Aspects of antibody-catalyzed primary amide hydrolysis.

Because there are many known C-terminally amidated peptides of biological importance, there is great potential in medicine and organic synthesis for antibodies that catalyze primary amide bond hydrolysis or formation. We characterized a catalytic antibody, 13D11, raised to a phosphinate hapten, that hydrolyzed the primary amide of a dansyl-alkylated derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a kcat of 1.65 x 10(-7) s-1 (kcat/kuncat = 132) and a Km of 432 microM, and was stereospecifically hapten-inhibited (Ki = 14.0 microM). Control experiments indicated that the catalytic activity was not the result of a contaminating protease. In accordance with the hapten being a transition-state analog of base hydrolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity declined rapidly suggesting the antibody was inactivated during the long incubation period. This work demonstrates the feasibility of generating catalytic antibodies to hydrolyze unactivated amide bonds without cofactor assistance.

Rapid synthesis of oligodeoxyribonucleotides. VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support.

A new kieselguhr-polydimethylacrylamide support has been used in a continuous flow, column system for solid phase synthesis of oligodeoxyribonucleotides by a phosphotriester procedure. Using only protected mononucleotides a 14-mer, 20-mer and 27-mer were assembled in high repetitive yield using a simple manually operated, bench top apparatus.

Improvements to solid phase phosphotriester synthesis of deoxyoligonucleotides.

A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.

Chicken histone H5: selection of a cDNA recombinant using an extended synthetic primer.

We describe the use of a synthetic primer to select a cDNA recombinant clone containing H5 coding sequences. The strategy used was as follows: 1. Prepare oligo(dT) cellulose-bound mRNA from chicken reticulocytes and select 11S-18S material from sucrose gradients. 2. Use this RNA fraction both to prepare a cDNA library and as a template for H5-specific cDNA synthesis using a synthetic primer. 3. Screen out most globin cDNA recombinants with oligo(dT)-primed globin cDNA. 4. Search for H5 recombinants using H5 specific cDNA and verify the identity by DNA sequencing. Our screening suggests an H5 mRNA abundance of about two parts per thousand in chicken reticulocyte poly(A)-containing RNA. The isolation of an H5 cDNA recombinant clone is an initial step in the study of H5 genes and their relationship to H1 and core histone genes.

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.

Rapid synthesis of oligodeoxyribonucleotides. V. Further studies in solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

The phosphotriester solid phase method of oligodeoxyribonucleotide synthesis on a polyamide support [M.J. Gait et al. (1980) Nucleic Acids Research 8, 1081-1096] has been applied to purine rich oligodeoxyribonucleotides of 10-12 units. Use of trichloroacetic acid as reagent for removal of terminal dimethoxytrityl groups reduced depurination during chain assembly. Improvements to reaction and isolation conditions for the preparation of monomer and dimer building blocks are also described. The new methods provide a simple, quick and efficient procedure for medium length oligodeoxyribonucleotide synthesis on a scale adequate for most requirements of molecular biology.