Biomedical subjects
Publications and source records attributed to B Rayner.
Prooligonucleotides with alternating S-Acyl-ThioEthyl (SATE) phosphotriester and phosphodiester linkages or a gap were synthesized by elimination of cyanoethyl group with non-nucleophilic base DBU. Their half-lives in the presence of Pig Liver Esterases (PLE) and in total cell extract were determined.
Here we report that the poor binding of methylphosphonate oligodeoxynucleosides (MP-ODNs) to their nucleic acid targets can be improved by additional inversion of the anomeric configuration (from beta to alpha) in the sugar moieties to give a new class of analogs, MP alpha-oligonucleosides. MP alpha-dT12and MP 5' alpha-d(TCTTAACCCACA) 3' were synthesized and their ability to form hybrids with complementary single stranded (ss)DNA and ssRNA, as well as with double stranded (ds)DNA, was evaluated. The thermal stability of hybrids formed with MP alpha-analogs was compared with the affinity of phosphodiester (PO) and phosphorothioate (PS) beta- and alpha-oligomers for their targets. Non-ionic MP alpha-oligonucleosides bound to their complementary DNA and RNA strands more tightly than their homologues with natural beta-anomeric configuration did. With DNA target, MP alpha-oligomers formed duplexes more stable than the corresponding natural PO beta-oligomer did. MP alpha-heteropolymer hybridized to RNA target better than PS beta-oligonucleotide did but the hybrid was less stable (DeltaTm-0.5 degrees C per mod.) than the hybrid formed with the natural PO beta-oligomer. Only MP alpha-dT12 bound to dsDNA target at low salt concentration (0.1 M NaCl).
A modified phosphoramidite method has been designed for the solid-phase synthesis of two dodecathymidine phosphotriesters and two dodecathymidine thionophosphotriesters. In these analogs, each internucleoside link bears an S -acyl-2-thioethyl (Me-SATE or tBu-SATE) group removable upon esterase activation. Efficient synthesis of these lipophilic analogs was achieved thanks to the use of a photolabile linker anchored to the solid support in combination with thymidine-3'- O -phosphoramidites having a SATE group in place of the regular 2-cyanoethyl one. Both dodecathymidine phosphotriester and thionophosphotriester having S -acetyl-2-thioethyl groups were found to be stable in the presence of snake venom and calf spleen phosphodiesterases whereas, upon incubation in CEM cell extracts, they were selectively hydrolyzed to the anionic parent dodecathymidylate and dodecathymidine phosphorothioate, respectively. In addition, Me-SATE-protected dodecathymidine thionophosphotriester was stable in mouse and human sera as well as in human gastric juice. These results depict the potential of SATE-protected oligonucleotides as prodrugs of antisense oligonucleotides.
All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.
The comparative stability of eight different triplexes constituted with 16-mer pyrimidine-modified oligodeoxynucleotides (wild-type ODN, PS-ODN, alpha-ODN, or alpha-PS-ODN) or oligoribonucleotides (wild-type ORN, alpha-ORN, 4'-thio-ORN, or 2'-O-MeORN) and a DNA hairpin, termed H36, was studied in five different buffers by UV melting curve analysis. The composition of buffers varied in pH (5.5 and 6.5), in salt concentration (100 mM and 1 M Na+), and in the presence or absence of divalent cation (0 or 3 mM Mg2+) or spermine (0 or 1 mM). At pH 5.5, the eight triplexes are formed with Tm values ranging from 24.7 degrees C to 50.9 degrees C (delta G298K between -8.1 and -16.8 kcal/mol). At pH 6.5, the triplexes are less stable, and thus 4'-thio-ORN and PS-ODN showed broad transitions that did not allow us to conclude triplex formation. An increase of salt concentration or the presence of spermine stabilizes the triplexes, whereas Mg2+ has a destabilizing effect (excepted for alpha-ORN). In general ORN:H36 and 2'-O-MeORN:H36 triplexes were the most stable. Finally, introduction of alpha-anomeric nucleosides led to an alpha-ORN analog that showed low binding with H36 and to alpha-ODN and alpha-PS-ODN analogs. Triplexes formed with alpha-ODN were slightly less stable than those formed with unmodified ODN. Surprisingly, introduction of phosphorothioate in an alpha analog led only to a low destabilization.
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Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.
