Small, efficient hammerhead ribozymes

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5′ and 3′ hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5′ arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix+loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5′ and the 3′ arms of a miniribozyme containing an optimized sequence for helix+loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of genelength RNA substrates may be best achieved by miniribozymes.