Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

13 Pages·1996·1 MB·English

by  Tsang, Joyce

NASA/CR- --_ __- 207598
Reprinted from J. Mol. Biol. (1996) 262, 31-42
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Specialization of the DNA-cleaving Activity of a
Group I Ribozyme Through In Vitro Evolution
Joyce Tsang and Gerald F. Joyce
J. MoL Biol. (1996) 262, 31-42
.]MB
Specialization of the DNA-cleaving Activity of a
Group I Ribozyme Through In Vitro Evolution
Joyce Tsang and Gerald F. Joyce*
Departments of Chemistry In an earlier study, an in vitro evolution procedure was applied to a large
and Molecular Biology and population of variants of the Tetrahymena group I ribozyme to obtain
The Skaggs Institute for individuals with a 105-fold improved ability to cleave a target
Chemical Biology, The single-stranded DNA substrate under simulated physiological conditions.
Scripps Research Institute The evolved ribozymes also showed a twofold improvement, compared to
10550 North Torrey Pines the wild-type, in their ability to cleave a single-stranded RNA substrate.
Road, La Jolla, CA 92037 Here, we report continuation of the in vitro evolution process using a new
USA selection strategy to achieve both enhanced DNA and diminished
RNA-cleavage activity. Our strategy combines a positive selection for
DNA cleavage with a negative selection against RNA binding. After 36
"'generations" of in vitro evolution, the evolved population showed an
,,,100-fold increase in the ratio of DNA to RNA-cleavage activity.
Site-directed mutagenesis experiments confirmed the selective advantage
of two covarying mutations within the catalytic core of the ribozyme that
are largely responsible for this modified behavior. The population of
ribozymes has now undergone a total of 63 successive generations of
evolution, resulting in an average of 28 mutations relative to the wild-type
that are responsible for the altered phenotype.
© 1996 Academic Press Limited
*Corresponding author Keyzoords: catalytic RNA; DNA cleavage; in vitro evolution; ribozyme
Introduction
study, a group I ribozyme that efficiently cleaves
RNA but has a barely detectable ability to cleave
In vitro evolution has proven to be a powerful DNA was evolved to cleave DNA efficiently, yet
technique for obtaining novel catalytic RNAs (for showed aslight improvement in its ability to cleave
reviews, see Breaker & Joyce, 1994; Lorsch & RNA (Beaudry &Joyce, 1992; Tsang & Joyce, 1994).
Szostak, 1996). One approach begins with a pool of Here we describe anovel in vitro evolution scheme
variants of an existing ribozyme that catalyzes a that simultaneously selects for the desired activity
reaction similar to the one desired. Repeated and against the existing one, leading to specializ-
rounds of selection, amplification, and mutation are ation rather than expansion of the enzyme's
carried out until the desired catalyst is obtained. capabilities.
Previous examples of this approach have demon- We began this study with variants of the
strated that the original catalytic activity usually is self-splicing group I intron of Tetrahymena ther-
retained and may even improve, suggesting that mophila, a ribozyme that catalyzes the sequence-
in vitro evolution has served to broaden rather than
specific cleavage of a single-stranded RNA
transform the activity of the starting enzyme. For substrate via a phosphoester transfer mechanism
example, a group I ribozyme that is active in the (Figure 1; Zaug &Cech, 1986; Zaug et al., 1986). The
presence of Mgz_ but not Caz* was evolved to ribozyme binds its substrate in two steps: first, a
function in the presence of Ca2÷,but did not lose its region at the 5' end of the ribozyme, termed the
ability to function in the presence of Mg2÷(Lehman "internal guide sequence" fiGS), forms Watson-
& Joyce, 1993). More pertinent to the present Crick base-pairs with the substrate; then the
ribozyme/substrate duplex (P1 helix) forms ter-
tiary interactions with residues in the catalytic core
Abbreviations used: IGS, internal guide sequence;
PCR, polymerase chain reaction; Gn, generation after of the ribozyme. This positions a specific phospho-
