On the use of phasing experiments to measure helical repeat and bulge loop-associated twist in RNA

Abstract

In a phasing experiment, two bends are introduced into a long duplex RNA or DNA and the number of base pairs between them varied. When electrophoresed in a gel, the set of molecules may show a periodic variation in mobility that contains information about the twist associated with the bends and the intervening helix. We show how a set of three phasing experiments can be used to extract this information, and apply it to an RNA helix bend at the bulge sequence A2. The bulge introduces a negative (left-handed) twist of ̃30°; at low temperatures, It is mostly confined to the 5′ side of the bulge. The apparent helical repeat of random sequence RNA measured In these experiments was 10.2±0.1 base pairs, an unexpectedly low value. It Is likely that moderate curvature of the RNA helix axis (30-40° over 80 bp) has affected the measurement. © 1994 Oxford University Press.