Directed Evolution of an RNA Enzyme
Author(s): Amber A. Beaudry and Gerald F. Joyce
Source: Science, New Series, Vol. 257, No. 5070 (Jul. 31, 1992), pp. 635-641
Published by: American Association for the Advancement of Science
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Directed
RNA
Evolution
Enzyme
of
an
AmberA. Beaudryand Gerald F. Joyce
An in vitro evolution procedure was used to obtain RNA enzymes with a particularcatalytic
function. A population of 1013 variants of the Tetrahymenaribozyme, 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 MgCI2concentration, 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 amplificationto maintain heterogeneity in the population.
This process was repeated for ten successive generations, resulting in enhanced (100
times) DNA cleavage activity.
Biological systemshave a remarkablecapacityto generatea diversearrayof macromolecularcatalysts.These catalysts,composedof either proteinor RNA, appearto
be the resultof Darwinianevolution operatingat the organismallevel. The sequence
and correspondingfunctionof a macromolecularcatalystis a reflectionof alternatives
that have been adoptedover evolutionary
historyand that conferselectiveadvantage
to the organism.
The need for catalyststhat operateoutside of their native context or catalyze
reactionsthat arenot representedin nature
has resultedin the developmentof "enzyme
engineering"technology. The usual route
takenin enzymeengineeringhas been sitedirected mutagenesis-alteration of the
gene that encodes the catalyst-usually
guidedby structuraland phylogeneticinformation (1). Site-directedmutagenesishas
been improvedby in vitro selective amplification techniques for generating large
numbersof mutantswith subsequentselection of some desirableproperty.Individual
macromoleculesare selected, and those selected are then amplified to generate a
progenydistributionof favorablemutants.
The process is repeated until only those
individualswith the most desirableproperties remain. This technique has been applied to RNA moleculesin solution (2-4),
RNA'sboundto a ligandthat is attachedto
a solid support (5), peptides attached directly to a solid support(6), and peptide
epitopesexpressedwithin a viral coat protein (7).
The processof Darwinianevolution,by
which enzymesarise in nature, does not
operateby generatinga diversepopulationof
variantsand harvestingthe most advantageousindividuals.In biologicalsystems,diDepartments of Chemistry and Molecular Biology, The
Scripps Research Institute, La Jolla, CA 92037.
versityis maintainedby ongoingmutations,
and the populationis shapedby selection.
Novel mutations augment existing variation, so that the evolutionarysearchis biased,in an appropriate
fashion,by selection
events that have alreadyoccurred(8). The
more advantageousmutants,which are relativelyabundantin the population,give rise
to largernumbersof novel variantscomparedto the less advantageous.
We now describeour systemof in vitro
evolutionas a methodforenzymeengineerriing. We began with the Tetrahymena
bozyme, an RNA enzyme that catalyzes
sequence-specificphosphoestertransferreactionsthat resultin cleavageor ligationof
RNA substrates(9-11). This enzymecan
be used to cleave a single-strandedDNA
substrate,albeit only under conditions of
high temperature(50?C) or high MgCI2
concentration (50 mM), or both (4). A
kinetic study (12) showed that, even at
50?C, this reactionis inefficientcompared
to the "native" reaction with an RNA
substrate. Under physiologic conditions
(37?C, 10 mM MgCI2),the DNA cleavage
reaction is almost undetectable. In our
study, we used directed evolution, maintaining a population of 1013ribozymes over
tagenic oligodeoxynucleotides(14, 15), or
inaccuratecopyingby a polymerase(16, 17).
The gene productcan be selected,forexample, by its abilityto bind a ligandor to carry
out a chemical reaction (2-5). The gene
that correspondsto the selectedgene product can be amplifiedby a reciprocalprimer
method, such as the polymerasechain reaction (PCR) (18). A majorobstacleto realizing Darwinianevolution in vitro is the
need to integratemutation and amplification, both of which are genotyperelated,
with selection, which is phenotyperelated.
In the case of RNA enzymes, for which
genotype and phenotypeare embodiedin
the samemolecule,the taskis simplified.
Using a combinationof two polymerase
enzymes,we can amplifyvirtuallyany RNA
(19). RNA is copied to a complementary
DNA (cDNA) with reverse transcriptase
(RT), and the resulting cDNA is transcribedto RNA with T7 RNA polymerase
(T7 Pol) (Fig. 1A). Amplificationoccurs
duringtranscriptionas a consequenceof the
ability of T7 RNA polymeraseto generate
200 to 1200 copies of RNA transcriptper
copy of cDNA template(20). The amplification reactionis done in a single test tube
at a constanttemperatureof 37?C,resulting
in an increase of 103 to 106 times the
originalinput of RNA after 1 hour (21).
The amplificationwas performedselectively in that individualRNA's in the populationwererequiredto catalyzea particular
chemicalreactionin orderto becomeeligible for amplification(3, 4). The selection
wasbasedon the abilityof groupI ribozymes
to catalyzea sequence-specific
phosphoester
transferreactioninvolving an oligonucleotide (or oligodeoxynucleotide)substrate
(Fig. 1B). The productof the reactionwasa
molecule that containedthe 3' portionof
the substrateattachedto the 3' end of the
ribozyme(EP). Selectionoccurredwhen an
oligodeoxynucleotide
primerwas hybridized
across the ligation junction and used to
initiatecDNA synthesis.The primerdidnot
bind to unreactedstartingmaterials(< 10-8
comparedto reactionproducts)and thusled
to selectiveamplificationof the catalytically
active RNA's.
Mutationswereintroducedin two ways.
First,at the outset, we used a set of mutathat contained
genic oligodeoxynucleotides
randomsubstitutionsat a fixedfrequencyof
occurrence.These partiallyrandomizedoligonucleotideswere producedon an automated DNA synthesizerwith nucleoside
3 '-phosphoramidite
solutionsthat had been
doped with a small percentageof each of
the three incorrect monomers (15). Sec-
ten successive generations, to obtain ribozymesthat cleave DNA with improved
efficiencyunderphysiologicconditions.We
have completeaccessto genotypicand phenotypic parametersfor the entire population over the course of its evolutionary
history.
