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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.

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.

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).

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

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.

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 BH 3 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.

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.

Chemically modified oligodeoxynucleotides (ODNs) are known to modulate gene expression by interacting with RNA. An efficient approach for synthesizing amino acid- or peptide-substituted triazolylphosphonate analogs (TP ODNs) has been developed to provide improved stability and cell uptake. The chemistry is quite general, as peptides can be introduced throughout the TP ODN at any preselected internucleotide linkage. These synthetic TP ODNs enter cells through endocytosis in the absence of transfection reagents and localize into perinuclear organelles. The entrapped ODNs are released into the cytoplasm by treatment with endosomal-releasing agents and several are then active as microRNA inhibitors., Competing Interests: The authors declare no conflict of interest.

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.

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.

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.

Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2'-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.

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.

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.

Oligodeoxyribonucleotides bearing boranephosphonate linkages (bpDNA) were shown to reduce a number of metal ions and form nanoparticles through a novel reaction pathway that leads to phosphate diesters or phosphate triesters in water or alcohols respectively. The synthetic utility of this reaction was further demonstrated through the synthesis of oligodeoxyribonucleotides containing phosphate triester linkages. This new reactivity also makes bpDNA promising for use in construction of DNA templated metallic nanostructures.

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.

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

An improved method for the chemical synthesis of RNA was developed utilizing a streamlined method for the preparation of phosphoramidite monomers and a single-step deprotection of the resulting oligoribonucleotide product using 1,2-diamines under anhydrous conditions. The process is compatible with most standard heterobase protection and employs a 2'-O-(1,1-dioxo-1λ(6)-thiomorpholine-4-carbothioate) as a unique 2'-hydroxyl protective group. Using this approach, it was demonstrated that the chemical synthesis of RNA can be as simple and robust as the chemical synthesis of DNA.

The major hurdle associated with utilizing oligodeoxyribonucleotides for therapeutic purposes is their poor delivery into cells coupled with high nuclease susceptibility. In an attempt to combine the nonionic nature and high nuclease stability of the P-C bond of methylphosphonates 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-BH(3)) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynucleotides (ODNs) 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-BH(3) 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.

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

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).

An accurate mass determination method utilizing electrospray ionization mass spectrometry is described for analysis of several different types of phosphoramidites that are extremely acid-sensitive compounds. An earlier method, which applied a LiCl/acetonitrile system, was extended for this special application by using polymeric standards including poly(ethylene glycol) (PEG), poly(ethylene glycol) dimethyl ether (PDE) and poly(propylene glycol) (PPG). Concentrations of standards, samples and LiCl were optimized and potential impurities that affect the analyses were also investigated.

Oligodeoxynucleotides containing internucleotide phosphonoacetate esters are taken up irreversibly by cells in culture in the absence of cationic lipids. These oligonucleotides also are active in stimulating RNase H and are stable toward nucleases.

A novel solid-phase phosphoramidite-based method has been developed for the synthesis of borane phosphonate DNA. Keys to this new approach are replacement of the common 5'-dimethoxytrityl blocking group with a 5'-silyl ether and the use of new protecting groups on the bases (adenine, N6-dimethoxytrityl; cytosine, N4-trimethoxytrityl; guanine, N2-[9-fluorenylmethoxycarbonyl]; thymine, N3-anisoyl). Because of these developments, it is now possible for the first time to synthesize oligodeoxynucleotides having any combination of the four 2'-deoxynucleosides and both phosphate and borane phosphonate internucleotide linkages (including oligomers having exclusively borane phosphonate linkages).

Phosphonoformate oligodeoxyribonucleotides were prepared via a solid phase synthesis strategy. The first step in the preparation of appropriate synthons was condensation of bis(N,N-diisopropylamino)phosphine and diphenylmethylsilylethyl chloroformate in the presence of sodium metal to yield formic acid, [bis(N,N-diisopropylamino)phosphino]-beta-(diphenylmethylsilylethyl) ester. The product of this reaction was then condensed with appropriately protected 2'-deoxynucleosides using 4,5-dicyanoimidazole to yield the 3'-O-phosphinoamidite reactive monomers. The exocyclic amines of cytosine, adenine, and guanine were protected with 9-fluorenylmethyloxycarbonyl, and oligodeoxyribonucleotides were synthesized on controlled pore glass using the hydroquinone-O,O'-diacetic acid linker. Synthons were sequentially added to this support using tetrazole as an activator, oxidized to phosphonoformate, and the transient 5'-protecting group was removed with acid. Following total synthesis of an oligomer, protecting groups were removed with TEMED.HF and products purified by HPLC. These analogues were resistant to nucleases, formed duplexes with complementary RNA (A-form), and, as chimeric oligomers containing phosphate at selected sites, stimulated RNase H1 activity.

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.

