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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]

Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases' inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO). [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.

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.

Morpholinos are six‐membered rings that may provide higher conformational rigidity when incorporated into an oligonucleotide (ODN) backbone. Phosphorodiamidate morpholinos are chemically modified ODNs containing morpholinos in place of 2'‐deoxyribose moieties throughout their backbone and have garnered much interest in recent years due to their ability to function as highly effective steric blockers in exon skipping therapy. To further explore the biophysical and biological properties of ODNs derived from morpholino nucleosides, we have replaced the 2'‐deoxyribonucleotides of phosphodiester DNA with morpholinonucleotides to generate phosphoramidate ODNs. Here, we evaluate the mechanistic pathways observed during the solution‐phase synthesis of morpholinonucleoside phosphoramidites, solid‐phase synthesis of morpholinonucleotide phosphoramidates of mA, mG, mC and mT (prefix 'm' represents morpholino) and our first attempts directed at the solid‐phase synthesis of chimeric DNA‐phosphoramidate ODNs, as well as fully modified 22‐mer phosphoramidate ODNs. [ABSTRACT FROM AUTHOR]

Phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras have been prepared on DNA synthesizers using phosphoramidite chemistry. This was possible by first generating boranephosphoroamidate morpholino internucleotide linkages followed by oxidative substitution with four different amines: N,N-dimethylamine, N-methylamine, ammonia, and morpholine. When compared to a natural DNA duplex, the amino modified PMO was found to have a higher melting temperature with either complementary DNA or RNA, whereas the remaining PMO analogues having morpholino, dimethylamino, or N-methylamino phosphorodiamidate linkages had melting temperatures that were either comparable or reduced. Additionally the N,N-dimethylamino PMO-DNA chimeras were found to stimulate RNaseH1 activity. Treatment of HeLa cells with fluorescently labeled PMO chimeras demonstrated that these analogues were efficiently taken up by cells in the presence of a lipid transfection reagent. Because of the simplistic synthesis procedures, various PMO analogues are now readily available and should therefore open new pathways for research into the antisense, diagnostic, and nanotechnology oligonucleotide fields. [ABSTRACT FROM AUTHOR]

Analogues of oligonucleotides and mononucleotides with hydrophobic and/or cationic phophotriester functionalities often generate an improvement in target affinity and cellular uptake. Here we report the synthesis of phosphotriester oligodeoxyribonucleotides (ODNs) that are stable to the conditions used for their preparation. The method has been demonstrated by introducing phosphoramidite synthons where N-benzyloxycarbonyl (Z) protected amino alcohols replace the cyanoethyl group. After synthesis these ODNs were found to be stable to the condition required to remove base labile protecting groups and the ODNs from the solid support. Moreover the use of 1-(4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde) in place of Z protection on the amino alcohol has allowed us to introduce cationic aminoethyl phosphotriester modifications into ODNs. Melting temperatures of duplexes containing cationic or hydrophobic Z modified ODNs indicate that the backbone-phosphotriester modifications minimally affect duplex stability. Nuclease stability assays demonstrate that these phosphotriesters are resistant toward 5′- and 3′-exonucleases. Fluorescently labeled 23-mer ODNs modified with four cationic or hydrophobic Z phosphotriester linkages show efficient cellular uptake during passive transfection in HeLa and Jurkat cells. [ABSTRACT FROM AUTHOR]

The chemical synthesis of DNA and RNA is universally carried out using nucleoside phosphoramidites or H-phosphonates as synthons. This review focuses on the phosphorus chemistry behind these synthons and how it has been developed to generate procedures whereby yields per condensation approach 100% with very few side products. Additionally the synthesis and properties of certain DNA and RNA analogs that are modified at phosphorus will also be discussed. These analogs include boranephosphonates, metallophosphonates, and alkylboranephosphines. [ABSTRACT FROM AUTHOR]

