-based smart materials.

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.