Automatedsequentialdegradationof Ribonucleicacids Materials and Methods

An instrument has been constructed that can automatically and selectively remove the terminal nucleotide residue from a ribonucleic acid. The instrument is designed to repeat the degradation cycle. This process involves two steps: (1) The base and sugar fragments are removed by the combined action of periodate and amine. (2) The phosphate residue, attached to the originally penultimate residue, is removed by enzymatic hydrolysis. Various techniques have been studied to establish the optimal methods for separating the residual polynucleotide chain from the various reagents and reaction products. In the most efficient and reproducible system, a membrane is used to separate the residual polynucleotide from the reaction products. The enzyme is bound in an active state to a plastic bead, so that simple filtration separates it from the dephosphorylated polynucleotide. All of the chemical and separation steps are quantitative (greater than 99% yield). The analyses are performed automatically by sampling the diffusate, and they are complete before the enzyme hydrolysis begins. The overall efficiency (number of cycles successfully completed) depends upon the stability of the instrument. We have performed up to six cycles with an average yield of 98%.