Single-stranded RNA molecular weight and shape determination by differential pressure capillary viscometry, sedimentation velocity, and gel electrophoresis

Abstract

Determination of the size of a population of nucleic acids can be achieved by several distinct methods. Most of these methods are cumbersome and require complicated equipment or techniques. We demonstrate here the use of a differential pressure capillary viscometer for the rapid and simple determination of RNA molecular weight. This highly sensitive viscometer allowed single viscosity determinations on dilute solutions of RNA, providing a direct measure of the intrinsic viscosity without the need to extrapolate from several concentrations. The molecular weights and conformations of the linear single-stranded RNA homopolymer poly(inosinic acid) (poly(I)) and the single-stranded RNA (ssRNA) copolymer poly(cytidylic acid:uridylic acid, 12:1) (poly(C 12,U)), were determined. The ssRNAs were synthesized in a range of sizes (100 to 100,000 bases). They were widely polydisperse. The Mandelkern-Flory equation (1952, J. Chem. Phys. 20, 212–214 ), which requires both the intrinsic viscosity and sedimentation coefficient of a macromolecule, was used to calculate the molecular weights. The molecular weights determined by agarose gel electrophoresis were compared to those determined by intrinsic viscosity plus sedimentation coefficient. The correlation between the molecular weights determined by these two methods was good, at R 2 ≥ 0.92. The conformations of the RNAs were determined by application of the Mark-Houwink equation. The Mark-Houwink exponents for poly(I) and poly(C 12,U) intrinsic viscosities were 0.90 and 0.84, respectively. When compared to other nucleic acid polymers, for which the conformations have been established by several methods, poly(I) and poly(C 12,U) are rigid, extended random coils, in a low-salt buffer (15 m m). These results demonstrate that differetial pressure capillary viscometry of dilute solutions of single-stranded RNA is a quick and accurate method for studying nucleic acid molecular weight, and, when combined with sedimentation coefficient, it is a good method for studying nucleic acid conformation.