n rounds of in vitro evolution; E, ribozyme; rP, RNA ester bond of the substrate in close proximity to the
product; dS, DNA substrate; dP, DNA product; wt, 3'-hydroxyl of a bound guanosine nucleoside,
wild-type; P1 helix, ribozyme/substrate duplex. including the guanosine that lies at the 3' end of the
0022-2836/96/360031-12 $18.00/0 _ 1996Academic PrL..ssLimited
32 Specialization of a DNA-cleaving Ribozyrne
ribozyme (Herschlag, 1992; Bevilacqua et al., 1992). and resulting in cleavage of the substrate with
A phosphoester transfer reaction ensues, involv- concomitant ligation of the 3' portion of the
ing nucleophilic attack by the guanosine 3'-OH, substrate to the 3' oxygen of guanosine (Michel
G--C
U A
A A
G.C
U*A P9.1a
G*C
A.U
A G
G G
340 "-_U A
A G
G*C
G,C
A.U
P9 U*G /_.2
U C*G U G
A GGGAG GAACUAAUU GUAUGC A
GA.c GAC"A_,?:1_!_ l ***** oo****** .0.*** I
A UCCUC UUUGAUUAG UAUAUG A
G•C _T_.:#', U :U U U A
C G
I U U'.*:A C G
A* U Q_;,C' I A
G*C P_
U*A G'Uu,'Gp5 a
o :t
CIG ,5"a
A*U U_._._,:G a ,OGU *• AG"*--'331143 P9.0
A I !1::_ _A G* t
U I A ':-A G* C
AA ] AIC::*G A AG** tc P/
U*A C'G: G*c
G*C A I --Q*c
G*C }>4 C".(3
APSc GUAU* A G* CA_ I,
C, Q .:".U: G*C P7
AC A•GeG• I A*U-':iJ': A*U
G UCCG--G A A_U.A
u
o,u::-u
165 G.C I_'A *U*-_
A*U cC°GA ,,AA--
PSb G*C A 'C: AUU G uo,:cU
U*A A G G G
U*G G*U A C 302--"A C* G
U*G U*A U*A ,A, C*O m
C*G Cc*.Ga AA*U c Y !':A"*U
A G --l.a.c
A--A U U C*G
A I C*G
A A A*U 8:8
G*U A*U
U*A A*U U*A P8
C*G I A U*A
P6b A*U U*A C*G
A*U U*A P2.I
C*G U*A
A*U C*G :AuG
U*'G .,
Aucu•GCu AG*CAA_ 289
,\
AAA*U
G*C
U*G
U*A
C*G
A*U
G*C
G*C
C A
A C
U-G
Figure 1. Secondary structure of the Tetrahymena ribozyme (Cech et al., 1994). Paired structural elements are
designated by Pi. Joining regions between paired elements i and j, referred to as Ji/j, are not labeled. Nucleotide
positions that were partially randomized in the initial population are indicated by shaded regions. The internal guide
sequence (IGS) is shown in bold face, and the DNA substrate is shown in lowercase letters. Nucleotide positions
discussed in the text are labeled. Positions that were subjected to site-directed mutagenesis are boxed.
Specialization of aDNA-cleaving Ribozyme 33
dS
E EoS
rP
Figure 2. Selection strategy. The
ribozyme population (E) was chal-
lenged to cleave the DNA substrate
(dS) in the presence of an inhibitor
corresponding to the 5' product
portion of the RNA-cleavage reac-
tion (rP). Ribozymes that cleaved
the DNA substrate became attached
to the 3' portion of the substrate,
which served as a tag for sub-
sequent selective amplification. Ri-
bozymes that bound the RNA
product were prevented from cleav-
E.rP E'odP ing the DNA substrate and were not
amplified.
et al., 1989; Been & Perotta, 1991; Mei & Herschlag, In asubsequent in vitro evolution study, Tsang &
1996).
Joyce (1994) set out to optimize both k_,tand K,, by
Certain 2'-hydroxyl groups within the RNA appropriate manipulation of the selection con-
substrate form tertiary contacts with residues in straints. First, the substrate concentration was
the catalytic core of the ribozyme during the reduced 50-fold during the target reaction to favor
second step of substrate binding (Pyle & Cech, ribozymes with high substrate binding affinity.
1991; Pyle et al., 1992, Bevilacqua & Turner; 1991). This resulted in a 25-fold improvement in both Kd
In addition, the 2'-hydroxyl of the ribonucleo- and K,, over nine generations of in vitro evolution.
tide at the cleavage site contributes ~103-fold Next, the time allowed for the cleavage reaction
to transition-state stabilization during the trans- was decreased by 12-fold to favor ribozymes with
esterification reaction (Herschlag et al., 1993). It a fast rate of reaction. This resulted in a 50-fold
is not surprising, therefore, that the wild-type improvement in kc,tover nine generations, with no
ribozyme cleaves a DNA substrate very ineffi- significant change in K.... Thus, after a total of 27
ciently, with a 1(P-fold reduction in substrate generations of in vitro evolution, ribozymes were
binding affinity and a1(P-fold reduction in catalytic obtained that cleaved DNA with a catalytic
rate compared to the reaction with an analogous efficiency, kc,t/Km, of 3.2 x 106M-1min -_, represent-
RNA substrate.
