Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method.

Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGpTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The synthesis were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3'-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.     

Derivatization of unprotected polynucleotides.

A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.     

Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with polynucleotides in vitro.

N-Acetoxy-4-acetamidostilbene (N-AcO-AAS) has been shown to react with mononucleosides to give numerous alkylation products [1]. In this work, homopolynucleotides, RNA and DNA were treated with N-[beta-14 C]-AcO-AAS, washed, degraded with S1 nuclease and acid phosphatase, and chromatographed on Sephadex LH-20. RNA prepared in vitro with 14C on cytosine, adenine or guanine was treated with non-radioactive N-AcO-AAS, then digested and chromatographed similarly. By this means, many of the adducts rising from nucleoside reactions were shown to result from treatment of nucleic acids with the same carcinogen, as well as a number of products which have not been matched to products of monomer alkylation. Labeled 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol was detected in the digest of RNA treated with radioactive N-AcO-AAS, suggesting that phosphate alkylation had taken place.     

Anti-complement activity of polynucleotides.

A biologic assay system, based on complement (C′) inhibition, is described to unravel structural differences among polynucleotides. The C′ system appears particularly suitable to distinguish (1) homo- from copoly-ribonucleotides, (2) deoxyribo- from 2′-OH and other 2′-modified polynucleotides, and (3) single homopolynucleotides from double- or triple-stranded complexes.From these studies a number of polynucleotides emerged with potent anti-C′ activities, worthy of further investigation. The most active polymers were (G)_n (polyguanylic acid), (dCc1)_n [poly(2′-chloro-2′deoxycytidylic acid)] and (dUz)_n [poly(2′-azido-2′-deoxyuridylic acid)].     

Interactions of 4'', 6-diamidine-2-phenylindole with synthetic polynucleotides.

4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs. With the exception of the four-stranded helical poly rI which exhibits considerable fluorescence enhancement upon binding of the ligand, none of the single- or multi- stranded polyribonucleotides and ribo-deoxyribonucleotide hybrid structures form fluorescent complexes with the dye. Poly rI is the only RNA which forms a DNA B-like structure (Arnott et al. (1974) Biochem. J. 141, 537). The B conformation of the helix and the absence of guanine appear to be the major determinants of the specificity of the fluorescent binding mode of the dye. Nonfluorescent interactions of the dye with polynucleotides are nonspecific; UV absorption and circular dichroic spectra demonstrate binding to synthetic single- and double-stranded DNA and RNA analogs, including those containing GC base pairs.     

Solid-phase synthesis of polynucleotides. II. Synthesis of polythymidylic acids by the block coupling phosphotriester method.

Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.     

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method.

A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.