Stefan Schürch

Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers.

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2′-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions.

A fluorescent metal sensor based on macrocyclic chelation

The ethylenediamine functionalized quinacridone derivatives 3a–c display an orange fluorescence (λem(max) = 558 nm) which is quenched upon addition of coordinating metal ions by formation of a macrocyclic chelate bringing metal ion and fluorophore in close proximity to one another.

LC‐MS/MS method for simultaneous analysis of uracil, 5,6‐dihydrouracil, 5‐fluorouracil and 5‐fluoro‐5,6‐dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 μm for U, 0.1-10 μm for UH(2) , 0.1-75 μm for 5-FU and 0.75-75 μm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei

Three novel glycine-rich peptides, named ctenidin 1–3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.

Furan‐Oxidation‐Triggered Inducible DNA Cross‐Linking: Acyclic Versus Cyclic Furan‐Containing Building Blocks—On the Benefit of Restoring the Cyclic Sugar Backbone

Oligodeoxynucleotides incorporating a reactive functionality can cause irreversible cross-linking to the target sequence and have been widely studied for their potential in inhibition of gene expression or development of diagnostic probes for gene analysis. Reactive oligonucleotides further show potential in a supramolecular context for the construction of nanometer-sized DNA-based objects. Inspired by the cytochrome P450 catalyzed transformation of furan into a reactive enal species, we recently introduced a furan-oxidation-based methodology for cross-linking of nucleic acids. Previous experiments using a simple acyclic building block equipped with a furan moiety for incorporation into oligodeoxynucleotides have shown that cross-linking occurs in a very fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated. Given the destabilization of duplexes observed upon introduction of the initially designed furan-modified building block into DNA duplexes, we explore here the potential benefits of two new building blocks featuring an extended aromatic system and a restored cyclic backbone. Thorough experimental analysis of cross-linking reactions in a series of contexts, combined with theoretical calculations, permit structural characterization of the formed species and allow assessment of the origin of the enhanced cross-link selectivity. Our experiments clearly show that the modular nature of the furan-modified building blocks used in the current cross-linking strategy allow for fine tuning of both yield and selectivity of the interstrand cross-linking reaction.

The influence of Cisplatin on the gas-phase dissociation of oligonucleotides studied by electrospray ionization tandem mass spectrometry.

Abstractcis-Diamminedichloroplatinum(II) (cisplatin, DDP) is a cornerstone of anticancer therapy and has become one of the most widely used drugs for the treatment of various epithelial malignancies. The cytotoxicity of cisplatin is mainly based upon its affinity to adjacent guanines in nucleic acids, resulting in the formation of 1,2-intrastrand adducts. In this study the gas-phase dissociation of DNA- and RNA-cisplatin adducts is investigated by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The fundamental mechanistic aspects of fragmentation are elucidated to provide the basis for the tandem mass spectrometric determination of binding motifs and binding sites of this important anticancer drug. It is shown that the binding of cisplatin to vicinal guanines drastically alters the gas-phase fragmentation behavior of oligonucleotides. The 3′-C-O bond adjacent to the GG base pair is preferentially cleaved, leading to extensive formation of the corresponding w-ion. This observation was even made for oligoribonucleotides, which usually tend to form c- and y-ions under CID conditions. The absence of complementary ions of equal abundance indicates that oligonucleotide-cisplatin adducts are following more than one dissociation pathway in the gas-phase. Several mechanisms that explain the increased cleavage of the 3′-C-O bond and the lack of the complementary a-ion are proposed. Results of additional MS/MS experiments on methylphosphonate-oligodeoxynucleotides confirm the proposed mechanisms.

Multicomponent venom of the spider Cupiennius salei: a bioanalytical investigation applying different strategies

The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI‐TOF‐MS and ESI‐MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP‐HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP‐HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP‐HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C‐terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys‐containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3‐ctenitoxin‐Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.

Remote control of bipyridine–metal coordination within a peptide dendrimer

The metal coordinating ability of a bipyridine ligand at the core of a peptide dendrimer was found to be controlled by the nature of amino acids placed at the dendrimer periphery, with coordination being promoted by anionic residues and inhibited by cationic residues; heterotrimers with mixed charges were preferentially formed.

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure.

Abstract. CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys6-Cys21, Cys13-Cys30, Cys20-Cys48, Cys32-Cys46). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail.