Directed Evolution of an RNA Enzyme

Abstract

An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme

“…Prior to performing NAIM with a complete series of analogues, it was necessary to identify a reaction condition that was selective, but had a minimum number of uninformative sites due to strong phosphorothioate interference+ Previous experiments with the Tetrahymena group I intron have shown that AaS incorporation can inhibit splicing activity (Deeney et al+, 1987), although the number and location of the detrimental sites varied with the experimental conditions (e+g+, temperature, salt concentrations, incubation time), and the category of ribozyme reaction being studied (39 splice site hydrolysis, 39 splicing by CU addition, or 59-exon cleavage by G; Waring, 1989;Christian & Yarus, 1992, 1993)+ Some of the apparent differences between these experimental results can be explained by experiments that allowed the splicing reaction to proceed too far to completion, which reduced the experimental signal (Waring, 1989), or by experiments that used primer extension to identify the sites of interference, which introduced excessive background noise into the data (Christian & Yarus, 1993)+ Nevertheless, there are some real, and possibly significant, differences in the phosphorothioate interference patterns observed for the first versus the second step of splicing, although it is still difficult to make conclusions about the importance of these differences+ For ease and efficiency of experimental analysis, we elected to study the 39-exon ligation reaction (Beaudry & Joyce, 1992;Mei & Herschlag, 1996)+ This reaction, wherein the 39-OH of the terminal G (G414) nucleophilically attacks an oligonucleotide substrate that mimics the 59-39 ligated exons, is analogous to the reverse of the second step of splicing+ The reaction transfers the 39-exon onto the 39 end of the RNA, and presents three very important advantages over previous efforts to map sites of phosphorothioate interference in the Tetrahymena group I intron+ (1) Using a 39-radiolabeled substrate, the active molecules in the ribozyme population become radioactively labeled during the ligation reaction+ No additional purification of the RNA is necessary prior to iodine treatment and gel electrophoresis, which makes it possible to directly visualize the interference pattern without using reverse transcriptase+ (2) Ease of the reaction makes it feasible to compare interference patterns under a variety of reaction conditions+ (3) Unlike the self-splicing reactions studied previously, the substrate in the reaction is a synthetic oligonucleotide that can be altered chemically to adjust the selectivity of the reaction+ We found the reaction to be maximally informative using a substrate with a 29-deoxy substitution at the cleavage site [dT(-1)S: CCCUCdTAAAAA] in a reaction buffer containing 3 mM MgCl 2 and 1 mM Mn(OAc) 2 + The 29-deoxy substitution reduces the rate of chemistry by more than 1,000-fold (Herschlag et al+, 1993b), which slows the reaction sufficiently that more subtle effects on activity can be detected+ The low metal concentration partially destabilizes the structure of the ribozyme, but the presence of a small amount of the thiophilic manganese ion minimizes the phosphorothioate effects that are present at several positions throughout the intron (Christian & Yarus, 1993)+ Under these conditions, ...…”

“…The Tetrahymena group I intron is one of several large catalytically active RNAs that folds into a compact globular structure (Fig+ 1;Cech, 1993)+ The intron catalyzes two transesterification reactions in the course of RNA self-splicing (Cech et al+, 1992)+ The first step consists of nucleophilic attack at the 59 splice site by the 39-OH of an exogenous guanosine cofactor+ In the second step, the 59-exon attacks the 39 splice site to produce ligated exons+ Under the appropriate conditions, the intron can also catalyze the reverse of either of these two reactions, resulting in exon ligation back onto the intron (Fig+ 2A;Woodson & Cech, 1989;Green et al+, 1990;Beaudry & Joyce, 1992)+ To understand the reaction specificity and catalytic rate enhancement achieved by this ribozyme, it is necessary to understand the structural basis of intron function+ Improved atomic resolution biochemical methods are needed to identify the specific chemical groups within the intron that are essential to its activity and, as a consequence, perhaps identify unique biochemical signatures for specific RNA structural motifs+…”

