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Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.

Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.

SummaryPulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPARγ-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.

Significance Autosomal recessive mutations or deletions of the gene Survival Motor Neuron 1 ( SMN1 ) cause spinal muscular atrophy, a neurodegenerative disorder. Transcriptional up-regulation of a nearly identical gene, SMN2 , can functionally compensate for the loss of SMN1 , resulting in increased SMN protein to ameliorate the disease severity. Here we demonstrate that the repressed state of SMN2 is reversible by interrupting the recruitment of a repressive epigenetic complex in disease-relevant cell types. Using chemically modified oligonucleotides to bind at a site of interaction on a long noncoding RNA that recruits the repressive complex, SMN2 is epigenetically altered to create a transcriptionally permissive state.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.

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.

To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.

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.

The molecular origins of fibrosis affecting multiple tissue beds remain incompletely defined. Previously, we delineated the critical role of the control of extracellular matrix (ECM) stiffening by the mechanosensitive microRNA-130/301 family, as activated by the YAP/TAZ co-transcription factors, in promoting pulmonary hypertension (PH). We hypothesized that similar mechanisms may dictate fibrosis in other tissue beds beyond the pulmonary vasculature. Employing an in silico combination of microRNA target prediction, transcriptomic analysis of 137 human diseases and physiologic states and advanced gene network modeling, we predicted the microRNA-130/301 family as a master regulator of fibrotic pathways across a cohort of seemingly disparate diseases and conditions. In two such diseases (pulmonary fibrosis and liver fibrosis), inhibition of microRNA-130/301 prevented the induction of ECM modification, YAP/TAZ and downstream tissue fibrosis. Thus, mechanical forces act through a central feedback circuit between microRNA-130/301 and YAP/TAZ to sustain a common fibrotic phenotype across a network of human physiologic and pathophysiologic states. Such re-conceptualization of interconnections based on shared systems of disease and non-disease gene networks may have broad implications for future convergent diagnostic and therapeutic strategies.

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.

Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2′- C -methyl-adenosine and 2′- O -methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2′- C -methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2′- O -methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2′- O -methyl-cytidine nucleoside to the active 5′-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of α- d -, β- d -, α- l -, and β- l -configuration were prepared, that included 5-methyl derivatives of both 5 R and 5 S configuration. Molecular mechanics calculations indicate that the 5 S and 5 R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.

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.

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.

Background: Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. Facets of PH have been characterized in isolation, but many have yet to be unified by a common upstream regulator. Methods & Results: We constructed a network in silico of 247 genes and 2,274 functional interactions involved in PH. Using this model and advanced network analysis, we predicted the miR-130/301 family to have the most diverse pool of PH-relevant target genes among conserved microRNAs (miRNAs). Guided by these predictions, we found that this family was up-regulated by hypoxia, inflammatory cytokines, and factors genetically linked to PH. MiR-130/301 family members were also up-regulated in remodeled pulmonary vessels in 7 animal models and 3 human subtypes of PH. Via gain- and loss-of-function experiments in vitro , we found that miR-130/301 suppressed a cohort of PPARgamma-related targets to regulate two pro-proliferative pathways (the apelin-miR-424/503-FGF2 axis in PAECs and the STAT3-miR-204-SRC axis in PASMCs); vasomotor tone ( e.g. , endothelin-1 and endothelial nitric oxide synthase); and extracellular matrix remodeling ( e.g. , the collagen crosslinking enzyme LOX). In turn, delivery of miR-130a mimic oligonucleotides in pulmonary vessels of mice altered expression of the predicted cohort of PPARgamma-related factors, leading to increased vascular remodeling (19.62±1.36 percent muscularized arterioles for miR-130a vs. 6.98±0.55 percent for scrambled control, p3 *dyn*s*cm -5 ] for miR-130a vs. 462±25.4 for control, pin vivo via antisense oligonucleotides mitigated these effects. Conclusions: We used network modeling to identify miR-130/301 as a master regulator of PH and a novel potential therapeutic target. These findings provide critical support for the evolving application of network modeling to discover the hidden molecular origins of PH and other human diseases.

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.

Synthesis of four methylene(methylimino) (MMI) linked dimers modifed 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, conformational study by NMR, and Tm analysis. Several chimeric antisense oligomers containing bis 2′-OMe dimers have been synthesized on a 10 μmol scale for in vivo studies.

Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.

