Immobilized small deoxyribozyme to distinguish RNA secondary structures.

Abstract

The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.