In vitro evolution. Darwinianevolution
requiresthe repeatedoperationof threeprocesses:(i) Introductionof geneticvariation;
(ii) selection of individualson the basisof
some fitnesscriterion;(iii) amplificationof ond, after each round of selective amplifithe selectedindividuals.Eachof these pro- cation, we introduced random mutations by
cessescan be realizedin vitro (3). A gene performing the PCR under mutagenic concan be mutagenizedby chemicalmodifica- ditions (17, 22). The RNA's obtained by
tion (13), incorporationof randomizedmu- selective amplification were subjected to
SCIENCE * VOL. 257 * 31 JULY 1992
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635
reversetranscription,the resultingcDNA's
werePCR amplified,and the PCRproducts
were transcribedto producea progenydistributionof mutantRNA's.
Integrationof the PCR with the selective RNA amplificationprocedurewas useful in three other ways. First, it increased
the overall amplificationby about 103
times. Second, it simplifiesthe processof
subcloning individualsfrom the evolving
population.Normally,only a smallportion
of the DNA in the RNA amplification
mixture is fully double-stranded,but with
the PCR the amount of double-stranded
DNA is greatlyincreased.Third, it returns
the RNA to a formthat can participatein
the RNA-catalyzedphosphoestertransfer
reaction. After phosphoestertransfer,the
ribozymehas the 3' portionof the substrate
attached to its 3' end, and after selective
RNA amplification,the substratesequence
remains attached (Fig. 1). However, by
subsequentuse of the PCR, followedby in
vitro transcription,the original3' end of
the ribozymeis restored.
We referto the entire seriesof events,
beginningwith a heterogeneouspopulation
of RNA's, proceedingwith RNA catalysis
in the targetreaction, selective amplification of catalyticallyactive RNA's, reverse
transcriptionof the selective amplification
products, mutagenic PCR, and in vitro
Fig. 1. Selective amplification of catalytic RNA.
(A) Procedure for amplification of RNA. Reaction conditions: 10-5 to 101 nM RNA, 1 pM
primers, 2 mM each nucleoside triphosphate
(NTP's), 0.2 mM each deoxynucleoside triphosphate (dNTP's), 10 mM MgCI2, 50 mM tris-HCI
(pH 7.5), 5 mM dithiothreitol (DTT), AMV reverse transcriptase at 0.5 U/,il, T7 RNA polymerase at 5 U/,ul; 370C, 1 hour. (B) Procedure
for selective amplification based on phosphoester transfer activity of a group I ribozyme. The
3' portion of the substrate, d(A3(TA3)3), was
transferred to the 3'-terminal guanosine of the
ribozyme. Reaction conditions for RNA-cata-
A
Primer1
RNA
T7 Po/
RT
efficiency (kcat/Km
prom
2
Primer
cleavage is low
B
lyzed DNA cleavage: 1 ,uM Tetrahymenari-
S
OH\
$
EP
E.S
E
and d(ATCGATAATACGACTCACTATAGGAG-
H
-
CIIJ
QUJ
Primer 1
RT
czJ
GGAAAAGTTATCAGGC)as primer 2. SubsecDNA
EP
quent selective cDNA synthesis with 0.2 pmol
EP.pnmrw1
of the selective amplification product under
conditions as in (A), but omitting primer 2 and T7 RNA polymerase. Subsequent PCR amplification
with 0.01 pmol of the selective cDNA synthesis product in a reaction mixture (100 ,ul volume)
0.2 jiM primer 2 (as above), 0.2 mM dNTP's,
containing 0.2 ,uMd(CGAGTACTCCAAAACTAATC),
50 mM KCI, 1.5 mM MgCI2, 10 mM tris-HCI (pH 8.3), 0.01 percent gelatin, and 2.5 U of Taq DNA
polymerase (Cetus), 30 cycles of 920C for 1 minute, 450C for 1 minute, 720C for 1 minute. PCR
products were purified by extraction with chloroform and isoamyl alcohol and by precipitation with
ethanol, and used to transcribe RNA as described in the legend to Fig. 2.
Fig. 2. Secondary structure of the
ribozyme (L-21 form)
Tetrahymena
showing those regions that were
randomly mutagenized (boxed
J
I
1
segments). Transcriptioncondi-
MgCl2,2 mM spermidine,5 mM
DTT, 50 mM tris-HCI (pH 7.5),
1500 U of T7 RNA polymerase; 60
,ul volume; 370C, 2 hours. RNA
was purified by electrophoresis in
a 5 percent polyacrylamide-8M
urea gel and subsequent column
chromatography
on
5
(5'
_-
=
P2
P1
_
_
P6
-
H1 1
P8
1
-
P7
,
I
11
-
-
G
_
-
J
Sephadex
G-50.
636
SCIENCE *VOL.
=
108 M-1 min-1)
(26).
As mentionedpreviously,the ribozymecan
endodeoxyriboalsoact as a sequence-specific
nuclease(4), althoughthe efficiencyof DNA
cDNA
dsDNA
R
bozyme (L-21 form), 10 ,M d(GGCCCTCTA3(TA3)3, 10 mM MgCI2,30 mM N-[2-hydroxymethyl]-piperazine-N'-[3-propanesulfonic acid]
(EPPS) (pH 7.5); 370C, 1 hour. Selective amplification as in (A), with d((T3A)3T3C)as primer 1
tions: 2 pmol of DNA template
(containing mutagenic oligodeoxynucleotides), 2 mM NTP's, 15 mM
transcriptionto producea progenydistribution of RNA's, as one "generation."Typically, a generationis completedin one to
two workingdays, excludingtime for analytic work. We referto the initial population of mutant RNA's as "generation0"
and to subsequentprogenypopulationsas
"generation 1," "generation 2," and so
forth. In principle,there is no limit to the
numberof successivegenerationsthat can
be obtained (23).