ing a -,-10S-fold improvement compared to the
Beaudry & Joyce (1992) used an in vitro evolution wild-type. However, as noted above, RNA-cleav-
procedure to improve the DNA-cleaving activity of age activity also improved, so that ribozymes
the Tetrahymena ribozyme by about 100-fold under obtained after the 27th generation still cleaved RNA
simulated physiological conditions. A pool of 10_3 about tenfold more efficiently than they cleaved
variants of the Tetrahymena ribozyme was gener- DNA. Thus, the in vitro evolution process had led
ated, and individuals with superior DNA-cleaving to a broadening of the catalytic abilities of the
activity were preferentially amplified on the basis ribozyme. Nature had evolved an RNA catalyst
of attachment of the 3' portion of the substrate to with the ability to cleave RNA; then, in the
the 3' end of the ribozyme, which occurred as a laboratory, this activity was generalized to include
consequence of the cleavage reaction. Amplified both RNA and DNA substrates.
molecules were utilized as templates for cDNA In this study, we devised a novel in vitro
synthesis, the resulting cDNA was amplified by a evolution strategy in order to improve the
polymerase chain reaction (PCR), and the PCR specificity of the ribozyme for DNA compared to
products were transcribed to generate a new pool RNA substrates. Our scheme combined a positive
of RNAs. This procedure was carried out for nine selection for DNA cleavage with a negative
successive generations, resulting in RNA molecules selection against RNA binding. As shown in Fig-
that cleaved a single-stranded DNA substrate with ure 2, ribozymes were challenged to cleave a DNA
a kc., of 0.06 min -j and Km of 2 IJM. This represents substrate in the presence of a large excess of an
an improvement of 30-fold and threefold, respect- RNA inhibitor that corresponds to the portion of an
ively, compared to the wild-type Tetrahymena RNA substrate that forms base-pairs with the IGS.
ribozyme. As in our previous selection procedure, ribozymes
34 Specialization ofa DNA-cleaving Ribozyme
that cleaved the DNA substrate became tagged by 2
attachment of the 3' portion of the substrate to their
3' end, and thus were selectively converted to
cDNA and amplified. On the other hand, ribozymes
that preferentially bound the RNA inhibitor were
prevented from binding and cleaving the DNA o
substrate, and thus could not be amplified.
Employing this selection scheme, we subjected °i1
the pool of ribozymes obtained after 27 generations
of in vitro evolution to an additional 36 generations,
F
selecting for DNA cleavage activity in the presence
i i i i i
of progressively increasing amounts of the RNA wt G27 G36 G45 G54 G63
inhibitor. Based on kinetic analysis of the resulting
generation
individuals, the population responded as expected.
Figure 3.Change in the catalytic efficiency of DNA and
The ratio of DNA to RNA-cleavage activity
RNA-cleavage reactions for the evolving population of
improved about 100-fold, with about a 50-fold
ribozymes. The kc.,,/K, values for DNA cleavage (filled)
increase in kcat/Km for DNA cleavage and a slight and RNA cleavage (open) were determined under
decrease in kc,t/Km for RNA cleavage, measured single-turnover conditions for the population as awhole,
for the population as a whole. A characteristic based on aleast-squares fit to the equation: k_ =(kc,i/
individual isolated from the evolved population K,)[E], where [El <<Kmand kob,is the initial rate of the
exhibited k_at/Km values of 1.6 x l07 M-' min -1 and reaction (see Materials and Methods). PCR mutagenesis
6.9 x 106M-1rain-I for the DNA and RNA-cleavage was performed twice at G36 and three times at G45 and
reactions, respectively. This altered behavior, G54 to increase the diversity of the evolving population,
as described in Materials and Methods.
relative to the DNA-cleaving ribozymes obtained
earlier, is largely due to two concerted mutations
that arose within the catalytic core of the ribozyme
over the course of the in vitro evolution process. the 5' RNA product provided additional negative
pressure against ribozymes that catalyzed a ligation
reaction resembling the reverse of the DNA-cleav-
Results
age reaction, in which the 5' RNA product bound
to a reacted DNA-cleaving ribozyme and attacked
Evolutionary Improvement in DNA the ligation junction between the 3' end of the
substrate specificity ribozyme and the 3' portion of the DNA substrate,
thereby releasing the tag that identifies the
Beginning with the population of DNA-cleaving ribozyme for selection.