We have explored different domains within the hepatitis C virus (HCV) 5' noncoding region as potential targets for inhibition of HCV translation by antisense oligodeoxynucleotides (ODNs). Inhibition assays were performed with two different cell-free systems, rabbit reticulocyte lysate and wheat germ extract, and three types of chemical structures for the ODNs were evaluated: natural phosphodiesters (beta-PO), alpha-anomer phosphodiesters (alpha-PO), and phosphorothioates (PS). A total of six original ODNs, displaying sequence-specific inhibition ranging from 62 to 96%, that mapped in the pyrimidine-rich tract (nucleotides [nt] 104 to 127) and in the initiator AUG codon (nt 338 to 357) were identified. Two ODNs, which were targeted at the initiatory AUG (nt 341 to 367 and 351 to 377) and which had been previously described as active against genotype 1b and 2a sequences, were shown to exhibit inhibition of expression (> 95%) of a type 1a sequence. Control experiments with the irrelevant chloramphenicol acetyltransferase sequence as a marker and randomized ODNs demonstrated that levels of inhibition associated with the use of PS compounds (of as much as 94%) were mainly due to nonspecific effects. Both alpha- and beta-PO ODNs were found equally active, and no difference could be seen in the activity of beta-PO when it was tested in either rabbit reticulocyte lysate or wheat germ extract, suggesting that RNase H-independent mechanisms may be involved in the inhibitions observed. However, specific RNA cleavage products generated from beta-PO inhibition experiments could be identified, indicating that, with these compounds, control of translation also involves RNase H-dependent mechanisms. This study further delimits the existence of favorable target sequences for the action of ODNs within the HCV 5' noncoding region and indicates the possibility of using nuclease-resistant alpha-PO compounds in cellular studies.
OBJECTIVE: To define the effect of intravenous recombinant human erythropoietin (rHuEPO) in patients with anaemia caused by chronic renal failure. Data collection included bone marrow response, ferrokinetics, red cell survival as well as the influence on red cell mass and plasma volume. DESIGN: A convenience sample of stable individuals on haemodialysis, with haemoglobin (Hb) levels of less than 80 g/l, was surveyed over a 12-week period while they were receiving the hormone. Blood counts and iron studies were monitored serially, while 51Cr-labelled red cells and 59Fe (III) citrate were used in the radionuclide investigations carried out before and after the course of replacement therapy was completed (pre and post injection). Bone marrow aspiration and trephine biopsy were performed at each of these times. RESULTS: The actual mean trial period was 14 weeks. Eight patients were enrolled but 1 was withdrawn after developing tuberculosis at the start of the study. Haemoglobin levels rose from 59 g/l (SD 8.8) to 96 g/l (SD 13.9) (P < 0.0001); mean red cell mass rose from 675 ml to 954 ml (P < 0.01). Concurrently, the serial studies in 6 patients showed prominent erythroid hyperplasia. Plasma iron turnover increased insignificantly from 42.5 to 118.9 mumol/l/day; erythron turnover increased from 42.5 to 87.3 mumol/l/day (P < 0.01). There was a marked shortening of the marrow transit time from 4.4 to 3.3 days (P < 0.03). Efficacy of erythropoiesis decreased from 1.17 to 0.65 (P < 0.05). Red cell survival was not altered. CONCLUSIONS: The patient with end-stage renal failure remains fully responsive to erythropoietin therapy. This is reflected in improved ferrokinetics, red cell hyperplasia in the marrow and rise in haemoglobin level without alteration in mean erythrocyte lifespan. Simple hormonal replacement is therefore a rational approach to management.
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Alpha-beta chimeric 17-mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha-beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell-free system to various extent, via the degradation of the RNA target by RNase H.
4'-Thio-beta-D-oligoribonucleotides (12 mer and 16 mer) containing a mixed base sequence were synthesized via the phosphoramidite solid support approach. These RNA analogs showed very good nuclease resistance as compared with wild-type RNA. Furthermore, 4'-thio-beta-D-oligoribonucleotides were shown to hybridize with a complementary DNA or RNA strand to form a duplex or with a DNA hairpin to form a triple helix. 4'-Thio-RNA binds more tightly to its complementary RNA strand than to its complementary DNA strand. A 4'-thio-RNA:RNA duplex is as stable as a 2'-O-methyl-RNA:RNA duplex. 4'-Thio-RNA, however, forms a 4'-thio-RNA:DNA:DNA triplex with a stability similar to the corresponding triplex with all wild-type DNA.