Improvedcatalyticfunction.The TetrahygroupI intron
menaribozymeis a self-splicing
derived from the large ribosomal RNA
thermophila.
of Tetrahymena
(rRNA)precursor
Its biologicalfunctionis to catalyzeits own
excision from precursorrRNA to produce
maturerRNA. This functionhas been expressedin vitro (9) and has been generalized
transferreacto includevariousphosphoester
(10, 24). For
tionsinvolvingRNA substrates
example, the ribozymehas been used as a
(11, 25),
endoribonuclease
sequence-specific
a reactionthat proceedswith high catalytic
(kcat/Km
= 200
M1 min-1,
determinedat 50?C, 10 mM MgCI2)(12).
DNA cleavThe efficiencyof RNA-catalyzed
ageunderphysiologicconditionsis evenlower
= 36 M1 min-', determinedat
(kcat/Km
370C, 10 mM MgC12;see below).
Our goal was to improve the catalytic
efficiencyof RNA-catalyzedDNA cleavage
under physiologicconditions and thereby
obtainribozymesthat couldcleave DNA in
vivo. It is not obvious how one should
ribozymeto conchange the Tetrahymena
vert it from an RNA-cleavingto a DNAcleaving enzyme. Thus, we turned to directed evolution as a means to acquirethe
desiredphenotype.
The ribozymeconsistsof 413 nucleotides
and assumesa well-definedsecondaryand
tertiarystructurethat is responsiblefor its
catalyticactivity(27-29). Phylogeneticanalmutagenby site-directed
ysis (30), supported
esis and deletion studies(31), points out a
distinctionbetweena conservedcatalyticcore
aboutone-thirdof the molecule)
(comprising
andsurrounding
stem-loopelementsthatoffer
structuralsupportbut are not essentialfor
catalyticactivity.To generatethe initialpopulationof ribozymevariants,we introduced
randommutationsthroughoutthe catalytic
coreof the molecule.Foursyntheticoligodewereprepared,each of which
oxynucleotides
35 nucleotidepositions
randomlymutagenizes
at an errorrateof 5 percentperposition(Fig.
2). The degenerate oligodeoxynucleotides
were incorporatedinto a DNA template that
encodes the ribozyme, and the template was
transcribed directly to produce the mutant
RNA's (32). We began with 20 pmol (1013
257 *1 31 JULY 1992
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RESEARCH ARTICLE
molecules)of material.Thus, the generation
0 populationwas expected to contain the
wild-typeribozyme,all possible1-, 2-, 3-, and
4-errormutants,anda samplingof the highererrormutants(Table1).
The evolution experimentspannedten
successivegenerations;each generationbegan with 20 pmol of RNA. The amountof
RNA was quantifiedafterselective amplification and after transcription(Fig. 3A).
DNA cleavage activity for the population
as a whole was monitoredby a gel electrophoresis assay involving cleavage of (5'to
32P)-labeled
d(GGCCCTCT-A3(TA3)3)
yield d(GGCCCTCT) (Fig. 3B). It is expectedthat anygiven mutationwouldmore
likely be detrimentalthan beneficial, although there may be a substantialnumber
of neutralmutations.Indeed, DNA cleavage activityfor the generation0 population
is less efficientthan for the wild type. The
generation 1 population, having been selected for DNA cleavage activity under
physiologic conditions, shows improved
catalyticactivitycomparedto generation0
and is slightlyimprovedover the wild type.
Through successive generations there is
continued improvementof phenotype. By
FIg. 3. Course of evolution over
ten successive generations. (A)
A
E
X10
Changes in RNApopulationsize
over time: 0, after transcription
(see legend to Fig. 2), quantitated
by [3H]uracil content; 0, at the
start of each generation, based
on 20-pmol portions; U, after reaction with substrate, estimated
by the assay shown in (C); 5,
after selective amplification (see
legend to Fig. 1), quantitated
by acid precipitation at 40C
of [a-32P]GTP-labeled progeny
RNA. (B) Cleavage of (5'-32P)labeled d(GGCCCTCT-A3(TA3)3)
substrate (S) in the absence of
enzyme (-), in the presence of
the wild-type Tetrahymena ribozyme (L-21 form), and in the
presence of the population of
RNA's obtained at each generation (Gn; n = 0,10). Reaction conditions: 0.5 1tMribozyme, 0.1 9?M
1
I0kKKAIAAIA A IAA
I
I
I
0
&
&
i
0a
,
1 ' 33-4
-5
6
F
Generation
Generation
B
-
A
lo
l
00
high
61
62
63
64
GS
66
67
GO
69
GIOI
low
s
the subsequentsite-specifichydrolysisreaction. As a consequence, the efficiencyof
the hydrolysisreactionrelativeto the initial
phosphoestertransferevent dropsfrom4.9
percentfor the wild type to 1.5 percentfor
the generation 10 population. There is
selection pressurefavoringaccuratecleavage of the DNA at the targetphosphodiester; inaccurate cleavage would result in
partial mismatch of the primer used to
initiateselectiveamplification.The accuracy of cleavage at first declines from 90
percentfor the wild type to 45 percentfor
the generation8 populationand then rises
to 60 percent for the generation10 population. There are some individualsin the
populationthat sacrificeaccuracyfor improvedcleavage activity in orderto enjoy
an overallselective advantage(see below).
Of course,the most favorablesolutionis an
individualthat has both high accuracyand
high cleavageactivity.
Evolutionaryhistory. Although evolution in natural populationsis an accomplishedfact, evolution in vitro is a workin
progressthat allows the experimenterto
accessany time in evolutionaryhistory.We
obtainedsubclonesfromthe evolving population at every generation (35). Generations 3, 6, and 9 were chosen for detailed
analysis.DNA was preparedfrom 25 subclones at generations3 and 6 and from50
subclonesat generation9. The nucleotide
sequence of the entire ribozymegene was
determinedforeach of these subclones(36)
and shows how genotypechangesover the
courseof evolutionaryhistory(Fig. 4).