ribozymes obtained after 27 generations of in vitro In order to assess the impact of our selection
evolution (Tsang & Joyce, 1994), we carried out 36 strategy, we determined the catalytic efficiency of
additional generations of the evolution process, both DNA and RNA cleavage for the population of
applying a new selection strategy that simul- ribozymes obtained after the 27th, 36th, 45th, 54th,
taneously selected for cleavage of a DNA substrate and 63rd generations of in vitro evolution (Figure 3).
and against binding of an RNA inhibitor (Figure 2). The preference for DNA over RNA, as determined
The RNA inhibitor, corresponding to the 5'product by the relative kc._,/Kmvalues for the reaction with
of the ribozyme-catalyzed RNA-cleavage reaction, DNA versus RNA substrates, increased steadily,
bound tightly to the IGS of the ribozyme improving 100-fold between generations 27 and 63.
(Ka < 1nM; Tsang & Joyce, 1994), thus preventing There was an overall 50-fold increase in DNA-
binding of the DNA substrate. In this way, cleavage activity and a twofold decrease in
ribozymes that preferentially bound RNA over RNA-cleavage activity. From generation 27 through
DNA were selectively disfavored. At the outset, the 36, there was a modest improvement in DNA-
concentration of DNA substrate and RNA inhibitor cleavage activity, but no significant change in
was 0.2 _M and 0.1 IJM, respectively. The concen- RNA-cleavage activity. Based on sequence analysis
tration of RNA inhibitor was increased gradually (see below), we concluded that the population at
over the course of in vitro evolution to 2_M in order that point in the evolution process lacked sufficient
to maintain strong selection pressure against RNA diversity for the desired phenotype to emerge.
binding. Accordingly, we subjected the population at
Selection against RNA binding also could have generation 36 to two successive rounds of
been accomplished using a full-length RNA mutagenic PCR, introducing random mutations at
substrate as the inhibitor. However, because the an expected frequency of 1.4% per nucleotide
chemical step of RNA cleavage by the wild-type position (Cadwell & Joyce, 1992). Following this
ribozyme is very fast and substrate binding is rate randomization procedure and an additional nine
limiting (Herschlag & Cech, 1990), we felt that the generations of in vitro evolution, RNA-cleavage
same negative selection pressure could be exerted activity declined significantly (Figure 3). Over the
using either the RNA substrate or the 5' RNA final 18 generations, from generations 45 through
product. Carrying out selection in the presence of 63, both DNA and RNA-cleavage activity rose
Specialization of a DNA-cleaving Ribozyme 35
Table 1. Catalytic properties and sugar-specificity of DNA-cleaving ribozymes
DNA RNA
Positions k,,,/ K,,, k,,t/ K,,u Specificity
Ribozyme Mutations 271/312 k,,t' (rain-_) Kin' [_tM) (107M-1min-1) (10: M-I min-_) (DNA/RNA) i
Wild-type" 0 U/G 2.4(+0.2) x 10-4 6.0 + 1.7 4.0(+1.6) x 10" 0.97 +_0.19 4.1(_+2.6) x 104
G2748 b 17 U/A 0.67 _+0.02 0.21 4- 0.03 0.32 + 0.05 2.6 4-0.05 0.12 4- 0.02
G45 8 21" C/AA 0.21 4-0.01 0.043 4- 0.007 0.48 + 0.1 0.60 4-0.02 0.81 4-0.2
G63 19 29_ C/AA 0.26 4-0.01 0.016 4- 0.002 1.6 _+0.2 0.69 4-0.02 2.4 4-0.3
Site-directed
mutants:
G63 19 29 C/AS 0.10 4-0.00 0.028 4- 0.003 0.35 + 0.05 0.43 4-0.02 0.82 + 0.2
G63 19 28 C/Gs 0.021 4-0.001 0.0092 _+0.002 0.23 4-0.05 0.30 + 0.07 0.75 -t-0.4
G63 19 28 U/AA" 1.5 4-0.1 0.33 4- 0.07 0.47 + 0.1 1.2 4-0.08 0.40 4- 0.2
G63 19 28 U/As 1.1 + 0.1 0.13 + 0.03 0.94 + 0.4 1.4 4-0.04 0.67 4-0.3
G63 19 27 U/GS 0.44 4-0.02 0.042 4- 0.005 13 -+0.2 1.3 + 0.03 0.82 4- 0.1
•Data obtained previously (Beaudry &loyce, 1992), modified slightly as aresult of subsequent statistical analysis.
bData obtained previously (Tsang &loyce, 1994).
