Minimal sequence requirements for ribozyme activity.

Abstract

The hammerhead ribozyme, as engineered by J. Haseloff and W. L. Gerlach [(1988) Nature (London) 334,[585][586][587][588][589][590][591], is an RNA molecule containing two regions of conserved nucleotides, a double helix, called helix H, which connects the two conserved regions, and flanking arms of variable sequence, which hybridize the ribozyme to its specific target. Here we show that this ribozyme may be reduced in size and still retain cleavage activity by replacing helix II with just a few nucleotides that cannot form Watson-Crick base pairs between themselves. Furthermore, the nucleotides replacing heli II and the nucleotides in the flanking arms may be substituted with DNA, and this small, DNA-containing ribozyme is fully as active as the original, full-size ribozyme. Cleavage activity of the minimized ribozyme depends on the number and sequence of the few nucleotides that replace helix II; optimal activity, thus far, is achieved by four or five deoxyribopyrimidines. The minimized ribozyme, or "mimnzyme," is active as a monomer, as shown by its nearly constant activity over a concentration range varying 25,000-fold, by the mobility of the minizyme-substrate complex in nondenaturing polyacrylamide gels as compared with other nucleic acid molecules of known size, and by other observations. These minizymes provide an excellent model system for studying the structure and mechanism of catalytic RNA; they might also be useful in a variety of biological applications.Ribozymes are RNA molecules that can cut or ligate other RNA or DNA molecules in a catalytic fashion (1,2). The hammerhead ribozyme, one of the best-known ribozymes, has been studied extensively in isolated chemical systems (3-7) and used in gene-control studies in living cells (8)(9)(10)(11). The essential features of a hammerhead ribozyme were determined initially by comparing the base sequences (3, 12) of a number of plant virusoids, linear satellite RNAs, and a viroid that can all cut themselves at unique locations (12-14); later they were refined in a comprehensive study of base mutations (15). These essential features are (i) three double helices of any base sequence, which meet at the cleavage site, (ii) the site of cleavage that has a sequence UX, with cleavage occurring on the 3' side of X, and (iii) two stretches of nucleotides of sequence 5'-CUGAZGA and 5'-GAAA, where X and Z are any base except guanine. It is possible to separate these essential features into two parts, a ribozyme and a substrate, in several ways (4, 5). A hammerhead ribozyme as defined by Haseloff and Gerlach (5) is shown in Fig. 1 at the top. This ribozyme contains the two stretches of conserved nucleotides and one of the three double helices (helix II), whereas the substrate forms double helices I and III in combination with the ribozyme. Because the base sequences of helices I and III are not conserved, the ribozyme may be designed to cut a substrate of virtually any sequence.Our primary aim in this work was to minimize the size of a Haseloff-Gerlach ribozym...

The ubiquitous hammerhead ribozyme

“…Similarly, a U10.1-U11.1 mismatch at the base of stem II, instead of the typical G10.1-C11.1 conserved base pair, illustrates the malleability of stem II. This result is not surprising, since that stem was known to be dispensable in vitro (McCall et al 1992;Tuschl and Eckstein 1993;Long and Uhlenbeck 1994) and explains the observation of many HHRs with a stem II composed of only 1 or 2 bp (Perreault et al 2011). Many ribozymes with short stem III have been observed as well, but in this case it was found that the ribozymes could fold into a dimeric configuration, permitted by a palindromic loop III, effectively yielding a stem III of 6-10 bp (Forster and Symons 1987a).…”

Hammerhead ribozyme kinetics

“…When the hammerhead helices are short, product oligonucleotides rapidly dissociate from the ribozyme after cleavage and thus do not influence the cleavage rate+ However, when the helices are sufficiently long for the products to remain stably bound, the overall cleavage rate is affected+ When ribozyme is in excess, stably bound products only slightly increase the rate of observed cleavage and slightly decrease the extent of reaction due to the slow reverse reaction+ However, when substrate is in excess, stable products can dramatically influence the cleavage rate because their release becomes rate-limiting+ Because product RNA duplexes as short as five base pairs can have dissociation rates at 25 8C slower than 1 min Ϫ1 , many hammerheads will show slower cleavage rates when measured in substrate excess than when measured in ribozyme excess+ Indeed, there are numerous examples in the literature where the multiple-turnover cleavage rate at saturation was not assigned to a specific step of the reaction and the rate determined probably reflected product release (Koizumi et al+, 1989;Goodchild, 1992;Hendry et al+, 1992;Paolella et al+, 1992;Slim & Gait, 1992;Taylor et al+, 1992;Hendrix et al+, 1995;Holm et al+, 1995)+ A burst experiment is useful for determining whether the rate-limiting step for a hammerhead reaction is k 2 or one of the product release steps+ A burst reflects the fast appearance of products during the first turnover of the ribozyme that is followed by a slower appearance of products representing the subsequent rate-determining step of the pathway+ Experimentally, a burst is easiest to detect when the substrate concentration is sufficiently high to be saturating throughout the experiment and a 3-10-fold excess of substrate over the ribozyme is used+ Figure 7 gives two simulated data sets for a hammerhead that is limited by product release when a ratio of substrate to ribozyme of 10:1 was used+ If all of the ribozyme can participate in the reaction, the formation of 10% product is equivalent to one turnover+ In Figure 7A, several time points were taken in the first turnover, clearly revealing the burst of product formation+ The rates of each phase as determined from this data set are 1+1 min Ϫ1 for the burst reflecting k 2 followed by 0+67 min Ϫ1 for subsequent turnovers reflecting release of one of the products+ This burst behavior is more difficult to detect if insufficient data points are taken during the first turnover (Fig+ 7B)+ Its existence can only be surmised by showing that the steady-state rate does not extrapolate to the origin at zero time+ Similarly, if the substrate is in greater excess, not all the ribozyme is active, or if saturation is not maintained throughout the reaction, the burst phase may also be difficult to detect+ This example stresses the importance of carefully defining the first turnover especially when the rate of product release may be close to the rate of the chemical step+…”

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA

“…A series of elegant experiments by Khvorova and colleagues demonstrated that a distal loop-loop interaction is required for activity under physiological conditions (Khvorova et al 2003;Penedo et al 2004;Saksmerprome et al 2004). However, because many different sequences can satisfy the requirements for the distal interaction, the relevant nucleotides were not included in the original active site specification (McCall et al 1992).…”