Fromgeneration0 to generation3, variation is discardedthroughoutmuch of the
catalyticcore of the ribozyme.The mean
numberof mutationsper subclonedecreased
from7.0 at generation0 to 2.7 at generation
3. By generation3, a smallnumberof muta-
.
p
10-
c
EPPS (pH 7.5); either 10 mM
MgCI2, 370C, 1 hour (low) or 50
generation 7, the populationas a whole
cleaves DNA more efficientlyat 370C and
10 mM MgCl2than does the wild type at
the high-temperature,high-MgCl2condition. Through generation 10 the rate of
improvementhas yet to level off.
RNA's fromeach generationwere purified by polyacrylamidegel electrophoresis
and Sephadexchromatography.
To provide
a more formalassayof DNA cleavage activity, we prepared d(GGCCCTCTA3(TA3)3[5'-32PIA)substrate (33) and
measuredformationof both the ribozymed(A3(TA3)3A)covalent intermediateand
the RNA-catalyzedsite-specifichydrolysis
product d(A3(TA3)3A) (Fig. 3C) (34).
The hydrolysisproductformseither by direct cleavageof the DNA substrateor by
cleavage of the ribozyme-d(A3(TA3)3A)
covalent intermediate.Together, these reactions account for less than 5 percent of
the cleavedsubstrate.
After ten generations, DNA cleavage
activityfor the populationas a whole is 30
times higher than that of the wild type.
Becauseselectionis basedon primerhybridization to the EP covalent intermediate
(Fig. 1B), thereis selectionpressureagainst
10
8
?
6-
6
-
4
mM MgCI2, 2 mM spermidine,
a.
500C, 1 hour (high). Reaction
21{
2
products were separated by electrophoresis in a 20 percent poly0 , -0
w-lo
S
wt-high 0
1
2
3
4
10
6
7
8
acrylamide-8 M urea gel, an au9
Generation
toradiogram of which is shown. P,
[5'-32P]d(GGCCCTCT); P+1, [5'32P]d(GGCCCTCTA). (C) Cleavage of [3'-32P]dA-Iabeled d(GGCCCTCT-A3(TA3)3[5'-32P]A)under reaction conditions as in (B). S,
EP, and P were separated by electrophoresis in a 20 percent polyacrylamide-8 M urea gel.
Individual bands were cut from the gel and quantitated by Cerenkov counting. Data points are the
average of five replicate experiments performed on three different days with two different
preparations of substrate. Errorbars correspond to +1 SD.
Table 1. Compositionof the initialpopulation
(generation0). The probabilityP of having k
errors in a doped oligonucleotideof length v
and degeneracy d is given by: P(k,vd) =
1-d)v-k. We randomly
mutagen[v!/(v-k)!k)Idk(
ized a total of 140 positions (v = 140) at a
degeneracy of 5 percent per position (d =
0.05). The numberof distinctk-errorsequences
of length vis given by: Nk= [v!/(v-k)!k!]3k.
The
expected number of copies per sequence is
based on a populationsize of 20 pmol (1.2 x
1013 molecules).
Errors Probability
Sequences
(%
0 (wt)
1
2
3
4
5
6
7+
0.1
0.6
2.1
5.0
9.0
12.8
15.2
55.4
SCIENCE * VOL.257 * 31 JULY1992
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1
420
9 x 104
1 x 107
1 x 109
1 x 1011
7 x 1012
Copies/
sequence
9 x 109
2 x 108
3 x106
5x104
9 x 102
15
0.3
637
tions outsideof the originalzone of random
mutationhave occurredbecauseof ongoing
mutationevents (Fig. 4B). The consensus
sequenceis still that of the wild type, although only one of 25 subcloneshas the
entirewild-typesequence.
Fromgeneration3 to generation6 the
dramaticaccumulationof mutationsat five
positions within the ribozyme coincides
with a threefoldimprovementin the phenotype of the populationas a whole. From
generation6 to generation9, these positions are furtheraccentuatedand aggregate
phenotypeimprovesanotherthreefold.The
mean number of mutations per subclone
rises to 4.6 at generation6 and to 5.9 at
generation9 as a largerproportionof subclones adoptthe commonmutationsand as
mutationsaccumulateoutsideof the original zone of randommutation.
The most frequentmutationis an AY
(Y = U or C) change at position 94
(94:A-*Y). This mutation is present, as
A-*U, in only 1 of 25 subclonesat generation 3. At generation6 there are 15 of 25
occurrences,12 as A->U and3 as A-*C; at
generation9 thereare35 of 50 occurrences,
22 as A-*U and 13 as A--C. Position94 is
unpairedin the secondarystructureof the
wild-typeribozyme(27). Consideringthe
60
t60
---40
effect of site-directedmutations made at
neighboringpositions (37), the 94:A-*Y
change may alter the orientation of ribozyme-boundsubstraterelativeto the catalytic core of the molecule.
Another frequent mutation, occurring
in 4 of 25 subclonesat generation3, 6 of 25
subclones at generation 6, and 22 of 50
subclones at generation 9, is a GA
changeat position215. This mutationconverts a G-U wobble pair to an AU Watson-Crickpair within the catalyticcore of
the ribozyme.Among 87 group I intron
sequencesthat have been analyzed,39 have
a G-U and 28 have a G-C, but only 4 have
B
40
1
20
20
94
-60
60
215
o
-~~
~
Cl40
~~~~~~~~~40
t:S~~~~~~~~~~~~~~~~~~~~~~~~~~6
-20
2
cI~~~~Uc~~~%l~~~ ~~313
k
/314
2
~~~~~~ci~~~~
0c~~~~~~~~~~~~~~~~0~~~0
Fig. 4. Sites at which mutationsoccurred over the course of evolution,
superimposed on the secondary structure of the Tetrahymenaribozyme. Box height corresponds to frequency of mutation(percent) at
each nucleotide position. Nonmutableprimerbinding sites are shaded;
substrate is shown in black. (A) Generation 0, based on expected
638
composition of the initial population (Table 1); (B) generation 3,
25-subclone resolution; (C) generation 6, 25-subclone resolution; (D)
generation 9, 50-subclone resolution. Labeled positions in (D) are sites
of frequent mutation, which were prepared individually by site-directed
mutagenesis.