'Determined under single-turnover conditions ([El >>[S]), as described in Materials and Methods. Data were fit to the equation:
k,_ = k,.,, [E]/([E] + Kin), which holds true when IS] <<Kin;k,,b,was obtained from the equation: y = I- e-k'_, where y = fraction
substrate cleaved at time t for a range of [E]spanning K,,.
dDetermined under single-turnover conditions, as described in Materials and Methods. Data were fit to the equation: k,,_=
(k_,t/Km) [E], where kot_is the initial rate of the reaction at varying [E] when [El <<Kin.
'44:G --*A, 51/52:CUAA insert, 86/87:UA _ del, 94:A _ U, 115/116:AG --*UA, 165:C --*U, 170:C _ U, 188:G_ A, 190/
191:UG _ AU, 205:U _ C, 215:G _ A, 239:U --*A, 271:U --*C, 289:U _ A, 312:G --*AA, 340:U --,A, 364:C _ U, and 366:G _ A.
f44:G --,A, 51:G ---,A, 51/52:CUAA insert, 86/87:UA --*del, 94:A _ U, 115/116:AG -* UA, 138:C _ A, 165:C _ U, 170:C --*U,
175:G --*A, 188:G--, A, 190/191:UG --,AU, 205:U --.C, 215:G --.A, 239:U --*A, 271:U _C, 288/289:GU _ AA, 292:U _ A,
312:G --*AA, 334:A _ G, 340:U --*A, 347:A _ G, 366:G --*A, 389:U --*C, and 390:A --,C.
sMutations introduced by template mutagenesis (see Materials and Methods).
hMutations introduced by nested PCR.
tRatio of k**,/K,, for DNA cleavage versus RNA cleavage.
steadily. By generation 63, ko,/K,, values had proceed to completion, reaching about 40% cleaved
reached 8 x 10°M -1 min -I and 9 x 106 M-1min -1 for for the G27 ribozyme and only about 15% cleaved
the DNA and RNA-cleavage reactions, respectively, for the G63 ribozyme (Figure 4).
measured for the population as a whole.
Evolutionary changes in genotype
Comparison of individual ribozymes
In order to determine which genotypic changes
Individual ribozymes were isolated from the were responsible for the improved phenotype of
population at nine-generation intervals from gener- the evolved population, sequence data were
ation 27 through 63 using a shotgun cloning obtained from 25 randomly chosen individuals,
procedure (see Materials and Methods). Represen- isolated from the population every ninth generation
tative individuals were chosen from generations 27, from G27 through G63, as summarized diagram-
45, and 63 for formal kinetic analysis (Table 1). matically in Figure 5. The mean number of
These ribozymes were representative of the mutations per cloned individual, relative to the
population from which they were cloned because wild-type, increased from 17 at G27 to 27 at G63,
they contained the typical number and constella- reflecting the continual accumulation of mutations
tion of mutations that occurred in the population. that occurred during the in vitro evolution process
The G6319 ribozyme catalyzed the DNA- (Figure 6(a)). However, the genetic diversity of the
cleavage reaction with a k_,t/K,, value of population, as measured by the Shannon entropy
1.6 x 107 M-_ min -_, about fivefold more efficiently statistic (see Materials and Methods), declined
than the G2748 ribozyme. This higher catalytic steadily between GO and G36. In order to increase
efficiency was due to a 13-fold lower K,, that was diversity, new mutations were introduced at an
partially offset by a 2.6-fold lower k_,t. In contrast, enhanced rate through successive mutagenic PCR
the G63 ribozyme catalyzed RNA cleavage about procedures, performed at G36, G45 and G54. This
fourfold less efficiently compared with the G27 resulted in increased genetic diversity between G45
ribozyme. The preference for DNA versus RNA and G63 (Figure 6(b)).
substrates, as determined by the ratio of k_,_/K,, Prior to generation 18, mutations accumulated in
values for the respective c]eavage reactions, both the phylogenetically conserved portions of the
improved about 20-fold, from 0.12 for the G27 molecule that were randomized in the initial
ribozyme to 2.4 for the G63 ribozyme. The population and the peripheral regions that were not
DNA-cleavage reaction proceeded to completion randomized (Figure 6(a)). After G18, however,
when carried out in the presence of an excess mutations tended to accumulate in the periphery
amount of either the G27 or G63 ribozyme. In but not in the conserved core. Accordingly, the
contrast, the RNA-cleavage reaction did not peripheral regions became more genetically diverse
36 Specialization of a DNA-cleaving Ribozyme
(a) mean number of mutations per cloned individual
continued to rise as a result of various low-
frequency changes that occurred in the P9 and L8
peripheral regions.
