Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing.

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2'-O-methyl and 2'-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5-20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.

Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis.

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.

Synthesis of Backbone-Modified Morpholino Oligonucleotides Using Phosphoramidite Chemistry.

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).

DNA Analogues Modified at the Nonlinking Positions of Phosphorus.

Chemically modified oligonucleotides are being developed as a new class of medicines for curing conditions that previously remained untreatable. Three primary classes of therapeutic oligonucleotides are single-stranded antisense oligonucleotides (ASOs), double stranded small interfering RNAs (siRNAs), and oligonucleotides that induce exon skipping. Recently, ASOs, siRNAs, and exon skipping oligonucleotides have been approved for patients with unmet medical needs, and many other candidates are being tested in late stage clinical trials. In coming years, therapeutic oligonucleotides may match the promise of small molecules and antibodies. Interestingly, in the 1980s when we developed chemical methods for synthesizing oligonucleotides, no one would have imagined that these highly charged macromolecules could become future medicines. Indeed, the anionic nature and poor metabolic stability of the natural phosphodiester backbone provided a major challenge for the use of oligonucleotides as therapeutic drugs. Thus, chemical modifications of oligonucleotides were essential in order to improve their pharmacokinetic properties. Keeping this view in mind, my laboratory has developed a series of novel oligonucleotides where one or both nonbridging oxygens in the phosphodiester backbone are replaced with an atom or molecule that introduces molecular properties that enhance biological activity. We followed two complementary approaches. One was the use of phosphoramidites that could act directly as synthons for the solid phase synthesis of oligonucleotide analogues. This approach sometimes was not feasible due to instability of various synthons toward the reagents used during synthesis of oligonucleotides. Therefore, using a complementary approach, we developed phosphoramidite synthons that can be incorporated into oligonucleotides with minimum changes in the solid phase DNA synthesis protocols but contain a handle for generating appropriate analogues postsynthetically.This Account summarizes our efforts toward preparing these types of analogues over the past three decades and discusses synthesis and properties of backbone modified oligonucleotides that originated from the Caruthers' laboratory. For example, by replacing one of the internucleotide oxygens with an acetate group, we obtained so-called phosphonoacetate oligonucleotides that were stable to nucleases and, when delivered as esters, entered into cells unaided. Alternatively oligonucleotides bearing borane phosphonate linkages were found to be RNase H active and compatible with the endogenous RNA induced silencing complex (RISC). Oligonucleotides containing an alkyne group directly linked to phosphorus in the backbone were prepared as well and used to attach molecules such as amino acids and peptides.

Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras.

This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacophores, namely, phosphorothioates (pS) and morpholinos, to create morpholino-pS hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino-pS moieties and therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogues that are hitherto unexplored in the oligotherapeutics arena. Exclusively TMO-modified ONs demonstrate high stability toward 3'-exonuclease. Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA-pS subunits exhibit higher binding affinity toward complementary RNA relative to the canonical DNA/RNA duplex (∼10 °C). Oligonucleotides that consist entirely of TMO linkages also show higher RNA binding affinity but do not recruit ribonuclease H1 (RNase H1). Chimeric TMO analogues demonstrate high gene silencing efficacy, comparable to that of a chimeric 2'-OMe-pS/pO control, during in vitro bioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.

Pyridinium Boranephosphonate Modified DNA Oligonucleotides.

The synthesis of previously unknown derivatives of boranephosphonate that contain amine substitutions at boron and the incorporation of these derivatives into the backbone of DNA oligonucleotides is described. These derivatives result from iodine-mediated replacement of one BH3 hydride of a boranephosphonate linkage by pyridine, various substituted pyridines, other aromatic amines, and certain unsaturated amines. Oligonucleotides containing these backbone modifications show enhanced uptake, relative to unmodified DNA, in mammalian cells. The redox behavior of the boranephosphonate and pyridinium boranephosphonate conjugated linkages has also been studied.

Oligodeoxynucleotides containing 2'-amino-LNA nucleotides as constrained morpholino phosphoramidate and phosphorodiamidate monomers.

Incorporation in a 2'→5' direction of a phosphorodiamidite 2'-amino-LNA-T nucleotide as the morpholino phosphoramidate and N,N-dimethylamino phosphorodiamidate monomers into six oligonucleotides is reported. Thermal denaturation studies showed that the novel 2'-amino-LNA-based morpholino monomers exert a destabilizing effects on duplexes formed with complementary DNA and RNA.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides and Their Chimeras Using Phosphoramidite Chemistry.

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.

Oxidative substitution of boranephosphonate diesters as a route to post-synthetically modified DNA.

The introduction of modifications into oligonucleotides is important for a large number of applications in the nucleic acids field. However, the method of solid-phase DNA synthesis presents significant challenges for incorporating many useful modifications that are unstable to the conditions for preparing synthetic DNA. Here we report that boranephosphonate diesters undergo facile nucleophilic substitution in a stereospecific manner upon activation by iodine. We have subsequently used this reactivity to post-synthetically introduce modifications including azides and fluorophores into DNA by first synthesizing boranephosphonate-linked 2'-deoxyoligonucleotides and then treating these oligomers with iodine and various nucleophiles. In addition, we show that this reaction is an attractive method for preparing stereodefined phosphorus-modified oligonucleotides. We have also examined the mechanism of this reaction and show that it proceeds via an iodophosphate intermediate. Beyond nucleic acids synthesis, due to the ubiquity of phosphate derivatives in natural compounds and therapeutics, this stereospecific reaction has many potential applications in organophosphorus chemistry.

Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate-Modified Nucleotides.

We investigate the efficiency of incorporation of boranephosphonate-modified nucleotides by phi29 DNA polymerase and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internucleotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single-stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5'-(α-P-borano)deoxynucleosidetriphosphate, only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides, it is possible to enzymatically synthesize a micrometer-sized, single-stranded DNA molecule with only boranephosphonate internucleotide linkages. Well-defined silver particles are then readily formed along the rolling circle product.

Solid-Phase Synthesis, Hybridizing Ability, Uptake, and Nuclease Resistant Profiles of Position-Selective Cationic and Hydrophobic Phosphotriester Oligonucleotides.

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.

The chemical synthesis of DNA/RNA: our gift to science.

It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs.

Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Silver nanoassemblies constructed from boranephosphonate DNA.

Spatially selective deposition of metal onto complex DNA assemblies is a promising approach for the preparation of metallic nanostructures with features that are smaller than what can be produced by top-down lithographic techniques. We have recently reported the ability of 2'-deoxyoligonucleotides containing boranephosphonate linkages (bpDNA) to reduce AuCl4(-), Ag(+), and PtCl4(2-) ions to the corresponding nanoparticles. Here we demonstrate incorporation of bpDNA oligomers into a two-dimensional DNA array comprised of tiles containing double crossover junctions. We further demonstrate the site-specific deposition of metallic silver onto this DNA structure which generates well-defined and preprogrammed arrays of silver nanoparticles. With this approach the size of the metallic features that can be produced is limited only by the underlying DNA template. These advances were enabled due to a new method for synthesizing bpDNA that uses a silyl protecting group on the DNA nucleobases during the solid-phase 2'-deoxyoligonucleotide synthesis.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

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.

Alkynyl phosphonate DNA: a versatile "click"able backbone for DNA-based biological applications.

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-modified 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-modified 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.