“…Primary sequence and secondary structure of the L-21 G414 version of the Tetrahymena group I intron (Cech et al+, 1994)+ This ribozyme binds the oligonucleotide CCCUCdTAAAAA and transfers the AAAAA onto the 39 end of the intron in a reaction analogous to the reverse of the second step of splicing+ Numbering of the nucleotides discussed in the text is shown, as are the names of the helical (P1-P9) and single-stranded regions (J6/6a, J8/7, etc+) of the RNA+ The three A-platforms in P4-P6 are shown as adjacent A's with a heavy underline+ The long thin lines indicate regions known to make tertiary interactions within the three-dimensional structure+ Thick lines designate connectivity of the RNA strand+ Cate et al+, 1996b)+ This motif involves a side by side alignment of two consecutive A's to form a pseudobase pair that serves as a platform for tertiary stacking interactions+ In one of the three occurrences, the A-platform makes tertiary interactions with another example of an A-rich motif, the GAAA tetraloop frequently found at the end of RNA hairpin loops (Woese et al+, 1990)+ The A's in this and related GNRA tetraloops FIGURE 2. A: Scheme for the reaction of the L-21 G414 ribozyme with oligonucleotide substrate+ This reaction is analogous to the reverse of the second step of splicing (Beaudry & Joyce, 1992;Mei & Herschlag, 1996)+ The ribozyme binds the substrate to form the P1 helix, which docks into the active site+ The terminal guanosine (G414) nucleophilically attacks the substrate and transfers the 39-terminus onto the 39 end of the intron+ The equilibrium constant for the chemical step of this reversible reaction is approximately 1 (Mei & Herschlag, 1996)+ B: Scheme for the identification of the chemical groups important for RNA activity by NAIM (Strobel & Shetty, 1997)+ The phosphorothioate-tagged nucleotide analogue (indicated as daS) is randomly incorporated into the transcript in place of A+ If the analogue does not interfere with function at a particular position (left side), then ribozymes with the analogue at that site perform the ligation reaction and become radiolabeled+ If the analogue disrupts activity (right side), then the subset of ribozymes that have the analogue incorporated at the susceptible site do not perform the ligation reaction and are not radiolabeled+ Cleavage of the phosphorothioate linkages by treatment with iodine and resolution of the cleavage products by PAGE produces a sequencing ladder with gaps that correspond to sites intolerant of analogue substitution+ AaS serves as a control to insure that loss of activity is not due to the phosphorothioate group+ Unreacted RNA is also 59 end-labeled to ensure that the gap in the sequencing ladder is not due to lack of analogue incorporation at a given site (not shown)+ appear to be widely utilized in tertiary interactions (Murphy & Cech, 1994;Pley et al+, 1994;Costa & Michel, 1995;Costa et al+, 1997)+ A third example of an A-rich motif in the P4-P6 structure is the asymmetric A-rich bulge (AAUAA; positions 183-187; Fig+ 1), which makes extensive tertiary interactions with the minor groove of the P4 helix (Cate et al+, 1996a)+ The A-rich bulge also has a corkscrew turn in the phosphate backbone that forms the binding site for two dival...…”

“…The importance of A nucleotides among this wide variety of structural motifs explains an observation made several years ago about the conservation and sequence distribution of A's within ribosomal RNA phylogeny+ A simple analysis of the 16S rRNA secondary structure revealed that there is a bias for unpaired A's (Gutell et al+, 1985)+ More than 60% of the A's are unpaired, whereas only 30% of the G, C, and U nucleotides were unpaired+ Among the unpaired A's, there is also a preference for the A's to occur in adjacent positions+ Furthermore, of the universally conserved nucleotides, more than 80% of the A's were unpaired, whereas only 50% of the U, G, and C's were unpaired+ At the time of this analysis (1985), there were no structural or functional explanations for this strong bias in favor of unpaired A's+ We now know that there are a number of structural motifs that utilize A's and require this pattern of sequence conservation+ Several chemical reagents are available that covalently modify functional groups on A and other nucleotides (Stern et al+, 1989)+ For example, dimethylsulfate (DMS) is used to explore the role of the N1 of A (von Ahsen & Noller, 1993) and diethylpyrocarbonate (DEPC) modifies A at the N7 position (Peattie, 1979)+ These reagents have proven valuable in biochemical structural and functional studies of RNA (Inoue & Cech, 1985;Moazed et al+, 1986;von Ahsen & Noller, 1993;Butcher & Burke, 1994;Beattie et al+, 1995)+ However, these chemical reagents have some severe limitations+ They leave several functional groups unmodified, and therefore untested+ They also probe the RNA by adding steric bulk to the nucleotide, which may not correlate with involvement of the unmodified functional group in the RNA structure+ Given the importance of A-rich motifs within the structure of the Tetrahymena intron and the prevalence of A residues in the unpaired regions of several different RNA molecules, we focused on developing improved biochemical methods to analyze these sequences+ Nucleotide Analog Interference Mapping (NAIM) is an efficient biochemical approach to identify individual functional groups and tertiary hydrogen bonds essential for RNA activity (Fig+ 2B;Strobel & Shetty, 1997)+ In this approach, a nucleotide analogue that contains an alteration of one functional group is covalently tagged with a 59-phosphorothioate linkage and randomly incorporated into an RNA transcript by T7 RNA polymerase at a level of approximately 5%+ The RNA transcripts are then separated into active and inactive fractions based upon their ability to perform a defined function+ For the group I intron experiments described here, this function is the ability to perform the 39-exon ligation reaction, which is analogous to the reverse of the second step of splicing (Fig+ 2A;Beaudry & Joyce, 1992;Mei & Herschlag, 1996)+ Cleavage of the phosphorothioate linkages with iodine (Gish & Eckstein, 1988) and resolution of the cleavage products by PAGE yields a sequencing ladder with gaps that correspond to sites where analogue substitution is detrimental to activity+ This interference approac...…”

The Effects of Stabilizing and Directional Selection on Phenotypic and Genotypic Variation in a Population of RNA Enzymes

“…In the first environment we supplied a ribozyme population with a "native" RNA oligonucleotide. Cleavage of this oligonucleotide results in the ligation of a portion the substrate to the 3'-end of the ribozyme, and we can exploit this sequence modification to selectively amplify catalytically successful molecules (Beaudry and Joyce 1992;Lehman and Joyce 1993). Molecules with higher activity are more likely to be replicated.…”