0.75 -
._ O.S-
Analysis of specific mutations
-- 0.25,
Two of the mutations within the catalytic core,
271:U _ C and 312:G --, AA, were chosen for more
0 detailed analysis. Both of these changes were
0 30 60 120 observed in all 25 clones that were obtained at G45
time {rnin) and G54 and in 24 of 25 clones that were obtained
at G63. No individual was found that contained
(b)
only one of these two mutations, suggesting that
they operate in concert to confer selective advan-
tage to the ribozyme. The single individual at G63
0.75- that lacked the two mutations contained instead the
wild-type nucleotide U at position 271 and aG _ A
O.5- change at position 312. Previous studies had shown
that the 312:G--,A mutation occurred in a
mutually exclusive fashion with regard to the
concerted mutations 313:G--* Y and 314:A _ G
o,r T, - ,
(Beaudry & Joyce, 1992; Tsang &Joyce, 1994). Over
the course of evolution, 313:G --, Y and 314:A _ G
0 0.5 1 1.5
nearly always occurred together, as their frequency
time(rr_n)
rose, then fell, then rose again, and finally fell to
Figure 4. Comparison of the extent of the DNA and extinction (Figure 7). The 312 mutation similarly
RNA-cleavage reactions catalyzed by the G2748 and rose and fell but in opposite phase relative to the
G63 19 ribozymes. Reactions were carried out in the 313:G _ Y and 314:A -* G mutations. Prior to G36,
presence of 2.5pM ribozyme and trace amounts (<1nM) the 312 mutation occurred as a G --*A change, but
of either RNA or DNA substrate. (O) DNA cleavage by following G36 it occurred as a G _ AA change.
G2748; (@) DNA cleavage by G63 19;(0) RNA cleavage Against the background of the G63 19 ribozyme,
by G27 48; (g) RNA cleavage by G63 19. (a)Time course we reverted the 271:U--, C mutation to the
over 120minutes. (b) Expanded view of the reaction over
wild-type U and the 312:G _ AA mutation to either
the first two minutes. Data for RNA cleavage and DNA
cleavage by G27 8 were fit to a biphasic exponential the wild-type G or a single A. All pairwise
equation with a fast phase followed by a slow phase. combinations of these mutations were prepared,
Data for DNA cleavage by G63 19 were fit to a single and their effect on the RNA-catalyzed phosphoester
exponential equation. All curve fits were based on the transfer reaction involving either an RNA or DNA
complete data set obtained over 120 minutes. substrate was analyzed (Table 1). Reversion of both
mutations to the wild-type state resulted in only a
slight decrease in the efficiency of DNA cleavage,
while the core became more homogenous (Fig- while reversion of either mutation alone led to
ure 6(b)). By generation 63, the majority of a more substantial decrease (Figure 8). The
mutations occurred in the periphery. 271:U _ C mutation, regardless of whether the 312
Two major genotypic changes first appeared in position was G, A or AA, resulted in lower
the population at G36. The first, a CUAA insert RNA-cleavage activity. The 271:U _ C mutation
located between nucleotide positions 51 and 52 also resulted in a lower kc._tvalue for DNA cleavage,
within the P2 stem (Figure 1), was present in >90% but this was compensated by a much lower K,
of the clones at G36, replacing the NGAA insert that value. The 312:G _ AA change resulted in a sig-
had occurred with a frequency of 30% at G27. The nificantly higher kc,t value for DNA cleavage,
second, a C _ U mutation at position 165 within regardless of whether the 271 position was U or C.
PSc was present in >90% of the clones at G36, However, for 271:U, but not for 271:C, this was
replacing a C _ A change at the adjacent position accompanied by a substantial increase in K._value.
166 that had occurred in nearly all of the clones at In summary, the 271:U-*C and 312:G--*AA
G27. Between G36 and G45, five new mutations mutations were synergistic: together they provided
became dominant (>90% frequency) in the popu- the greatest improvement in DNA-cleavage activity
lation. Three of these mutations, 271:U--* C, while diminishing RNA-cleavage activity.