Major hurdles associated with DNA-based biological applications include, among others, targeted cell delivery, undesirable nonspecific effects, toxicity associated with various analogues or the reagents used to deliver oligonucleotides to cells, and stability toward intracellular enzymes. Although a plethora of diverse analogues have been investigated, a versatile methodology that can systematically address these challenges has not been developed. In this contribution, we present a new, Clickable, and versatile chemistry that can be used to rapidly introduce diverse functionality for studying these various problems. As a demonstration of the approach, we synthesized the core analogue, which is useful for introducing additional functionality, the triazolylphosphonate, and present preliminary data on its biological properties. We have developed a new phosphoramidite synthon-the alkynyl phosphinoamidite, which is compatible with conventional solid-phase oligonucleotide synthesis. Postsynthesis, the alkynylphosphonate can be functionalized via "Click" chemistry, to generate the 1,2,3-triazolyl or substituted 1,2,3-triazolyl phosphonate-2'-deoxyribonucleotide internucleotide linkage. This manuscript describes the automated, solid-phase synthesis of mixed backbone oligodeoxyribonucleotides (ODNs) having 1,2,3-triazolylphosphonate (TP) as well as phosphate or thiophosphate internucleotide linkages and also 2'-OMe ribonucleotides and locked nucleic acids (LNAs) at selected sites. Nuclease stability assays demonstrate that the TP linkage is highly resistant toward 5'- and 3'-exonucleases, whereas melting studies indicate a slight destabilization when a TP-modifled ODN is hybridized to its complementary RNA. A fluorescently labeled 16-mer ODN modified with two TP linkages shows efficient cellular uptake during passive transfection. Of particular interest, the subcellular distribution of TP-modifled ODNs is highly dependent on cell type; a significant nuclear uptake is observed in HeLa cells, whereas diffuse cytoplasmic fluorescence is found in the WM-239A cell line. Cytoplasmic distribution is also present in human neuroblastoma cells (SK-N-F1), but Jurkat cells show both diffuse and punctate cytoplasmic uptake. Our results demonstrate that triazolylphosphonate ODNs are versatile additions to the oligonucleotide chemist's toolbox relative to designing new biological research reagents. [ABSTRACT FROM AUTHOR]

The major hurdle associated with utilizing oligo-deoxyribonucleotides for therapeutic purposes is their poor delivery into cells coupled with high nudease susceptibility. In an attempt to combine the nonionic nature and high nuclease stability of the P-C bond ofmethylphosphonates with the high membrane permeability, low toxicity, and improved gene silencing ability of borane phosphonates, we have focused our research on the relatively unexplored methylborane phosphine (Me.-P-BH3) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynucleotides (ObNs) consisting of methylborane phosphine and phosphate or thiophosphate linkages (16-mers). Nuclease stability assays show that methylborane phosphine ODNs are highly resistant to 5′ and 3′ exonucleases. When hybridized to a complementary strand, the ODN:RNA duplex was more stable than its corresponding ODN:DNA duplex. The binding affinity of ODN:RNA duplex increased at lower salt concentration and approached that of a native DNA:RNA duplex under conditions close to physiological saline, indicating that the Me-P-BH3 linkage is positively charged. Cellular uptake measurements indicate that these ODNs are efficiently taken up by cells even when the strand is 13% modified. Treatment of HeLa cells and WM-239A cells with fluorescently labeled ODNs shows significant cytoplasmic fluorescence when viewed under a microscope. Our results suggest that methylborane phosphine ODNs may prove very valuable as potential candidates in antisense research and RNAi. [ABSTRACT FROM AUTHOR]

Current methodologies used to synthesize DNA and RNA are reviewed. These focus on using controlled pore glass and microarrays on glass slides. [ABSTRACT FROM AUTHOR]

Borane phosphonate oligodeoxyribonucleotides are synthesized from 5'-O-[benzhydroxybis(trimethylsilyloxy)silyl]-2'-deoxyribonucleoside-3'-O-methylphosphoramidites. The exocyclic amine functions of adenine, guanine, and cytosine are protected with trimethoxytrityl, and thymine is unprotected. Using these synthons and under standard conditions via activation with S-ethylthiotetrazole, condensations on a highly crosslinked polystyrene support are in excess of 99%. Following the complete synthesis of the oligodeoxyribonucleotide phosphite triester, oxidation with THF·BH3 yields the oligodeoxyribonucleotide borane phosphonate. Further treatment with 80% aqueous acetic acid followed by disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate removes trimethoxytrityl from the 2'-deoxyribonucleoside bases and the methyl protecting group from the internucleotide phosphate triester, respectively. Cleavage from the support with ammonium hydroxide and purification by reverse phase HPLC affords the pure oligodeoxyribonucleotide borane phosphonate. These oligomers are taken up by cells in the absence of cationic lipids and transport biologically active interfering RNA into cells. [ABSTRACT FROM AUTHOR]