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-RESEARCH
an AU at this location (29).
The most remarkablemutations are a
G->U changeat position313 and an A->G
change at position 314 that alwaysoccur
together. These mutations are absent at
generation3, but are present in 5 of 25
subclones at generation 6 and 16 of 50
subclones at generation 9. The GA sequence normallypresentat positions 313314 is thought to form a short duplex
structure(the 5' half of the P9.0 pairing)
that drawsthe 3'-terminalguanosineresidue of the ribozymeinto the catalyticcore
(38). We utilize the 3'-terminalguanosine
as the nucleophile in the targetphosphoester transferreaction.The 313-314 mutations are expected to destroy the P9.0
pairing,yet conferselectiveadvantagewith
respectto DNA cleavage (see below).
There is a frequentG -> A change at
position312 that occursonly if the 313-314
mutationsarenot present.The 312:G -> A
change is present in 4 of 25 subclonesat
generation 3 and 8 of 25 subclones at
generation6, but only 5 of 50 subclonesat
generation9. In terms of populationfrequency, the 312:G -> A mutationdeclines
as the 313-314:GA -> UG mutationsbecome more abundant.
Activity of evolved individuals. DNA
from14 subclonesat generation9 was transcribedto produceindividualRNA's, which
gel electrowere purifiedby polyacrylamide
phoresis and Sephadex chromatography.
The catalyticbehaviorof these RNA's was
studiedwith (5t_32p)- and (3'-32P)-labeled
and (5t_32p)- and [ac-32PJDNA substrates
ATP-labeled RNA substrateshaving the
sequence GGCCCTCTC-A3(TA3)3. The
kineticparametermostrelevantto ourselecFig. 5. Catalyticactivityof
14 individual ribozymes
obtained at generation 9.
Ribozymes were transcribed as described
viduals; - - -, average cata-
lytic behaviorof the 14 in-dividuals (corresponds to
catalytic behavior of the
generation9 populationas
a whole, P > 0.99);
tivitywith DNA substrate at
"low" to activity with either
hghor or
DNAsubtrte
DNA
substrateatt "high"
0.3
0.2-
o
0.1
0.00.000
(+.57)
(+.92)
0.005
0.010
0.020
0.015
8'
B
-~6
4
100
(+.34)
0
:
80 -
80
0.0
0.5
1.0
1.5
2.0
VJ[S] (1 03 min1)
i
?
B
60 -
60
+
X
40
t
I
.
+
40 -
Fig. 6. Eadie-Hofsteeplots (42) used to determine Km(negativeslope) and Vmax(y-intercept)
forcleavage of (5'-32P)-labeledd(GGCCCTCTby (A)wildtype; (B) clones 29 and 23
A3(TA3)3)
from generation 9. Reaction conditions:1 ,uM
ribozyme,10 mMMgCl2,30 mMEPPS(pH7.5),
370C; for wild type, 10, 20, 40, and 80 ,uM
L
catalytic behavior of the
rentheses are correlation
coefficients(r) relatingac-
termined for the reaction with (5'-32P)-la-
substrate
beled d(GGCCCTCT-A3(TA3)3)
at 37?C and 10 mM MgCI2,with 1 ,uM
ribozymeand excess substrate.An EadieHofsteeplot of vo as a functionof vo/[S]was
usedto obtainVmaxand Km(Fig. 6). From
werecalculated.For
thisdata,kcatandkcatlKm
the wild-typeribozyme,Km= 6.6 ,uMand
= 36 M-1
kcat= 0.0002 min-' (kcat/Km
min-'). This comparesto Km= 30 ,uMand
kcat= 0.006 min-', previouslyreportedfor
in a relatedreactionat
the wild-typeribozyme
50?Cand 10 mM MgCI2(12). Forclone 29,
Km = 2.0 FtM and kcat= 0.007 min-1
= 3600 M` min-'); for clone 23,
(kcat/Km
Km = 1.9 FiM and kcat= 0.005 min-'
= 2700 M` min-') (dataobtained
(kcat/Km
at 37?C and 10 mM MgCI2).Thus, the
catalyticefficiencyof the two evolvedRNA's
wasincreasedandwasabout100timesgreater
(-.93)
,
wild type. Numbers in pa-
the 313-314:GA->UGmutations,while all
five membersof the latter grouplack these
changes.
We chose clones 29 and 23 for more
detailedkineticanalysis,in comparisonwith
the wild-typeribozyme.Initialrateswerede-
2
100 -
in
the legend to Fig. 2 and
assayed as described in
the legend to Fig. 3. 0,
catalytic behavior of indi-
tion criterionis the proportionof ribozyme
molecules that become joined to the 3'
portionof the DNA substrateafter1 hourat
37?C and 10 mM MgCI2.These data and
dataconcerningreactionswith a
comparable
DNA substrateat 50?Cand 50 mM MgCI2
and with an RNA substrateat 37?Cand 10
mM MgCI2 are presented in Fig. 5.
among
heterogeneity
Thereis considerable
the 14individualRNA'swithrespectto DNA
cleavageactivityin the targetreaction.All
aremoreactivethan the wild type,with the
best (clones29 and 23) beingabout60 times
moreactive.The fivemostactiveindividuals
aremoreactiveunderphysiologicconditions
andhigh-MgCI2
thanat the high-temperature
condition.All 14 individualsshowimproved
activitywiththe RNA substrate,even though
the populationhas never been challenged
with RNA. ImprovedRNA cleavageactivity
is largelydue to enhancedactivity in the
reaction(r = +0.93),
site-specifichydrolysis
whichallowsenhancedturnover.
As mentionedpreviously,there is selection pressureagainstsite-specifichydrolysis
of the EP covalent intermediatein the
reactionwith a DNA substrate.In fact, all
but one of the 14 individualsshow decreasedhydrolyticcleavageof the attached
DNA comparedto the wild type. All but
one of the individualsshow increasedhydrolyticcleavagewith the RNA substrate.
