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Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Pathogenic variations in the fused in sarcoma (FUS) gene are associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS). As FUS-ALS is a dominant disease, a targeted, allele-selective approach to FUS knockdown is most suitable. Antisense oligonucleotides (AOs) are a promising therapeutic platform for treating such diseases. In this study, we have explored the potential for allele-selective knockdown of FUS. Gapmer-type AOs targeted to two common neutral polymorphisms in FUS were designed and evaluated in human fibroblasts. AOs had either methoxyethyl (MOE) or thiomorpholino (TMO) modifications. We found that the TMO modification improved allele selectivity and efficacy for the lead sequences when compared to the MOE counterparts. After TMO-modified gapmer knockdown of the target allele, up to 93% of FUS transcripts detected were from the non-target allele. Compared to MOE-modified AOs, the TMO-modified AOs also demonstrated reduced formation of structured nuclear inclusions and SFPQ aggregation that can be triggered by phosphorothioate-containing AOs. How overall length and gap length of the TMO-modified AOs affected allele selectivity, efficiency and off-target gene knockdown was also evaluated. We have shown that allele-selective knockdown of FUS may be a viable therapeutic strategy for treating FUS-ALS and demonstrated the benefits of the TMO modification for allele-selective applications. [ABSTRACT FROM AUTHOR]

The method to form a carbonyl phosphorus bond by the use of a P(III) phosphorus nulceophile on a carbon electrophile to produce phosphinoformic acid diamidites is presented. The results demonstrate that phosphonoformate oligodeoxyribonucleotides could be successfully synthesized and are potentially useful in various biochemical systems.

A new two-step solid-phase phosphoramidite based oligodeoxynucleotide (ODN) synthesis method, which is developed by replacing the 5'-dimethoxytrityl blocking group with an aryloxycarbonyl and using N-dimethoxytrityl protection for the exocyclic amines of adenine and cytosine is described. It is observed that the cyclical removal of 5'-protecting group is nonreversible and quantitative.

The phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides are made through the solid-phase synthesis process. Both the analogues are quite consistent in the direction of hydrolysis with snake poison phosphodiesterase and activated RNase H1 activity.

The solid-phase synthesis of two novel classes of phosphopeptide mimetics, O-boranophosphopeptides and O-dithiophosphopeptides, derivatized on tyrosine, serine and threonine is depicted. These peptide analogues that mimic the binding properties of phosphoproteins are essential for the in vitro and in vivo study of signal transduction cascades.

Research has been conducted on the transition state interactions in the Tetrahymena ribozyme reaction. The oxygen atoms near the reaction center have been examined in investigating the effect of these interactions on the catalysis, and the use of the new type DNA substrate where 5'-oxygen is replaced with a methylene has been described.

Research has been conducted on the hammerhead ribozyme with a phosporodithioate linkage. The role of the metal ion in the catalysis has been investigated.

An approach for the synthesis of 5'-dithiophosphoryl deoxyoligonucleotides is discussed. It involves the reaction of O-(9-fluorenemethyl)H-phosphonothioate with a protected deoxyoligonucleotide and the oxidation of phosphorus with sulfur. Oxidation of the 5'-dithiophosphoryl moiety results in the formation of deoxyoligonucleotide dimers and deoxyoligonucleotides' cysteine unit.

DNA oligomers with phosphorodithioate are resistant to nuclease degradation and effectively direct RNase H degradation of target RNA. An evaluation in HeLa cell nuclear and cytoplasmic extracts indicates that oligomers with more than one unmodified linkage separating phosphorodithioates are degraded by DNase I. Oligomers with alternating phosphate and phosphorodithioate are however, resistant to degradation in all systems. Phosphorodithioate oligomers have low melting points and form stable duplexes with unmodified DNA.

Deoxynucleoside 3'-phosphorothioamidites with beta-(benzoylmercapto)ethyl protecting groups on sulfur rapidly produce phosphorodithioate DNA in high yields with minimum side products. The synthesis of phosphorodithioate DNA involves the formation of deoxynucleoside 3'-phosphorothioamidites from deoxynucleoside, tris(pyrrolidino)phosphine and ethanedithiol monobenzoate. The activation of the synthons with tetrazole, condensation with a deoxynucleoside on a polymer support, followed by oxidation with sulfur, acylation and removal of the 5'-protecting group completes the synthesis.

The paper describes the specific downregulation of ERBB2 protein and mRNa in the breast cancer cell line SK-BR-3, using antisense DNA phosphorothioates. A novel approach was developed to examine antisense effects that allows simultaneous measurement of antisense dose and gene specific regulation on an individual cell basis. A fluorescein isothiocynate end labeled tracer oligonucleotide along with antisense DNA followed by immunofluorescent staining for and ERBB2 protein expression was utilized. After 2 days of ERBB2 suppression, an accumulation of cancer cells in the G1 phase of the cell cycle was noticed.

A novel technique enables the synthesis of deoxyadenosine 5'- and 3'-dithiophosphate and thymidine 5'- and 3'-dithiophosphate. The technique couples deoxynucleoside H-phosphonodithioates and 9-fluorenemethanol in an oxidative environment, which yields 9-fluorenemethyl phosphoradithioates. Deprotection of 9-fluorenemethyl phosphoradithioates by ammonium hydroxide, followed by purification yields the dithiophosphates. In vitro studies on these compounds as inhibitor of enzyme activity for potential therapeutic use show no inhibitory effects.

Catalytic RNA of Tetrahymena thermophila is a metalloenzyme. This ribozyme has an absolute requirement for one of the divalent metal ions Mg2+ or Mn2+ to catalyze cleavage of exogenous RNA or DNA substrate. The presence of Mg2+ alone slows this cleavage while the addition of Mn2+ or Zn2+ speeds it about 1,000 times. The presence of Zn2+ and Mn2+ is thought to speed the reaction via coordination of sulphur.

Outlines the chemistry of the rapid synthesis of relatively large DNA fragments (100-200 monomers each) with yields exceeding 99 percent per coupling. DNA synthesis methodologies are outlined and a polymer-supported synthesis of DNA using deoxynucleoside phosphoramidites is described with structural formulas. (YP)

A new strategy that generates mixed sequence borane phosphonate DNA of high yield and purity is reported. This includes the use of borane phosphonate oligodeoxyribonucleotides for various diagnostic and therapeutic applications as well as for development of borane phosphonate oligomers, having among others, phosphorothioate, phosphorodithioate, and methyl phosphonate internucleotide linkages.