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The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

As part of a program aimed at exploring the structure- activity relationships of 2',4'-bridged nucleic acid (BNA) containing antisense oligonucleotides (ASOs), we report the synthesis and biophysical and biological properties of R- and S-5'-Me LNA modified oligonucleotides. We show that introduction of a methyl group in the (S) configuration at the 5'-position is compatible with the high affinity recognition of complementary nucleic acids observed with LNA. In contrast, introduction of a methyl group in the (R) configuration reversed the stabilization effect of LNA. NMR studies indicated that the R-5'-Me group changes the orientation around torsion angle γ from the +sc to the ap range at the nucleoside level, and this may in part be responsible for the poor hybridization behavior exhibited by this modification. In animal experiments, S-5'-Me-LNA modified gapmer antisense olignucleotides showed slightly reduced potency relative to the sequence matched LNA ASOs while improving the therapeutic profile.

We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.

Chemically modified antisense oligonucleotides are currently progressing in multiple clinical trials. Among several chemical modifications made, modification of the 2'-position has proved most successful. Second generation antisense oligonucleotides incorporating these 2'-modifications exhibit high binding affinity to target RNA, enhanced metabolic stability, and improved pharmacokinetic and toxicity profiles. This is, in part, due to the enhanced biophysical properties of second generation antisense oligonucleotides. 2'-Modifications that influence the sugar to adopt a 3'-endo sugar pucker can improve properties such as affinity. 2'-Modifications that provide a gauche effect and/or a charge effect can play a significant role in the level of nuclease resistance. The heterocyclic base modifications such as 2-thiothymine provides additive effect on the affinity of 2'-F and 2'-O-MOE modifications. This review summarizes the structural and biophysical properties of selected 2'-modified nucleosides which are candidates for use in oligonucleotide therapeutics.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.

Human RNase H1 cleaves RNA exclusively in an RNA/DNA duplex; neither double-strand DNA nor double-strand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2'-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2'-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra- and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.

A stereoselective synthesis of 3′-deoxy-3′-C-formyl-5-methyluridine 2 is described via the dithiane 4 as the key intermediate. Compound 2 was coupled with 3 into a novel ribo-MMI dimer 1. The dimer was then incorporated into antisense oligonucleotides which were found to have high binding affinity to the target RNA.

Synthetic small interfering RNA (siRNA) mediated silencing of a specific gene is emerging as a powerful tool for gene regulation. However, their utility is limited for therapeutic applications primarily due to poor stability. The 2',5'-linked oligonucleotides are known to be more stable to nucleolytic degradation than 3',5'-linked oligonucleotides. The 2',5'-linkage is tolerated in the sense strand of the siRNA duplex. However, the 2',5'-linkage is not tolerated in the antisense strand of the siRNA duplex.

N -Benzoyltetrazole has been developed as a mild and selective reagent for monobenzoylation of the exocyclic amino group in nucleic acid bases. Its usefulness is demonstrated by protection of adenine and cytosine bases, an important procedure in the nucleic acid chemistry field.

The facile preparation of two novel classes of nucleoside analogs for the inclusion as dimeric non-phosphorous containing subunits in chimeric backbones has been accomplished. The concise preparation of 3′-formylnucleosides and 5′-O-(N-methylhydroxylamino)-nucleosides is reported.

Methylene(methylimino) or MMI linkage is a novel backbone modification that has enormous potential in the oligonucleotide-based antisense therapeutics as a replacement for the natural phosphodiester linkage. This presentation synopsis covers the rationale, detailed SAR on the optimization process of this linkage vs. others, various synthetic strategies to construct MMI linkage and a brief discussion on the biological properties of the modified oligonucleotides.

We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.

The facile preparation of two novel classes of nucleoside analogs for the inclusion as dimeric non-phosphorous containing subunits in chimeric backbones has been accomplished. The concise preparation of 3′-formylnucleosides and 5′-O-(N-methylhydroxylamino)-nucleosides is reported.

In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2 ′-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2 ′-O-methyl-2-thiouridine residues could lead to modified siRNA with activity in mammalian cells.

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.

A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2‘-O-methyl and 2‘-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

The synthesis of a series of methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the 2′‐position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2′‐OMe MMI dimer was selected for further studies based on its synthetic accessibility and the increased thermodynamic stability conferred upon oligonucleotides incorporating this modification. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

We would like to comment on a recent study that analyzed the role of microRNA-21 (miR-21) in a mouse model of cardiac disease (1). Using miR-21–deficient mice and novel, very short, 8-nucleotide anti–miR-21 oligonucleotides, the authors failed to detect any modulation of pressure overload-induced myocardial hypertrophy and fibrosis and concluded that miR-21 plays no role in cardiac disease. In contrast, we and others reported that inhibition of miR-21 with highly specific, 22- and 15-nucleotide-long anti–miR-21 oligonucleotides effectively inhibits myocardial and pulmonary fibrosis (2, 3). While genetic deletion of a target may lead to compensation during development and is often different from pharmacological inhibition of this target in the adult organism, the discrepancy between the therapeutic trials using long versus short 8-mer oligonucleo­tides is striking. We therefore carried out a direct head-to-head comparison of three different oligonucleotide chemistries (Figure ​(Figure1A)1A) in the same model of pressure overload-induced cardiac hypertrophy (transaortic constriction [TAC]). The two 22-mer oligonucleotides were complementary to the full-length miR-21, while the 8-mer was complementary to nucleotides 2 to 9 of miR-21, locked nucleic acid modified (LNA modified), and identical to the oligonucleotide used in the report by Patrick et al. (Figure ​(Figure1A). 1A). Figure 1 Comparison of different miR-21 oligonucleotide inhibitors.

MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.

Synthesis of a novel ribo-MMI dimer with 2′-OH and 2′-OMe in 5′- and 3′-nucleosides, respectively is presented. The synthesis was accomplished by reductive coupling of 3′-deoxy-3′-C-formyluridine and 2′-O-methyl-5′-O-methylaminouridine via a thioacetal as the key intermediate for the top part of the dimer. Incorporation of ribo- MMI dimers into oligonucleotides increased binding affinity for target RNA.

A stereoselective synthesis of 3′-deoxy-3′-C-formyl-5-methyluridine 2 is described via the dithiane 4 as the key intermediate. Compound 2 was coupled with 3 into a novel ribo-MMI dimer 1. The dimer was then incorporated into antisense oligonucleotides which were found to have high binding affinity to the target RNA.

Phosphorothioate antisense oligodeoxyribonucleotides (PS-ASOs) have proven to be useful first generation antisense tools for in vitro and in vivo uses and now show great promise as human therapeutic agents. However, there are two characteristics of PS-ASOs that make it desirable to continue to attempt to improve their biophysical characteristics through chemical modification. First, PS-ASOs have been reported, at very high concentrations, to have some nonspecific activities, both in vitro and in vivo, usually attributed to their protein binding properties. Second, while significantly more stable than their phosphodiester analogues, the in vivo stability of phosphorothioate oligonucleotides can still be improved. This instability is primarily due to 3′ exonucleases, 5′ exonucleases, and to a lesser degree, endonucleases. There is a strong rationale for exploring backbone modifications that can reduce the P=S content and maintain or increase nuclease resistance of antisense oligonucleotides. One su...

Antisense drug discovery technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.(1) We have previously demonstrated that replacing 2'O-methoxyethyl (MOE, 2) residues in second generation antisense oligonucleotides (ASOs) with LNA (3) nucleosides improves the potency of some ASOs in animals. However, this was accompanied with a significant increase in the risk for hepatotoxicity.(2) We hypothesized that replacing LNA with novel nucleoside monomers that combine the structural elements of MOE and LNA might mitigate the toxicity of LNA while maintaining potency. To this end we designed and prepared novel nucleoside analogs 4 (S-constrained MOE, S-cMOE) and 5 (R-constrained MOE, R-cMOE) where the ethyl chain of the 2'O-MOE moiety is constrained back to the 4' position of the furanose ring. As part of the SAR series, we also prepared nucleoside analogs 7 (S-constrained ethyl, S-cEt) and 8 (R-constrained Ethyl, R-cEt) where the methoxymethyl group in the cMOE nucleosides was replaced with a methyl substituent. A highly efficient synthesis of the nucleoside phosphoramidites with minimal chromatography purifications was developed starting from cheap commercially available starting materials. Biophysical evaluation revealed that the cMOE and cEt modifications hybridize complementary nucleic acids with the same affinity as LNA while greatly increasing nuclease stability. Biological evaluation of oligonucleotides containing the cMOE and cEt modification in animals indicated that all of them possessed superior potency as compared to second generation MOE ASOs and a greatly improved toxicity profile as compared to LNA.

Antisense oligonucleotides complementary to specific mRNA sequences are widely used inhibitors of gene expression in vitro and in vivo . In vitro cationic lipids have been demonstrated to increase the pharmacological activity of antisense oligonucleotides by increasing cellular uptake and facilitating nuclear accumulation. We have investigated the intracellular fate of oligonucleotide/cationic lipid complexes using fluorescently labeled lipids and oligonucleotides targeted to protein kinase C-alpha. After addition to cells the lipids initially co-localized with the oligonucleotide on the cell surface and in fine punctate structures within the cytoplasm. At later times the oligonucleotide began to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cytoplasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appearance of a smaller mRNA transcript believed to result from RNase H cleavage of protein kinase C-alpha mRNA. These data suggest that although cationic lipids facilitate uptake of oligonucleotides, the complex must disassociate before the oligonucleotide can gain access to the nucleus and induce degradation of targeted mRNA.

micro-RNA-21 is frequently over-expressed and has been shown to correlate with poor outcome in multiple cancer types. Its genomic locus is amplified in some cancer types and miR-21 is also transactivated by several known cancer drivers (e.g. STAT3 and Ras). We have used a publically available data set from 86 patients to show that miR-21 is over-expressed in hepatocellular carcinoma (HCC). The treatment options for HCC are limited, thus making it the third leading cause of cancer related deaths worldwide. Additional treatments for HCC are greatly needed. We investigated the therapeutic potential of inhibiting miR-21 with chemically modified oligonucleotides known as anti-miRs using models of HCC, including a genetically engineered mouse model of HCC driven by an inducible, activated form of Ras. Short term treatment with the anti-miR led to a reduction in tumor formation. Furthermore, inhibition of miR-21 was clearly demonstrated by analysis of genome wide mRNA expression data from treated versus untreated tumors. This data showed that genes containing the mir-21 octamer sequence were the most significantly over-enriched among up-regulated genes compared to all of the other 65,535 possible octamers. We also determined the effect of long-term anti-miR-21 treatment on the survival in this mouse model of HCC. anti-miR-21 treated mice demonstrated a highly significant survival advantage (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2011-4708