Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

Tuning of conformational preorganization in model 2',5'- and 3',5'-linked oligonucleotides by 3'- and 2'-O-methoxyethyl modification

Conformational properties of trimeric and tetrameric 2′,5′-linked oligonucleotides, 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2), and their 3′,5′-linked analogs, 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4), were examined with the use of heteronuclear NMR spectroscopy. The temperature-dependent ³J_HH, ³J_HP and ³J_CP coupling constants, acquired in the range of 273–343 K, gave insight into the conformation of sugar rings in terms of a two-state North ↔ South (N ↔ S) pseudorotational equilibrium and into the conformation of the sugar–phosphate backbone in the model antisense oligonucleotides 1–4. 2′,5′-linked oligomers 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) show preference for N-type conformers and indication of A-type conformational features, which is prerequisite for antisense hybridization. The drive of N ↔ S equilibrium in 1–4 has been rationalized with the competing gauche effects of 2′/3′-phosphodiester and 3′/2′-MOE groups, anomeric and steric effects. Furthermore, the pairwise comparisons of 3′-MOE with 3′-OH and 3′-deoxy 2′,5′-linked adenine trimers emphasized the fine tuning of N ↔ S equilibrium in 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) by the steric effects of 3′-MOE group and the possibility of water-mediated H-bonds with vicinal phosphodiester functionality. In full correspondence, the drive of N ↔ S equilibrium towards N by 2′-MOE in 3′,5′-linked analogs 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4) is weaker in comparison with 3′-OH group in the corresponding ribo analogs. β^t, γ⁺ and ε^– rotamers are preferred in both 2′,5′- and in 3′,5′-linked oligonucleotides 1–4.     

In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.     

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.     

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.     

Human telomerase RNA degradation by 2'-5'-linked oligoadenylate antisense chimeras in a cell-free system, cultured tumor cells, and murine xenograft models

Ribonuclease L (RNase L) is a latent single-stranded RNA-directed endoribonuclease that is activated on binding to short 2'-5'-linked oligoadenylates (2-5A), a feature that has led to its use in antisense therapeutic strategies. By attaching a 2-5A moiety to the 5' terminus of standard antisense oligonucleotides, it is possible to activate RNase L and guide it to specific RNAs for degradation. These 2-5A antisense chimeras have been used successfully to target a variety of cellular and viral RNAs. Telomerase is a nuclear ribonucleoprotein complex that elongates telomeric DNA and contributes to cellular immortalization. Telomerase is composed of a protein catalytic subunit and an RNA (hTR or TERC) component, both of which are critical for holoenzyme activity. We describe the characterization of 2-5A antisense chimeras targeting the hTR component of telomerase (2-5A antihTR). Newly designed 2-5A anti-hTR molecules were assayed for their abilities to selectively degrade hTR in a cell-free system. Of the five chimeras tested, one (RBI011) degraded hTR by 97%, and two others (RBI013 and RBI009) were also found to be highly active (73-76% degradation). The ability of transfected RBI011, and its homolog RBI254, to degrade hTR in cultured tumor cells was assessed by real-time RT-PCR. In these studies, RBI011 and RBI254 effectively degraded hTR in a variety of hTR-positive tumor cell lines. The hTR degradation studies were extended to growth assays to determine whether hTR ablation affected tumor cell viability or proliferation. RBI254 treatment resulted in reduced tumor cell viability over the course of 4-day growth assays, effects that were augmented by cotreatment with interferon-beta. To extend these results to an in vivo system, nude mice were implanted subcutaneously or orthotopically with hTR-positive prostate tumors and treated with RBI254. RBI254-treated mice exhibited enhanced tumor cell apoptosis and reduced tumor volume as compared with controls. These findings demonstrated the effectiveness of highly active forms of 2-5A antisense against hTR, and also highlight the usefulness of the cell-free system in predicting chimera efficacy before to inception of cell-based and in vivo studies.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.     

Synthesis and binding properties of a homopyrimidine 2',5'-linked xylose nucleic acid (2',5'-XNA)

The synthesis and hybridization properties of pyrimidine 2',5'-RNA and 2',5'-Xylose Nucleic Acid (2',5'-XNA) are described.     

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.     

NMR conformational analysis of p-tolyl furanopyrimidine 2'-deoxyribonucleoside and crystal structure of its 3',5'-di-O-acetyl derivative

The conformation of a representative molecule of a new, potent class of antiviral-active modified nucleosides is determined. A bicyclic nucleoside, 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, shows C2'-endo and C3'-endo ribose conformations in solution (63:37, 37 degrees C; DMSO-d6), as determined by 1H NMR studies. The crystal structure of a 3',5'-di-O-acetyl-protected derivative (monoclinic, P21, a/b/c= 6.666(1)/12.225(1)/24.676(2) A, beta=90.24(1) degrees , Z=4) shows exclusively C2'-endo deoxyribose puckering. The base is found in the anti position both in solution and in crystalline form.     

Kinetics and activation parameter analyses of hydrolysis and interconversion of 2',5'- and 3',5'-linked dinucleoside monophosphate at extremely high temperatures

Kinetic analysis of hydrolytic stability of 2',5'- and 3',5'-linked dinucleoside monophosphate (N(2)'pN and N(3)'pN) was successfully performed in aqueous solution at 175-240 degrees C using a new real-time monitoring method for rapid hydrothermal reactions. The half-lives of NpN were in the range 2-8 s at 240 degrees C and apparent activation energy decreases in the order U(2)'pU>A(2)'pA>G(2)'pG>U(3)pU approximately C(3)'pC>A(3)pA. The stability of phosphodiester bond was dependent on the types of base moiety and phosphodiester linkages, but no systematic correlation was found between the structure and stability. The interconversion of 2',5'-adenylyladenosine monophosphate (A(2)'pA) and 3',5'-adenylyladenosine monophosphate (A(3)'pA) was enhanced in the presence of D- or L-histidine. The rate constants of degradation of NpN were dissected into the rate constants of hydrolysis and interconversion between N(2)'pN and N(3)'pN using a computer program SIMFIT. Kinetic analysis supports the mechanism that imidazolium ion and imidazole catalyze interconversion and hydrolysis even under hydrothermal environments. The activation parameters for the hydrolysis and interconversion of NpN were systematically determined for the first time from the temperature dependence of the rate constants, where both DeltaH(app)( not equal ) and DeltaS(app)( not equal ) for 2',5'-linked NpN are larger than those for 3',5'-linked NpN. These parameters support the pseudorotation mechanism through pentacoordinate intermediate from 2',5'- and 3',5'-linked NpN, where the average value of DeltaH( not equal ) (pseudorotation) was estimated to be 30+/-18 kJ mol(-1) at 175-240 degrees C.     

Biologically active antisense phosphorothioate oligodeoxyribonucleotides: synthesis, characterization, and studies of 3'-terminal phosphorothioate monoester analogues

Multiple phosphorothioate oligonucleotides containing a 3'-terminal negative charge were synthesized and characterized. Influence of the added negative charge on activation of duplexes by RNase H was investigated. No additional help in recruitment of RNase H was observed.     

Functional characterization of 2',5'-linked oligoadenylate binding determinant of human RNase L

RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.     

Structural basis for recognition of 2',5'-linked oligoadenylates by human ribonuclease L

An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5'-phosphorylated 2',5'-linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2-4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L.