289:U _ A, and 312:G _ AA, occurred within the This mutational analysis clearly demonstrates the
catalytic core of the ribozyme and two, 340:U _ A selective advantage of the concerted nucleotide
and 364:C _ U, occurred in the peripheral P9 mutations at positions 271 and 312. These two
stem. Between G45 and G63, no new mutations changes, however, do not explain all of the
rose to dominance in the population. Yet, the improvement in DNA versus RNA-cleavage activity
A
|
38 Specialization of a DNA-cleaving Ribozyme
that occurred between G27 and G63. Even when (a)
both mutations were reverted to the G27 state 30-
(271:U; 312:A) on the G63 19 background, the ratio
of DNA to RNA-cleavage activity was still
significantly greater than that of the G2748
20"
ribozyme. Therefore, other mutations that arose
between G27 and G63 must contribute to the
10"
enhanced substrate specificity of the evolved
ribozymes, such as those that occurred in the P9
and L8 regions.
O"
wt 9 18 27 36 45 54 63
Discussion
generation
Preference for DNA versus RNA substrates (b)
0.3
We have described a novel in vitro evolution
strategy that simultaneously selects for cleavage of
DNA and against binding of RNA, leading to 0.2
specialization of the DNA-cleaving activity of a
group I ribozyme. Beginning with a heterogeneous
population of ribozymes that cleave both RNA and
DNA substrates, we applied this strategy for 36 t
generations of in vitro evolution and obtained a
50-fold increase in DNA-cleavage activity and a 0
twofold decrease in RNA-cleavage activity, corre- 0 9 18 27 36 45 54 63
sponding to an overall increase of 100-fold in the generatbn
preference for DNA over RNA substrates. From the
Figure 6. Genotypic change over 63 generations of
evolved population at G63, we isolated a ribozyme in vitro evolution. (a) Mean number of mutations per
that cleaves DNA more efficiently than it cleaves individual, pertaining to the entire ribozyme (0) or the
RNA, with a catalytic efficiency, kc,_/K,,, of 140 nucleotides within the catalytic core that were
2x l07 M-1min-: and 7x 106M-Imin -1for the DNA partially randomized in the initial population (O). Data
and RNA-cleavage reactions, respectively. Site-di- forG9 arefrom Beaudry &Joyce (1992); data forG18 and
rected mutagenesis experiments confirm the selec- G27 are from Tsang &Joyce (1994). (b)Shannon diversity
tive advantage of two covarying mutations at of randomized core (O) and peripheral regions if-l),
calculated as described in Materials and Methods.
nucleotide positions 271 and 312 within the
catalytic core of the ribozyme that are largely
responsible for this modified behavior.
Our selection regime required that the ribozymes G27 and G63 ribozymes: G27 48 cleaves RNA to a
cleave a DNA substrate in the presence of an RNA maximal extent of ,,-40%, while G6319 reaches
inhibitor that corresponds to the 5' product only ,,-15%. For the wild-type Tetrahymena ri-
generated by the RNA-catalyzed RNA-cleavage bozyme, incomplete cleavage of the RNA substrate
reaction. Ribozymes with enhanced affinity for the occurs because an internal equilibrium is estab-
lished between the forward and reverse reactions
DNA substrate were more likely to avoid inhibition
by the RNA product. Under pseudo-first-order, due to slow dissociation of the product (Mei &
single-turnover conditions (see Materials and Herschlag, 1996). A similar situation may exist for
Methods), substrate binding is reflected by K,,, the the G27 and G63 ribozymes. If the rate of the
concentration of ribozyme that is required to obtain reverse reaction is similar for these two ribozymes,
the half-maximal first-order rate of reaction. The then the lesser extent of RNA cleavage by G63 19
G6319 ribozyme catalyzed the DNA-cleavage compared with G27 48 is likely due to the slower
reaction with a K,, value that is 13-fold lower than rate of forward reaction for the former.
that of the G27 ribozyme. Thus, the G63 ribozyme's
enhanced preference for DNA versus RNA sub- Genetic basis for the evolved phenotype
strates compared to the G27 ribozyme may be due,
at least in part, to the G63 ribozyme's greater Site-directed mutagenesis experiments indicate
affinity for the DNA substrate. that covarying mutations at positions 271 and 312
Ribozymes that bound the RNA product more in the G63 19 ribozyme contribute to its preference
slowly also would have had an increased likelihood for DNA versus RNA substrates (Table 1). Both of
of escaping inhibition by the RNA product under these mutations disrupt phylogenetically conserved
our selection conditions, assuming that the rate of base-pairing interactions within the catalytic core
DNA substrate binding did not decrease as well. (see Figure 1). The 271:U--* C mutation disrupts
Changes in RNA binding rates may explain the what is thought to be a reverse Hoogsteen pair
differing maximal extent of RNA cleavage by the between 103A and 271U, located at the end of the
Specialization of a DNA-cleaving Ribozyme 39
31.3"314 312
GA--)CG G**A4
100
d
312 .c 1.5-
A 75. G-_A t '7,E "I-- _[_
313/314 _'/'
50. \
¢P
E
25- J 0.5
ttL_
_._
O, i o , ! I
0 9 18 27 45 _ 63 wt/wt C/AA
generation position271/312
Figure 7.Frequency of mutually exclusive mutations at Figure 8. Co-dependence of the 271:U--, C and
positions 312 (@) and 313/314 ((3) over the course of 312:G _ AA mutations with regard to enhanced prefer-
invitro evolution. Data obtained at discrete times were fit ence for DNA versus RNA substrates. The ko,/Km values
to a spline curve. The 312:G--* A mutation was not for the DNA (filled) and RNA (open) cleavage reactions
detected between G27 and G36, but reappeared were determined for ribozymes containing neither,
beginning atG45 as312:G -* AA. The 313/314:GA --,UG either, or both of the 271and 312 mutations in the context
mutation was replaced with 313/314:GA --,CG begin- of the G63 19 ribozyme.