For the first time a new, two-step method is described for synthesizing deoxyribonucleic acid. The approach uses 5'-carbonate protected 2'-deoxynucleoside-3'-phosphoramidites as synthons and a peroxy anion buffer that removes the carbonate protecting group and oxidizes the internucleotide linkage. Following synthesis via this two-step cycle, oligomers are isolated by standard procedures.

Oligodeoxyribonucleotides with phosphonoacetate or thiophosphonoacetate internucleotide linkages can be made in high yield by solid-phase synthesis and possess many advantages. They are highly stable to nucleases, water-soluble, and anionic at neutral pH. They form stable duplexes with DNA and RNA, and stimulate RNase H degradation of complementary RNA. The preparation of the N,N-(diisopropylamino)phosphinyl acetate monomers from standard protected nucleosides is described here, followed by the synthesis of phosphonoacetate and thiophosphonoate oligodeoxyribonucleotides, as well as chimeric oligomers that have these modified linkages in combination with natural or phosphorothioate linkages. Purification and characterization of these oligomers is also presented.

A novel solid-phase phosphoramidite based oligodeoxynucleotide two-step synthesis method has been developed. Keys to this method are replacement of the 5'-dimethoxytrityl blocking group with an aryloxycarbonyl and the use of N-dimethoxytrityl protection for the exocyclic amines of adenine and cytosine. With these modifications, coupling of each 2'-deoxynucleoside 3'-phosphoramidite to the growing oligodeoxynucleotide on the solid support can be followed by treatment with an aqueous mixture of peroxy anions buffered at pH 9.6. This reagent effectively removes the carbonate protecting group and simultaneously oxidizes the phosphite internucleotide linkage. As a consequence a new two-step synthesis cycle is possible. Oligodeoxynucleotides synthesized using this approach are identical to authentic samples when tested by a variety of analytical techniques.

Phosphorus-modified phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides were chemically synthesized and their biochemical properties evaluated. Under physiological pH, these DNA analogs possess negative charge and form stable, complementary A-like DNA:RNA heteroduplexes when analyzed via circular dichroism spectroscopy. Phosphonoacetate and thiophosphonoacetate oligomers were found to stimulate RNase H activity and to be completely resistant to degradation by snake venom phosphodiesterase, DNase I and HeLa cell nuclear extract. Further research has demonstrated that neutral, esterified forms of these analogs can be taken up by cells. Phosphonoacetate and thiophosphonoacetate oligomers therefore represent a new class of oligodeoxyribonucleotide analogs having phosphorus- carbon bonds with considerable potential for use in biological research.

Phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides were prepared via a solid-phase synthesis strategy. Under Reformatsky reaction conditions, novel esterified acetic acid phosphinodiamidites were synthesized and condensed with appropriately protected 5'-O-(4, 4'-dimethoxytrityl)-2'-deoxynucleosides to yield 3'-O-phosphinoamidite reactive monomers. These synthons when activated with tetrazole were used with an automated DNA synthesizer to prepare phosphonoacetic acid modified internucleotide linkages on controlled pore glass. The phosphinoacetate coupling products were quantitatively oxidized at each step with (1S)-(+)-(10-camphorsulfonyl)oxaziridine or 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce mixed sequence phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides with an average per cycle coupling efficiency of greater than 97%. Completely deprotected, modified oligodeoxynucleotides were purified by reverse-phase HPLC and characterized by ion exchange HPLC, (31)P NMR, and MALDI/TOF mass spectroscopy. Both analogues were stable toward hydrolysis with snake venom phosphodiesterase and stimulated RNase H1 activity.

Signal transduction cascades maintain control over important cellular processes such as cell growth and differentiation by orchestrating protein phosphorylation and dephosphorylation. Specific control of these processes in vivo and in vitro can be achieved with peptide analogues that mimic the binding properties of phosphoproteins. We present here the solid-phase synthesis of two novel classes of phosphopeptide mimetics, O-boranophosphopeptides and O-dithiophosphopeptides, derivatized on tyrosine, serine, and threonine. The use of H-phosphonate and H-phosphonothioate monoesters containing the base labile 9-fluorenemethyl protecting group was key to the synthesis of both phosphopeptide mimetics. O-Boranophosphopeptides were synthesized by condensing O-(9-fluorenemethyl)-H-phosphonate to the peptide hydroxylic component (tyr, ser, or thr) followed by oxidation with borane complexes. Similarly, the synthesis of O-dithiophosphopeptides used the O-(9-fluorenemethyl)-H-phosphonothioate synthon and oxidation with elemental sulfur. Base elimination of the Fmol protecting group and cleavage from the solid support with concentrated ammonium hydroxide afforded the boranophosphopeptide and dithiophosphopeptide target compounds. Ac-YIIPLPG-NH2, having either dithiophosphoryl tyrosine or boranophosphoryltyrosine but no sequence specificity for Yersinia protein tyrosine phosphatase (PTP), was found to competitively inhibit this enzyme with KI values of 430 +/- 50 and 670 +/- 50 microM, respectively. In addition, both phosphopeptide analogues were resistant toward Yersinia PTP enzymatic hydrolysis. Under conditions (pH 8.0) where the phosphopeptide was rapidly dephosphorylated, the boranophosphopeptide hydrolyzed slowly (t1/2 = 15 h) and the dithiophosphopeptide was completely stable over 24 h.

