Capillary electrophoretic analysis of synthetic short-chain oligoribonucleotides.

Abstract

Thirty synthetic oligoribonucleotides, 3 to 18 nucleotides (nt) long, were analyzed by capillary electrophoresis, under nondenaturing conditions, using a commercial kit. The migration time t(m) was dependent on nt length and composition, capillary length, operating temperature, and type of sieving polymer. Under fixed experimental conditions, the t(m) proved predictable by the equation: t(m) = [0.22(n-1) + 6.14A/n + 6.86G/n + 3.61 (C+U)/n] min, for n>3, where A/n, G/n, C/n, U/n is the frequency of each type of nt within the oligonucleotide (ONT). The equation accounts for the influence of charge-to-mass ratio on t(m), but not for structural effects, if present. This approximation is acceptable for short ONTs. The possibility of detecting n+1, n-1, n-2 impurities, having predicted the t(m), is of crucial importance in assessing the purity of synthetic ONTs dedicated to structural studies. This appears to be feasible. High resolution was shown among homologous series of ONTs of increasing length, and in some cases, even within groups of ONTs of the same length but different composition. The addition of 7 M urea to the buffer, as denaturing agent, accelerates the t(m) and significantly lowers the resolution for the shortest ONTs. It was also possible to monitor the state of association of mixtures of RNA and DNA sequence-complementary strands.