ning at G27, but was not detected at G45 and thereafter.
groups in the DNA substrate, specific nucleotides
P3 stem (Kim & Cech, 1987; Green et al., 1990). The within ]8/7 appear to be critical for DNA-cleavage
312:G--* AA mutation disrupts the 262C.312G activity.
Watson-Crick pair, which lies within the P7 stem Although the G6319 ribozyme contains 29
adjacent to the 264G.311C base-pair that forms a mutations relative to the wild-type sequence
base-triple with the bound guanosine nucleophile and cleaves RNA fourfold less efficiently than the
(Michel et al., 1989). By disrupting the local G27 48 ribozyme, it still cleaves RNA quite readily,
structure near the G-binding site, the 271 and 312 with a kot/K_ value of 6.9 x 106M-_min -_, which is
mutations may alter the orientation of the attacking similar to the wild-type level of 9.7 x 106M-_min-L
3'-terminal guanosine relative to the target phos- The ribozyme's ability to tolerate a large number of
phate, differentially affecting the DNA versus mutations suggests that it is overspecified with
RNA-cleavage reactions. Although covarying mu- respect to RNA-cleavage activity. More dramatic
tations are often observed between two interacting sequence or structural alterations may be necessary
components of an enzyme, the nucleotides at in order to achieve a substantial reduction in this
positions 271 and 312 are unlikely to interact activity. However, elimination of RNA-cleavage
directly because they are located at opposite ends activity without significant reduction of DNA-
of the P7 stem. Their codependent behavior more cleavage activity is likely to be difficult to achieve
likely results from indirect interactions. Other for several reasons. Firstly, the ribose at the
mutations that contribute to the altered phenotype, cleavage site is inherently more reactive than
including those in the L8 and P9 regions, occur deoxyribose, contributing, in the case of the
outside the catalytic core of the ribozyme, wild-type Tetrahymena ribozyme, _,10_-fold
suggesting that they too exert their influence via towards stabilization of the transition state com-
indirect or long range effects. pared with deoxyribose (Herschlag et al., 1993).
Residues in the J8/7 region of the wild-type Secondly, RNA/RNA homoduplexes are more
Tetrahymena ribozyme have been shown to interact stable than DNA/RNA heteroduplexes, resulting
directly with Z-hydroxyl groups of the substrate in inherently greater binding affinity for RNA
strand of the P1 duplex (Pyle et al., 1992). Thus, we compared to DNA substrates (Roberts & Crothers,
expected that selection against RNA binding would 1993). Thirdly• both RNA/RNA and RNA/DNA
lead to mutations in this region. Yet, J8/7 proved duplexes tend to adopt A-form helices (Wang et al.,
to be highly resistant to mutation over the course 1980; Sanger, 1984; Egli et al., 1992), making it
of in vitro evolution. Not a single individual among difficult for the ribozyme to distinguish them,
the 100 that were isolated from generations 36, 45, especially if the ribozyme is to maintain generality
54, and 63 had a mutation in this region. with respect to the sequence of the ribozyme/sub-
Furthermore, when we intentionally mutated strate pairing. Fourthly, steric conflicts involving
nucleotide position 302, which in the wild-type 2'-hydroxyl groups, which might reduce RNA-
ribozyme interacts with a particular 2'-hydroxyl of cleavage activity, may not be substantial due to
the RI_IA substrate, we observed a near-complete the small size of these groups. Considering the
loss of DNA-cleavage activity (data not shown). inherent advantages of a ribose compared to
Therefore, despite the absence of T-hydroxyl deoxyribose-containing substrate, it is remarkable