Synthetic Oligoribonucleotides: A Messenger RNA and Purification of Isoleucine Transfer RNA by Affinity Chromatography

Abstract

<p>Chemical synthesis of oligoribonucleotides has been applied to two problems of biochemical interest. The synthesis of a model messenger RNA coding for N-formylmethionylangiotensin and use of a synthetic tetranucleotide (ApUpCpA) bound to cellulose for affinity chromatography of isoleucine tRNA were proposed.</p> <p>Synthesis of the required tri- and tetranucleotide blocks for assembly of the thirty nucleotide messenger sequence was carried out by the phosphotriester method developed by Neilson. In this method, a nucleoside destined to become the 5'-terminal residue, protected with the acid labile 2'-0-tetrahydropyranyl group, and except for uridine an N-benzoyl group, is further protected by a 5'-triphenylmethoxyacetyl group which can later be removed selectively. This protected nucleoside is phosphorylated at the 3'-hydroxyl with 2,2,2-trichloroethylphosphate which is subsequently coupled to the 5'-hydroxyl of the second nucleoside, protected as the original one. Addition of further nucleosides is accomplished by the same two step procedure. Yields of coupled products appeared to depend upon the sequence and nucleoside being coupled, and ranged from 20-70%.</p> <p>Relative rates of hydrolysis of the 5'-protecting group and N-benzoyl groups also depended upon the sequence of nucleotides used and an undesirable extent of loss of N-benzoyl groups was found particularly in sequences containing adjacent adenosine and cytidine residues or cytidine and cytidine residues. Results obtained in these reactions were adequate to allow two block coupling reactions to be attempted, and the products of these two coupling reactions were isolated. However, on completely deprotecting these products, the yields of the expected oligonucleotides was 0%, precluding further characterization. After complete deprotection of the shorter oligonucleotides, synthesized by the stepwise addition of nucleosides, low but adequate yields were obtained which allowed contribution of their structures.</p> <p>These unexpected difficulties with the apparently unique properties of different protected oligonucleotides together with the length of the synthetic pathways involved resulted in the decision not to continue towards the objective sequence. The sterically hindered mechanism of coupling and possible binding to and degradation by Zn/Cu couple used in complete deprotection steps were discussed to account for these difficulties, especially with longer sequences. Potential solutions, based on these hypotheses were proposed.</p> <p>The decision was also made to proceed with the application to affinity chromatography.</p> <p>Model studies demonstrated feasibility of incorporating 5'-phosphates or 3'-phosphate-linked decane diol as possible sites for attachment of the affinity site to cellulose. Coupling of protected sequences to cellulose via phosphodiester or triester linkages were not suitable, but coupling to cellulose via phosphodiester after deprotection was demonstrated. Affinity chromatography of isoleucine labelled and leucine labelled bulk-tRNA did not indicate evidence for specific binding.</p> <p>Theoretical treatment of the affinity experiment showed the validity of the idea and suggested that availability of binding sites was the critical parameter. Nuclease digestion of the oligonucleotide cellulose confirmed this hypothesis. Consideration of other matrices, spacers and coupling methods, as well as improvement of yields on deprotection were recommended in order to increase the availability of binding sites.</p>