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by <5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.

The phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.

Under standard reaction conditions, a hammerhead ribozyme with a phosphorodithioate linkage at the cleavage site cleaved to the expected products with a rate about 500-fold slower than the corresponding phosphodiester linkage. When the greater stability of the dithioate linkage to nonenzymatic nucleophilic attack is taken into account, the hammerhead is remarkably effective at cleaving the dithioate linkage considering that the R(P)-phosphoromonothioate linkage is virtually inactive. On the basis of experiments determining the Mg(2+) concentration dependence of the cleavage rate and the stimulation of cleavage by thiophilic Cd(2+) ion, the lesser catalytic rate enhancement of the dithioate linkage is primarily due to the loss of a single Mg(2+) ion bound near the cleavage site. These results are qualitatively similar to, but quantitatively different from, similar experiments examining the hammerhead cleavage properties of the R(P)-phosphoromonothioate linkage. The dithioate linkage thus promises to be a valuable alternative phosphate analogue to the monothioate linkage in studying the mechanisms of RNA catalysis.

Chemical modification of backbone structures has been an important strategy in designing oligonucleotides capable of improved antisense effects. However, altered backbone chemistry may also affect the binding of oligonucleotides to key cellular proteins, and thus may impact on the overall biological action of antisense agents. In this study we have examined the binding of oligonucleotides having four different backbone chemistries to single-strand binding protein (SSB), a protein having a key role in DNA repair and replication. The oligomers tested had the same sequence, while the internucleoside linkages were phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), or methylphosphonate (MP). We found that both PS and PS2 oligomers bound to SSB with higher affinity than PO oligonucleotides, while MP oligonucleotides did not bind appreciably at the concentrations tested. Oligonucleotide length was also an important factor in binding to SSB, but sequence was less critical. These observations indicate that backbone chemistry is an important factor in interactions between oligonucleotides and critical cellular proteins, and thus may be a key determinant of the biological effects of antisense oligonucleotides.

Antisense activity against erbB-2 of a variety of sulfur-modified oligonucleotides was examined in a breast cancer cell line which overexpresses this oncogene. Using a 15 base anti-erbB-2 sequence previously shown to be effective, various backbone configurations containing phosphoromonothioate or phosphorodithioate linkages were evaluated for antisense activity by a two-color flow cytometric assay. This sequence was effective in inhibiting the production of erbB-2 protein when it was configured as a monothioate at each linkage and as an alternating dithioate/phosphodiester. Both of these compounds were also able to specifically inhibit erbB-2 mRNA expression, indicative of RNase H-mediated activity. The same sequence protected by either three dithioate or three monothioate linkages at each end was ineffective as an antisense reagent, suggesting that endonuclease activity is a significant determinant of the stability of oligonucleotides. Finally, the erbB-2 sequence target was shifted in an effort to improve antisense activity. A new lead sequence was identified that was significantly more effective in inhibiting erbB-2 protein levels and retained activity at lower concentrations.

The biochemical and physicochemical properties of DNA oligomers containing phosphorodithioate linkages in various configurations were evaluated. Duplex stability studies, which were carried out by thermal denaturation analysis with complementary unmodified DNA, indicated a highly cooperative process similar to completely unmodified duplexes. Oligomers containing phosphorodithioate linkages were found to have reduced melting temperatures relative to unmodified duplexes, with the degree of Tm depression paralleling the percent phosphorodithioate composition of the oligomer. Relative to activation of RNase H, DNA oligomers containing up to 50% phosphorodithioate linkages were able to direct RNase H degradation with the same efficiency as unmodified DNA while those containing from 50 to 100% acted with somewhat reduced efficiency. At limiting concentrations, an oligomer containing alternating phosphorodithioate and phosphate linkages was able to direct RNase H degradation of the target RNA in an extended incubation, while an unmodified oligomer did not. The nuclease resistance of phosphorodithioate-containing oligomers was evaluated in HeLa cell nuclear and cytoplasmic extracts, in human serum, and with nucleases S1 and DNase I. Oligomers containing alternating phosphorodithioate and phosphate were highly resistant to degradation in all systems. However, oligomers having more than one unmodified linkage separating phosphorodithioates were degraded rapidly by DNase I, while demonstrating stability to degradation in all other systems tested. These results indicate that phosphorodithioate-containing DNA oligomers are highly nuclease-resistant, are able to form stable duplexes with complementary nucleic acid sequences, and efficiently direct RNase H degradation of target RNA.