Furthermore,there is a strong negative
correlation(r = -0.93) betweenhydrolytic
cleavageof DNA and RNA. The population is clearly divided into two groups:
those with low DNA and high RNA hydrolysisactivity and those with high DNA
and low RNA hydrolysisactivity (Fig. 5).
All nine membersof the formergroupcarry
ARTICLE
.
20 -
20
.
.
,
.
DNA
DNA
low
.
.*
DNA?RNA
RNA
DNA
low
high
DNA
DNA
low
DNA
DNA
high
RNA
RNA
low
RNA substrate at "low."
Hydrolysis
Phosphoester transfer
RNA substrate was prepared by in vitrotranscriptionwitha syntheticoligodeoxynucleotidetemplate (41); reactionconditionswere as described in
at 0.003 ,uCi/pmolto labelthe 3' portionof the substrate.
the legend to Fig.2, but include [(X-32P]ATP
substrate at 0.25, 30, 60, 120, 180, and 240
minutes;for clone 29, 2.5, 5, 10, and 20 ,uM
substrate at 0.25, 5, 10, and 15 minutes;for
clone 23, 2.5, 5, 10, and 20 ,uMsubstrate at
0.25, 5, 10, 20, and 30 minutes.Ribozymeand
substratewere firstincubated separately in 10
mM MgCI2,30 mM EPPS (pH 7.5), 370C, for 15
minutes, then mixed to start the reaction. 0,
wild type; *, clone 29; A, clone 23; each data
point is the average of three independent determinationsof initialvelocity.The extent of the
reactionwas linearover the chosen time interval (rm,n= 0.94, ravg =0.99).
SCIENCE o VOL. 257 * 31 JULY 1992
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639
Table 2. Genotype and phenotype of 14 individuals from generation 9. Genotype is represented as
a binary matrix (shown in brackets). Phenotype is represented as a column vector b,, with values
normalized to wild type = 1.0. DNA cleavage and hydrolysis activity were determined with
(3'-32P)-labeled DNA substrate under physiologic conditions, as described in the legend to Fig. 3C.
Accuracy was determined with (5'-32P)-labeled DNA substrate under physiologic conditions,
measuring the fraction of substrate cleavage that occurs at the target phosphodiester bond;
reaction conditions were as described in the legend to Fig. 3B. Avg., the average of the 14
individuals. G9 genotype is the average of the 50 subclones obtained from the 9th generation; G9
phenotype is the behavior of the G9 population as a whole.
t
1t
tt
-
low
hgh
t.u
ca;;;;
G
:
*
:dJ::
.M
.
:i~
S
*.
0
I
Clone
<
Errors
(N)
>
u
II
I
I ~~~~~~~
I
0
co~
29
23
30
43
5
37
28
2
42
8
40
12
27
11
wt
avg.
G9
O
O
DNA
clDegs<
D
nD
0
R
C
-
co
I
LO
co
cJ
cO
co
CO
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
1
1
0
1
0
0
1
0
1
0
0
0
0
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
LO)
0
Cm
~-
'
0)
o
6
7
8
8
7
4
5
5
6
8
1
6
2
6
0
1
1
1
1
1
0
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
x1:
x2:
X3:
10
0.4
0.3
2 -18
0.7 0.2
0.6 0.4
13
0.4
0.3
18
-0.4
0.2
13
-0.2
-0.1
-9
-0.1
-0.3
5.6
5.9
0.6
0.7
0.2
0.1
0.5
0.4
0.6
0.3
0.1
0.1
0.1
0.2
0.1
0.2
than that of the wild type, becauseof improvementin both Kmand kct.
Correlating genotype and phenotype.
The relationbetweengenotypeand phenotypein the context of an RNA-basedevolving system can now be formalized.Genotype can be representedas a matrixA, the
rows correspondingto individualsin the
populationand the columnscorresponding
to functionallysignificantpositionswithin
the nucleotidesequence (Table2). Phenotype can be representedas a columnvector
b, whoseentriesaresome measureof fitness
(catalyticbehavior)of the variousindividuals. We then seek a row vector x that
providesa best fit to the equation:Ax = b,
that is, providesa best fit linearestimation
of the relationbetween genotypeand phenotype. The solution that minimizesthe
least-squareserroris: x = (A*A) l A*b,
whereA* is the transposeof A. In this way,
we obtain a weighting vector x that provides an estimate of phenotype for any
given genotype (Table 2).
The dataobtainedfrom 14 individualsis
not sufficientto providea meaningfulsolution
to the relationof genotypeto phenotype,
even for those nucleotidepositionsthat are
knownto be most significantbasedon their
highfrequencyof acceptedmutation.We use
640
D
cleav-
Hyydsrsol-
Arcacu-
yss
accu
65
57
48
32
22
21
15
11
7
3
3
3
3
3
1
0.1
0.1
0.1
0.0
0.1
0.1
0.0
0.0
0.7
0.2
0.9
0.7
0.8
1.2
1.0
0.7
0.6
0.8
0.4
0.4
0.6
0.7
0.8
0.8
1.1
0.6
0.8
0.6
0.8
1.0
21
21
0.3
0.3
0.7
0.6
age
b1
~~~~b2
Hydr
rc
b3
the weightingvector x as a guide to help
impordecidewhichmutationsaresufficiently
tant to warrantindividualstudy.
The followingindividualmutationswere
preparedby site-directedmutagenesis:94:A
-)U, 94:A--C, 215:G--A, 313:G-3U,
314:A--G, and 313-314:GA--UG (39).
Catalyticactivity was studiedwith d(GG'-32P]A) substrate.
