Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Ester modification at the 3' end of anti-microRNA oligonucleotides increases potency of microRNA inhibition.

MicroRNAs (miRNAs) are short noncoding RNAs that play a fundamental role in gene regulation. Deregulation of miRNA expression has a strong correlation with disease and antisense oligonucleotides that bind and inhibit miRNAs associated with disease have therapeutic potential. Current research on the chemical modification of anti-miRNA oligonucleotides (anti-miRs) is focused on alterations of the phosphodiester-ribose backbone to improve nuclease resistance and binding affinity to miRNA strands. Here we describe a structure-guided approach for modification of the 3'-end of anti-miRs by screening for modifications compatible with a nucleotide-binding pocket present on human Argonaute2 (hAgo2). We computationally screened a library of 190 triazole-modified nucleoside analogs for complementarity to the t1A-binding pocket of hAgo2. Seventeen top scoring triazoles were then incorporated into the 3' end of anti-miR21 and potency was evaluated for each in a cell-based assay for anti-miR activity. Four triazole-modified anti-miRs showed higher potency than anti-miR21 bearing a 3' adenosine. In particular, a triazole-modified nucleoside bearing an ester substituent imparted a nine-fold and five-fold increase in activity for both anti-miR21 and anti-miR122 at 300 and 5 nM, respectively. The ester group was shown to be critical as a similar carboxylic acid and amide were inactive. Furthermore, anti-miR 3' end modification with triazole-modified nucleoside analogs improved resistance to snake venom phosphodiesterase, a 3'-exonuclease. Thus, the modifications described here are good candidates for improvement of anti-miR activity.

DNA-RNA Heteroduplex Oligonucleotide for Highly Efficient Gene Silencing.

Heteroduplex oligonucleotides (HDOs) were a novel type of nucleic acid drugs based on an antisense oligonucleotide (ASO) strand and its complementary RNA (cRNA ) strand. HDOs were originally designed to improve the properties of RNase H-dependent ASOs and we reported in our first paper that HDOs conjugated with an α-tocopherol ligand (Toc-HDO ) based on a gapmer ASO showed 20 times higher silencing effect to liver apolipoprotein B (apoB) mRNA in vivo than the parent ASO. Thereafter the HDO strategy was found to be also effective for improving the properties of ASOs modulating blood-brain barrier function and ASO antimiRs which are RNase H-independent ASOs. Therefore, the HDO strategy has been shown to be versatile technology platform to develop effective nucleic acid drugs.

Progress towards drug discovery for Friedreich's Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein.

Friedreich's Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of "gapmer" oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.

Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro.

Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2'-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2'-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2'-O-methyl (2'-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2'-F nucleotides induced efficient exon-23 skipping, with LNA/2'-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2'-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2'-OMe/2'-F chimeras. Overall, our findings certainly expand the scope of constructing 2'-F modified AOs in splice modulation by incorporating 2'-OMe and LNA modifications.

G-Quadruplex Structure Improves the Immunostimulatory Effects of CpG Oligonucleotides.

Single-strand oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) are recognized by the toll-like receptor 9, a component of the innate immunity. Therefore, they could act as immunotherapeutic agents. Chemically modified CpG ODNs containing a phosphorothioate backbone instead of phosphodiester (PD) were developed as immunotherapeutic agents resistant to nuclease degradation. However, they cause adverse side effects, and so there is a necessity to generate novel CpG ODNs. In the present study, we designed a nuclease-resistant nonmodified CpG ODN that forms G-quadruplex structures. G-quadruplex formation in CpG ODNs increased nuclease resistance and cellular uptake. The CpG ODNs designed in this study induced interleukin-6 production in a human B lymphocyte cell line and human peripheral blood mononuclear cells. These results indicate that G-quadruplex formation can be used to increase the immunostimulatory activity of CpG ODNs having a natural PD backbone.

Vitamin B12 transports modified RNA into E. coli and S. Typhimurium cells.

Specifically designed, antisense oligonucleotides are promising candidates for antibacterial drugs. They suppress the correct expression of bacterial genes by complementary binding to essential sequences of bacterial DNA or RNA. The main obstacle in fully utilizing their potential as therapeutic agents comes from the fact that bacteria do not uptake oligonucleotides from their environment. Herein, we report that vitamin B12 can transport oligonucleotides into Escherichia coli and Salmonella typhimurium cells. 5'-Aminocobalamin with an alkyne linker and azide-modified oligonucleotides enabled the synthesis of vitamin B12-2'OMeRNA conjugates using an efficient "click" methodology. Inhibition of protein expression in E. coli and S. Typhimurium cells indicates an unprecedented transport of 2'OMeRNA oligomers into bacterial cells via the vitamin B12 delivery pathway.

A NanoFlare-Based Strategy for In Situ Tumor Margin Demarcation and Neoadjuvant Gene/Photothermal Therapy.

