Biomedical subjects

A N Manning

Publications and source records attributed to A N Manning.

3 recordsLinked to original sources

PubMed1997

Effects of synthetic oligonucleotides on human complement and coagulation.

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for prothrombin fragment PF(1+2) and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than prothrombin time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement fragment C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in internucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying internucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of internucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.

Blood↗

PubMed1995

Single strand targeted triplex formation: targeting purine-pyrimidine mixed sequences using abasic linkers.

Foldback triplex-forming oligonucleotides (FTFOs) that contain an abasic linker, [2-(4-aminobutyr-1-yl)-1,3-propanediol] (APD linker), in the Hoogsteen domain against pyrimidine bases of a C:G and a T:A base pair were studied for their relative stability and sequence specificity of triplex formation. In general, the APD linker has less destabilizing effect against a C:G base pair than a T:A base pair. Incorporation of three APD linker moieties resulted in decreased binding to the target, which was comparable to results observed with three imperfectly matched natural base triplets. The APD linker incorporation did not result in the loss of sequence specificity of FTFOs, unlike in the case of normal triplex-forming oligonucleotides (TFOs). The introduction of a positively charged abasic linker, however, resulted in decreased stability of the triplex, because of loss of hydrogen bonding and stacking interactions in the major groove. The results of a molecular modeling study show that APD linker can be readily incorporated without any change in the conformation of the natural sugar-phosphate backbone conserving overall triple helix geometry. Further, the modeling study suggests a hydrogen bond formation between the amino group of linker and N4 of cytosine mediated by a solvent molecule (water) in the floor of the base triplet in addition to a contribution from the positive charge on the APD linker amino group. Either a direct or water-mediated hydrogen bond between the amino group of the APD linker and the O4 of thymine is unlikely when the linker is placed against a T:A base pair.

Base Composition↗

PubMed1995

Single strand targeted triplex formation: strand displacement of duplex DNA by foldback triplex-forming oligonucleotides.

Linear oligonucleotides that bind to single-stranded nucleic acid targets by formation of both Watson-Crick (duplex) and Hoogsteen (triplex) hydrogen bonds simultaneously (foldback triplex-forming oligonucleotides; FTFOs) were studied for their ability to disrupt duplex DNA. Recently, we reported that FTFOs interfere with quadruplex forming ability of guanine rich RNA and DNA sequences and indicated that they might also disrupt duplex structures binding to the purine target strand by foldback triplex formation (Kandimalla and Agrawal, Nucleic Acids Res. (1995) 23, 1068-1074). We now obtained evidence for strand displacement of duplex DNA by FTFOs using nuclease assays and thermal melting studies. UV melting studies revealed that complementary strands of 16 to 31 bases long were completely displaced. Results of DNase I assays showed that the FTFOs bound to purine site by strand displacement probably by preassociating with the duplex DNA in the major groove via Hoogsteen hydrogen bonding and subsequently displacing the complementary strand. Experiments with S1 nuclease, an enzyme specific for single-stranded nucleic acids, confirmed the strand displacement ability of the FTFOs.

Base Sequence↗