CCCTCT-A3(TA3)3[5
The individual mutations result in improvedactivitycomparedto the wild type,
but they do not resultin activityexceeding
that of the generation9 populationas a
whole (Fig. 7). Activity in the 94:A-+U
mutant is seven times greaterand in the
94:A-*C mutant it is two times greater
than in the wild type. The 313314:GA-*UG double mutant is more active than either the 313:G-*U or
314:A--G single mutant, explainingwhy
the 313-314 mutations occur together
among the evolved individuals that we
have studied.As predictedfromthe analysis of 14 individualsat generation9, the
313-314:GA--UG mutationsresult in diminishedsite-specifichydrolysisof the DNA
substratecomparedto the wild type. These
mutationsconfer both enhancedphosphoester transferactivity and diminishedsitespecifichydrolysisactivity,and thusarewell
Fig. 7. DNA cleavage
activity of individuals
obtained by site-directed mutagenesis. Reaction conditions as described in the legend to
Fig. 3C.-, no enzyme; G9, generation9 population as a whole. Reaction products were
separated in a 20 percent polyacrylamide-8M
urea gel, an autoradiogramof which is shown.
suited to meet the imposedselection constraintwhich dependson availabilityof the
EP covalentintermediate.
The evolutionary frontier. As an in
vitro modelof Darwinianevolution, a popcatalystswas diulationof macromolecular
rectedtowardthe expressionof novel catalytic function. In our study, we wantedto
develop ribozymesthat cleave DNA with
improvedefficiencyunderphysiologicconditions. In a related study, we used these
evolved RNA's to cleave a targetDNA in
vivo; ribozymesobtainedfromgeneration9
were expressed in E. coli and shown to
prevent infection by M13 single-stranded
DNA bacteriophage(40).
We arecontinuingthe presentsuccessful
phylogenybeyondthe tenth generation,but
only after decreasingthe concentrationof
DNA substrate in the target reaction.
Throughthe first ten generationsthe substrate concentrationwas 10 ,uM, roughly
matchingthe Kmforthe wildtype.Now that
the evolved individualshave attaineda Km
of about2 P,M,the substrateconcentration
levels to
must be reducedto subsaturating
promotefurtherimprovementin substrate
binding. In addition,we are attemptingto
improve catalytic tumover in the DNA
cleavagereactionby selectingforboth phosphoestertransferactivity, which generates
the EP covalent intermediate,and subsequentRNA-catalyzedsite-specifichydrolysis
activity,which freesthe ribozymeto act on
anothersubstratemolecule.
The selectionschemethat we usedcould
be appliedto varioussubstratesof the form:
whereQlrefersto
d(CCCTCfQ-A3(TA3)3),
somenucleotideanalogueand the ribozyme
is selected for its ability to cleave the
phosphodiester bond following the se-
SCIENCE * VOL.257 * 31 JULY1992
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All use subject to JSTOR Terms and Conditions
-RESEARCH
quenceCCCTCQ. The substrateneed not
be a nucleotide or nucleotide analogue.
The only requirementis that RNA's that
react with the substratebecome taggedin
some way so that they can be distinguished
fromnonreactivemoleculeswith respectto
the amplificationprocess.Forexample,reactive RNA's could become joined to a
portionof the substratethat is attachedto a
solidsupport.NonreactiveRNA'swouldbe
washedaway, leaving the boundRNA's to
be selectivelyamplified.
The step from an RNA-cleaving ribozymeto a DNA-cleaving ribozymeis a
modestone. It seemsreasonablethat RNA
is capableof catalyzinga broaderrangeof
phosphoestertransferreactions,but it is not
clearwhich of these activitiesareaccessible
fromexisting ribozymesvia directedevolution. In some cases, it maybe necessaryto
evolve a successionof ribozymesthat lead
progressivelytoward the desired catalytic
behavior.An importantmilestone will be
the evolution of a ribozymethat performs
novel chemistry, that is, catalyzessome
reactionother than phosphoestercleavage
or ligation. Considering the functional
groupsthat exist within RNA, there are a
numberof plausibleavenuesto be explored.
16. R. A. Zakourand L. A. Loeb, Nature295, 708
(1982); P. M. Lehtovaara,A. K. Koivula,J. Bamford,J. K. C. Knowles,ProteinEng. 2, 63 (1988).
17. D. W. Leung,E. Chen, D. V. Goeddel, Technique
1, 11 (1989); Y. Zhou, X. Zhang, R. H. Ebright,
Nucleic Acids Res. 19, 6052 (1991).
18. R. K. Saikiet al., Science 230, 1350 (1985);R. K.
Saiki et al., ibid. 239, 487 (1988).
19. D. Y. Kwoh et al., Proc. Natl. Acad. Sci. U.S.A. 86,
1173 (1989);G. F. Joyce, in MolecularBiologyof
RNA: UCLA Symposia on Molecular and Cellular
Biology,T. R. Cech, Ed. (Liss, New York,1989),
pp. 361-371.
20. M.Chamberlin
and T. Ryan,in TheEnzymes,P. D.
Boyer, Ed. (Academic Press, New York,1982),
vol. 15, pp. 87-108. 21. J. C. Guatelli et al., Proc. Natl. Acad. Sci. U.S.A.
22.
23.
24.
REFERENCESAND NOTES
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2. D. R. Mills,R. L. Peterson,S. Spiegelman,Proc.
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Burke,ibid.354, 320 (1991).
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(1990).
5. C. Tuerkand L.Gold,Science 249, 505 (1990);A.
D. Ellingtonand J. W. Szostak, Nature346, 818
(1990).
6. K. S. Lametal., Nature354, 82 (1991).
7. J. K. Scott and G. P. Smith, Science 249, 386
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9. K. Krugeretal., Cell31, 147 (1982).
10. A. J. Zaug and T. R. Cech, Science 231, 470
(1986); P. S. Kayand T. Inoue, Nature327, 343
(1987).
11. A. J. Zaug, M. D. Been, T. R. Cech, Nature324,
429 (1986).
12. D. Herschlag and T. R. Cech, ibid. 344, 405
25.
Sci. U.S.A. 86, 9218 (1989).
26. D. Herschlagand T. R. Cech, Biochemistry29,
10159 (1990).
27. J. M. Burke et al., Nucleic Acids Res. 15, 7217
(1987).