A "phosphorothioate triester method" was investigated for the solution-phase synthesis of phosphorothioate oligonucleosides. Using fully protected 3'-phosphorothiolate thymidine bearing O-cyanoethyl and S-2,4-dichlorobenzyl groups as phosphorothioate protecting groups, decathymidine nonaphosphorothioate was efficiently assembled through a blockwise procedure. Two side reactions occurred during the deprotection steps: breakage of internucleoside linkages (1.8% per linkage) and formation of phosphate diester linkages (0.9%). Substitution of the dichlorobenzyl group by the more labile 4-nitrobenzyl S-protecting group reduced the extent of internucleoside bond breakage by one-half.
Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected human CEM cells. Encapsulation in antibody-targeted liposomes (immunoliposomes) permitted intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. Our results are consistent with four mechanisms of antiviral activity for these antisense oligonucleotides: (i) interference with virus-mediated cell fusion by free but not liposome-encapsulated phosphorothioate oligonucleotides of any sequence; (ii) interference with reverse transcription in a sequence non-specific manner by phosphorothioate oligonucleotides in alpha and beta configurations; (iii) interference with viral reverse transcription in a sequence-specific and RNase-H-independent manner by alpha and beta phosphodiester oligonucleotides; (iv) interference with viral mRNA in a sequence-specific and RNase-H-dependent manner by beta-phosphorothioate oligonucleotides.
Two-dimensional NMR methods were used to model the possible solution structure of an intercalative complex of 9-aminoellipticine (Aell), a polycyclic pyridocarbazolamine, covalently bound to an apurinic ring-opened deoxyribose site of a duplex DNA fragment in the reduced Schiff base form. The required oligonucleotide single strand containing covalently attached aminoellipticine was obtained by reductive amination in the presence of sodium cyanoborohydride. The combined NMR-energy minimization methods were employed to refine the model structures of two distinct forms, intrahelical and extrahelical, of a control 9-mer duplex DNA, d(CGTG.dr.GTGC).d(GCACTCACG), which contains an apurinic site positioned opposite a dT residue on the complementary strand. The model structure of an aminoellipticine conjugate with the same DNA sequence, derivatized via the aforementioned covalent attachment, was also obtained by incorporating intermolecular drug-DNA and intra- and internucleotide NOE-derived proton-proton distance estimates as restraints in energy minimization routines. The indole ring system of aminoellipticine, which is inserted at the apurinic site, intercalates between and is parallel to flanking GC base pairs. The pyridinic ring of aminoellipticine, in protonated form, also stacks between cytidine and thymidine bases on the complementary strand, which is consistent with the observation that the normal sequential NOE connectivity at the 5'-C13-T14 step is broken and indeed diverted through the ellipticine moiety, e.g., C13-Aell-T14 connectivities through the Aell-H4/C5Me protons. Interestingly, the partial stacking of the pyridinic ring is observed only between the 5'-CT step vs an adjacent 5'-TC step, owing to inherently weak stacking interactions associated with the former. In the absence of any potential groups that can participate in electrostatic or hydrogen-bonding interactions with the nucleic acid, pi-pi stacking and hydrophobic contacts at the intercalation site appear to be the important factors in determining stability and conformation of the aminoellipticine-DNA conjugate. Stacking interactions in such a bistranded intercalative complexation of aminoellipticine apparently govern the formation of a single intrahelical form of a right-handed B-type DNA duplex. The overall structural features lead us to propose working models for an enzyme-like DNA cleavage activity of 9-aminoellipticine and the observed inhibition of the AP endonuclease-dependent DNA excision-repair pathway.
The potential use of alpha-beta-anomeric duplex oligonucleotides to inhibit transcription factor activity by the decoy approach is investigated in this report. Indeed, several alpha-beta-anomeric heteroduplexes display a sequence-specific interaction with the p50 subunit of the transcription factor NF kappa B. Used in a decoy approach, these duplexes interact strongly enough with this transcription factor to modulate the expression of a reporter gene, under the control of NF kappa B. However, all the alpha-beta-anomeric heteroduplexes do not interact with the p50 subunit; the sequence of the chirally natural beta-anomeric strand may explain the different recognition properties of the protein. The analysis of the appropriate beta-anomeric sequences is consistent with a preferential interaction of the p50 subunit with one strand of double-stranded DNA.
The phosphorothioate oligonucleotide T2G4T2 was identified as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gp120 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection.
12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).