A highly convergent free radical coupling of alkyl iodides and oximes, mediated by bis(trimethylstannyl) benzopinacolate (8), has been utilized to prepare a series of dimeric nucleosides as mimics of natural nucleic acids. The systematic optimization of the reaction conditions allowed for the single-step conversion of the appropriate iodides and oximes into the 2'-deoxy dimers 9 in moderate to excellent yields. For example, the reaction of 3'-deoxy-3'-iodo-5'-(triphenylmethyl)thymidine (6a) with 3'-O-(tert-butyldiphenylsilyl)-5'-O-(methyleneimino)thymidine (7a) in the presence of 8 in degassed benzene gave an 81% yield of 3'-de(oxyphosphinico)-3'-(methyleneimino)-5'-O-(triphenylmethyl)thymidylyl-(3'--5')-3'-O-(tert-butyldiphenylsilyl)thymidine (9a). Similarly prepared were dimers containing both pyrimidine (thymine, 5-methylcytosine) and purine (adenine, guanine) bases. The reaction was highly stereoselective, giving only a single dimeric species having the ribo-configuration of the newly introduced C-3'-branched methylene moiety. Also prepared were dimers 16, incorporating 2'-O-methyl ribonucleosides in both halves of the dimer. This required the synthesis of 3'-deoxy-3'-iodo-2'-O-methyl nucleosides 12 as well as 2'-O-methyl-5'-O-methyleneimino nucleosides 15. For example, 5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-iodo-2'-O-methyl-5-methyluridine (12e) was prepared in 80% yield by displacement of the corresponding triflate with Bu(4)NI. Also prepared were the suitably protected 3'-deoxy-3'-iodo adenosine and guanosine derivatives. Compounds 15 were prepared in high yield by a regioselective Mitsunobu reaction to give the corresponding 5'-O-phthalimido nucleosides 13, which were subsequently converted to the requisite oximes 15. In the 2'-O-methyl series, the pinacolate coupling reaction proceeded with efficiency equal to that observed for the 2'-deoxy series 9, but with slightly less stereoselectivity, giving predominantly the C-3'ribo products 16, contaminated with 5-25% of the epimeric material. Mixed base dimers containing both pyrimidine and purine bases at all possible positions, including purine-purine dimers were prepared. The hydroxylamine or methyleneimino (MI) backbone of several representative dimers so prepared was converted via methylation to give the corresponding methylenemethylimino (MMI)-linked compounds, which are novel phosphate surrogates for use in antisense oligonucleotides.

Deoxyadenylic acid and deoxythymidilic acid have been attached to a covalently-linked cross section, dA dT, on the same side (proximal) and on opposite sides (distal) by synthetic sequences involving various combined protection/deprotection steps. The structures in aqueous solution buffered at pH 7.0 have been examined by 2D-NOESY NMR spectroscopy. The covalently-linked cross section provides a template that stabilizes dA·dT base pairing in the proximal isomer at 2 °C. In the distal isomer, it contributes to favorable conformations for intermolecular association.

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.

Covalently linked cross sections refer to structures that mimic hydrogen-bonded purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine duplexes. Cross sections dA [symbol:see text] U and A [symbol: see text] dT, which have been synthesized chemically, have molecular dimensions similar to purine-pyrimidine base pairs in a double helix. We propose that antibodies to such covalent cross sections might facilitate the study of the pathogenesis of specific diseases or of biochemical processes in which base pair involvement is suspected and/or demonstrated. We have made polyclonal antibodies against "A:U" and "A:T" cross sections by immunizing rabbits with dA [symbol: see text] U and A [symbol: see text] dT, each conjugated to keyhole limpet hemocyanin (KLH). The antibodies were found to be highly specific for the cross sections and to cross react minimally to single nucleosides. Hybridomas secreting monoclonal antibodies to "A:T" were then generated from spleen cells of mice immunized with A [symbol: see text] dT conjugated to KLH. The MAbs produced were also found to be highly specific for "A:T" among various nucleosides. In fact, the binding of most of the monoclonal antibodies to "A:T" was only partially inhibited by high concentrations of adenosine or thymidine. All monoclonal antibodies to "A:T" cross react, but with lower affinity, to "A:U." Selected MAbs showed greater inhibition of binding to "A:T"-BSA by A + T than by A or T alone.

L -[2,4,5,6,7- 2 H 5 ]Tryptophan was incorporated into roquefortine 2 by Penicillium roqueforti and aszonalenin 3 by Aspergillus zonatus with retention in each case of five deuterium atoms; that 5a-hydrogen of both metabolites is derived from the 2-hydrogen of tryptophan

X-ray crystal structures have been determined and compared for fluorescent 1,3,4,6-tetraazapentalenes that are syn and anti disubstituted with pyrimidinone rings or with a combination of pyrimidinone and pyridine rings. Syn-disubstituted tetraazapentalenes were synthesized by a three-step sequence from 1-ethylcytosine or O-protected cytidine (also O-protected adenosine), chloroketene diethyl acetal, and either 2-aminopyridine or another cytosine or cytidine unit. The final step was an oxidative cyclization by means of an I III compound. Anti-disubstituted tetraazapentalenes bearing terminal pyrimidinone rings or pyrimidinone and pyridine rings were obtained from the corresponding syn compounds by treatment with ammonia or sodium methoxide in methanol. The anti tetracyclic ring system based on two cytidine units attached to the 1,3,4,6-tetraazapentalene central moiety provides a covalently linked cross section that is a model for base pairing in a double-helical polynucleotide having parallel rather than antiparallel strands

A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2′-fluoro or 2′-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2′-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2′-F/2′-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson–Crick base pairs with additional 3′ UU overhangs and a 5′ phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.

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.