Accurate tumor margin demarcation in situ remains a paramount challenge. Herein, a NanoFlare (also known as spherical-nucleic-acid technology) based strategy is reported for in situ tumor margin delineation by transforming and amplifying the pathophysiological redox signals of tumor microenvironment. The NanoFlare designed (named AuNS-ASON) is based on gold nanostar (AuNS) coated with a dense shell of disulfide bridge-inserted and cyanine dyes-labeled antisense oligonucleotides (ASON) targeting survivin mRNA. The unique anisotropic ASON-spike nanostructure endows the AuNS-ASON with universal cellular internalization of tumor cells, while the disulfide bridge inserted confers response specificity toward redox activation. In vitro experiments demonstrate that the AuNS-ASON can discriminate tumor cells rapidly with activated fluorescence signals (>100-fold) in 2 h, and further achieve synergistic gene/photothermal tumor cells ablation upon near-infrared laser irradiation. Remarkably, in situ tumor margin delineation with high accuracy and outstanding spatial resolution (<100 µm) in mice bearing different tumors is obtained based on the AuNS-ASON, providing intraoperative guidance for tumor resection. Moreover, the AuNS-ASON can enable efficient neoadjuvant gene/photothermal therapy before surgery to reduce tumor extent and increase resectability. The concept of NanoFlare-based microenvironment signal transformation and amplification could be used as a general strategy to guide the design of activatable nanoprobes for cancer theranostics.

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted "click" conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400-2650 cm-1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 µM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Managing the sequence-specificity of antisense oligonucleotides in drug discovery.

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.

Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens.

There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis.

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

The interactions of amphiphilic antisense oligonucleotides with serum proteins and their effects on in vitro silencing activity.

Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.

Encapsulation of water-soluble drugs by an o/o/o-solvent extraction microencapsulation method.

A new o/o/o-solvent extraction microencapsulation method based on less toxic solvents is presented in this study. The drug is dissolved/dispersed into a poly(D,L-lactide)/or poly (D,L-lactide-co-glycolide) (PLGA) solution in a water-miscible organic solvent (e.g., dimethylsulfoxide or 2-pyrrolidone) (o(1)), followed by emulsification into an oil phase (o(2)) (e.g., peanut oil). This emulsion is added to the external phase (o(3)) to solidify the drug-containing polymer droplets. The polymer solvent and the oil are extracted in an external phase (o(3)) (e.g., ethanol), which is a nonsolvent for the polymer and miscible with both the polymer solvent and the oil. One major advantage of this method is the reduced amount of solvent/nonsolvent volumes. In addition, very high encapsulation efficiencies were achieved at polymer concentration of 20%, w/w for all investigated polymers and o(1)/o(2) phase ratios with ethanol as the external (o(3)) phase. The encapsulation efficiency was very low (<20%) with water as external phase. The particle size of the microparticles increased with increasing polymer concentration and o(1)/o(2) phase ratio and larger microparticles were obtained with 2-pyrrolidone compared to dimethylsulfoxide as polymer solvent (o(1)). After an initial burst, in vitro drug release from the microparticles increased for the investigated polymer as follows: Resomer(®) RG 506>RG 756>R 206. A third more rapid release phase was observed after 6 weeks with Resomer(®) RG 506 due to polymer degradation. Similar drug release patterns were obtained with the o/o/o and w/o/w multiple emulsion methods because of similar porous structures. This new method has the advantages of less toxic solvents, much lower preparation volume and solvent consumption and high encapsulation efficiencies when compared to the classical w/o/w method.

Double sugar and phosphate backbone-constrained nucleotides: synthesis, structure, stability, and their incorporation into oligodeoxynucleotides.

Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S(p))- or (R(p))-D(2)-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio calculations showed that in (S(p))- and (R(p))-D(2)-CNA the following occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) = 54 degrees ; (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)], epsilon (cis), zeta [anticlinal(+)] for (S(p))-D(2)-CNA, and delta [gauche(+)], epsilon (cis), zeta [anticlinal(-)] for (R(p))-D(2)-CNA. It has been found that F(-) ion can catalyze the isomerization of pure (S(p))-D(2)-CNA or (R(p))-D(2)-CNA to give an equilibrium mixture (K = 1.94). It turned out that at equilibrium concentration the (S(p))-D(2)-CNA isomer is preferred over the (R(p))-D(2)-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered dioxaphosphorinane ring in D(2)-CNA was found to be dependent on the internucleotidic phosphate stereochemistry. Thus, both (S(p))- and (R(p))-D(2)-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximately 70-90%, carba-LNA-3',5'-phosphodiester (22) approximately 10%, and carba-LNA-6',3'-phosphodiester (23) <10%. In contrast, the (S(p))-D(2)-CNA was about 2 times more stable than (R(p))-D(2)-CNA under hydrazine hydrate/pyridine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S(p))-D(2)-CNA-incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S(p))-D(2)-CNA-modified AONs by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S(p))-D(2)-CNA modification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes the AON/DNA and AON/RNA duplex by about -6 to -9 degrees C/modification. Treatment of (S(p))-D(2)-CNA-modified AON with concentrated aqueous ammonia gave carba-LNA-6',5'-phosphodiester modified AON ( approximately 80%) plus a small amount of carba-LNA-3',5'-phosphodiester-modified AON ( approximately 20%). It is noteworthy that Carba-LNA-3',5'-phosphodiester modification stabilized the AON/RNA duplex by +4 degrees C/modification (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6', 5'-phosphodiester modification destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 degrees C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6',5'-phosphodiester modified AONs are much more weakly stacked than carba-LNA-3',5'-phosphodiester-modified AONs.

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases.

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A(16)). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.

Fine tuning of electrostatics around the internucleotidic phosphate through incorporation of modified 2',4'-carbocyclic-LNAs and -ENAs leads to significant modulation of antisense properties.

In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the internucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic part and their integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH(3)) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resistance without loss of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreased cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or C8') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.