Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

Preparation and properties of 2',5'-linked oligonucleotide analogues containing 3'-O,4'-C-methyleneribonucleosides

Bicyclic nucleoside analogues, 3'-O,4'-C-methyleneuridine and -5-methyluridine, were successfully incorporated into oligonucleotides via connection with 2',5'-phosphodiester linkage, and hybridization behavior and nuclease stability of the modified oligonucleotides were investigated.     

Modification of end phosphate gruops in mono- and oligonucleotides.

A method is described for selective activation of phosphomonoester end groups of oligonucleotides and nucleosidedi-(tri) phosphates via mixed anhydrides with mesitoic acid. Mixed anhydrides are synthesized in high yield and isolated by paper or DEAE-cellulose column chromatography. The ability of such anhydrides to phosphorylate different nucleophilic agents was used for synthesis of amidates, imidazolidates, esters, thioesters and pyrophosphates of mono- and oligonucleotides. Mixed anhydrides mono-, oligonucleotides and nucleosidedi-(tri)phosphates and mesitoic acid were also applied to achieve immobilization of the mono- and oligonucleotides via their end groups on hexamethylenediamine - Sepharose support. Mixed anhydrides studied may be efficiently used for affinity labeling of proteins and nucleic acids and also as material for preparating reagents for template reactions.     

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

Synthesis of oligonucleotides carrying 5'-5' linkages using copper-catalyzed cycloaddition reactions

There is considerable interest in coupling oligonucleotides to molecules and surfaces. Although amino- and thiol-containing oligonucleotides are being successfully used for this purpose, cycloaddition reactions may offer greater advantages due to their higher chemoselectivity and speed. In this study, copper-catalyzed 1,3-dipolar cycloaddition reactions between oligonucleotides carrying azido and alkyne groups are examined. For this purpose, several protocols for the preparation of oligonucleotides carrying these two groups are described. The non-templated chemical ligation of two oligonucleotides via copper-catalyzed [3+2] cycloaddition is described. By solid-phase methodology, oligonucleotides carrying 5'-5' linkages can be obtained in good yields.     

Oligonucleotides with strongly fluorescent groups pi-conjugated to a nucleobase: syntheses,melting temperatures,and conformation

Phosphoramidite 1 was prepared, incorporated into oligonucleotides, and these were studied via thermal denaturation and circular dichroism.     

Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis.

5'-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5'-->3' direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5'-ends while the free 3'-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

Synthesis and characterization of covalently linked single-stranded DNA oligonucleotide-dendron conjugates

A solution-phase synthesis and characterization of covalent DNA-dendron conjugates is presented. Thiol-terminated 12-base oligonucleotides were added to second- and third-generation triazine-based dendrons via thiol/disulfide exchange chemistry. Single-stranded DNA oligonucleotides were successfully attached to dendrons at the core, the periphery, and both. Proof of structure for these architectures is derived primarily from mass spectrometry and polyacrylamide gel electrophoresis and complemented by labeling analysis using Ellman's reagent and degradation analysis using a reducing agent.     

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.     

Novel method for the covalent immobilization of oligonucleotides via Diels-Alder bioconjugation

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.     

Synthesis and bioconjugation of diene-modified oligonucleotides

The preparation of diene-modified oligonucleotides as well as their properties and further derivatization are described. Self-complementary oligonucleotides containing a diene moiety in the loop region form stable, hairpin-like secondary structures. These hairpin mimics can be further derivatized via the Diels-Alder reaction. Diene modification in the stem region leads, in contrast, to a marked destabilization of the hairpin structure. No further reduction in stability is observed, however, upon conjugation of the stem-modified derivatives via the Diels-Alder reaction with an N-substituted maleimide dienophile.     

A rapid method for the construction of oligonucleotide arrays

A simple method has been devised to construct oligonucleotide array on a variety of surfaces using commonly available reagents and chemistry with good efficiency and accuracy. The method involves the generation of hydroxyl functionalities on glass, polypropylene, polyethylene, and commonly used surfaces for construction of oligonucleotide arrays followed by their activation with trifluoroethanesulfonyl chloride (tresyl chloride). The activated surface in the subsequent reaction is used to covalently immobilize oligonucleotides in regioselective fashion to create an oligonucleotide array. The surface bound tresyl sulfonate esters allow the immobilization of oligonucleotides specifically via their 3'- or 5'-end having mercaptohexyl- or aminohexyl functionalities. The constructed oligonucleotide arrays were successfully used to analyze oligonucleotides by hybridization technique.     

Reaction of DNA oligonucleotides with [Pt(dien)GSMe]2+ (GSMe =S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+: evidence of oligonucleotide platination via sulfur-coordinated platinum intermediates

To study the possibility of DNA platination via platinum-sulfur coordinated intermediates, the reactions of the complexes [Pt(dien)GSMe]2+ (GSMe=S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+ with the synthetic oligonucleotides d(ATATGCATAT), d(ATTACCGGTAAT), and d(ATCCTATTTTTTTTAGGAT) have been investigated. The reactions were studied using FPLC, NMR, and mass spectrometry. It was found that the sulfur atom of the platinum-thioether adduct is substituted by these oligonucleotides. For the reactions with [Pt(dien)GSMe]2+ at 310 K, half-lives were determined to be t 1/2 =147+/-7 h for d(ATATGCATAT), t 1/2 =84+/-4 h) for d(ATTACCGGTAAT), and t 1/2 = 21+/-1 h for d(ATCCTATTTTTTTTAGGAT. This study clearly shows that it is indeed possible for oligonucleotides to be platinated via Pt-thioether coordinated intermediates. The rates at which such substitutions occur, however, makes it improbable that such a mechanism contributes significantly to the antitumor activity of cisplatin.     

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.

A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.     

Transformation from block-type to graft-type oligonucleotide-glycopolymer conjugates by self-organization with half-sliding complementary oligonucleotides and their lectin recognition

Block-type oligonucleotide-glycopolymer conjugates bearing alpha-mannosides and beta-galactosides were prepared by coupling 5'-thiol-modified oligonucleotides with iodoacetamidated glycopolymers that were synthesized by telomerization. The conjugates minimally affected the DNA conformation and melting behavior of the duplex. Their self-organization via hybridization with the half-sliding complementary oligonucleotides produced graft-type conjugates or macromolecular gapped DNA duplexes grafted with glycopolymers at regular intervals, which was confirmed using size exclusion chromatography and electrophoresis. The binding affinity of block-type and self-organized graft-type conjugates to lectins was investigated using fluorometry. The affinity of the graft-type duplex assembly bearing mannosides to Con A was approximately 2 times stronger than that of block-type single-stranded or double-stranded conjugates with full complementary oligonucleotides. The organization strategy of DNA-glycopolymer conjugates might be useful for constructing novel glyco-clusters and also for developing a new methodology for gene therapy.     

Oligonucleotide Conjugates Derived from an Electrophilic Site: Conjugation to Baseless Carbohydrate Residue. Synthesis,Hybridization and Modeling Studies

Abstract

Conjugation of electrophiles to oligonucleotides via tethers camying nucleophilic sites well-known. However, for the rever.se reaction, the availahlc methods to generate electrophilic sites in oligonucleotides are not many: (e.g., periodate oxidation of terminal rihose sugar followed by reductive amination).