28. S.-H. Kim and T. R. Cech, Proc. Natl. Acad. Sci.
U.S.A.84, 8788 (1987);D. W. Celanderand T. R.
Cech, Science 251, 401 (1991).
29. F. Micheland E. Westhof,J. Mol.Biol.216, 585
(1990).
30. R.W. Davies,R. B. Waring,J. A. Ray,T.A. Brown,
C. Scazzocchio, Nature 300, 719 (1982); F.
Michel,A. Jacquier,B. Dujon,Biochimie64, 867
(1982); F. Micheland B. Dujon,EMBOJ. 2, 33
(1983);T. R. Cech, Gene 73, 259 (1988).
31. J. V. Price,G. L. Kieft,J. R. Kent,E. L. Sievers,T.
R. Cech, Nucleic Acids Res. 13, 1871 (1985); J.
W. Szostak, Nature322, 83 (1986); G. F. Joyce
and T. Inoue,NucleicAcids Res. 15, 9825 (1987);
E. T. Barfodand T. R. Cech, Genes Dev. 2, 652
(1988); G. F. Joyce, G. van der Horst,T. Inoue,
Nucleic Acids Res. 17, 7879 (1989); S. Couture et
32.
33.
(1990).
13. C.-T.Chu,D. S. Parris,R.A. F. Dixon,F. E. Farber,
P. A. Schaffer,Virology98, 168 (1979);D. Shortle
and D. Botstein, Methods Enzymol. 100, 457
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Acids Res. 11, 3113 (1983); J. A. Wells, M.
Vasser, D. B. Powers,Gene 34, 315 (1985).
15. J. B. McNeiland M.Smith,Mol.Cell.Biol.5, 3545
(1985);C. A. Hutchison,S. K. Nordeen,K.Vogt,
M.H. Edgell,Proc.Natl.Acad. Sci. U.S.A.83, 710
(1986); K. M. Derbyshire,J. J. Salvo, N. D. F.
Grindley,Gene 46, 145 (1986).
87, 1874 (1990); G. F. Joyce, in Antisense RNA
and DNA,J. A. H. Murray,Ed. (Wiley-Liss,New
York,1992), pp. 353-372.
In our study, the PCRwas performedunder our
standardreactionconditions(see legend to Fig.
1), resultingin an errorrate of approximately0.1
percent per position per generation. We have
developed a mutagenicPCRprocedurethat provides an errorrate of 0.66 + 0.13 percent per
position(95 percentconfidence level) (R.C. Cadwell and G. F. Joyce, PCR MethodsAppl. 2, 28
(1992).
In practice,there is always the danger of developing a "parasite"that circumventsthe selection
criterionand is amplifiedmoreefficientlythanthe
most reactive species (21). In our study, for
example, a sequence arose that allowed false
of one of the amplification
hybridization
primersat
an internalsite, generating a species with a
53-nucleotidedeletion that was amplifiedmore
efficientlythan the full-lengthribozyme.
M. D. Been and T. R. Cech, Science 239, 1412
(1988); S. A. Woodson and T. R. Cech, Cell 57,
335 (1989); J. A. Doudna and J. W. Szostak,
Nature339, 519 (1989).
F. L. Murphyand T. R. Cech, Proc. Natl.Acad.
34.
35.
ARTICLE
TC-3' and 5'-CTGCAGAATTCTAATACGACTCACTATAGGAGGGAAAAGTTATCAGGC-3',
producing a 435-bp (base pair)fragmentwithunique
restrictionsites at its ends. The fragmentwas
digested withEco RIand HindIIIand ligated into
a pUC18 vectorthathad been linearizedwithEco
RI and Hind IlIland purifiedin a 1.5 percent
agarose gel. The resultingplasmid DNA'swere
used to transformcompetent DH5a-F'cells [D.
Hanahan, in DNA Cloning: A Practical Approach,
D. M. Glover,Ed. (IRLPress, Oxford,1985), pp.
109-135], which were then grown on ampicillincontainingplates. Individualcolonies were chosen at random and grown overnight in liquid
media. DNAwas prepared by the boiling, lysis
method [D. S. Holmes and M. Quigley, Anal.
Biochem.114, 193 (1981)] and screened for the
insertby restrictiondigestion.
36. Cloned individualswere sequenced by dideoxy
chain-termination[F. Sanger, S. Nicklen,A. R.
Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463
(1977);R. Zagursky,K. Baumeister,K. N. Lomax,
M.L.Berman,GeneAnal. Tech.2, 89 (1985)]with
reciprocalprimers5'-GTAAAACGACGGCCAGT3' and 5'-CATGATTACGAATTCTA-3',
which are
compatible with the pUC plasmid. Sequencing
reactions utilizedmodifiedT7 DNA polymerase
(Sequenase, USB) and [35S](a-thiosphate)
dATP
and were analyzed by electrophoresis in a 6
percentpolyacrylamide-8 Mureagel. Nucleotide
sequences of individualsubclones are available
on request.
37. B. Young,D. Herschlag,T. R. Cech, Cell67, 1007
(1991).
38. F. Michel,M. Hanna,R. Green,D. P. Bartel,J. W.
Szostak, Nature342, 391 (1989); F. Michel, P.
Netter,M.-Q.Xu, D. A. Shub, Genes Dev. 4, 777
(1990).
39. Site-directed mutagenesis was carried out as
described [Y. Morinaga, T. Franceschini, S.
Inouye,M. Inouye,Biotechnology2, 636 (1984)].
PlasmidpT7L-21[A.J. Zaug, C. A. Grosshans,T.
R. Cech, Biochemistry27, 8924 (1988)] was digested witheither(i)Eco RIand HindIlIl
to remove
the ribozymecoding region,or (ii)Bsa I and Xmn
I to remove the ampicillin-resistancegene. The
resultingfragmentswere purifiedin a 1 percent
agarose gel and cross-hybridizedinthe presence
of a 5'-phosphorylatedsynthetic oligodeoxynucleotidethatintroducesthe desired mutation.The
annealingmixturecontained 0.06 pmol of pT7L21 (AEco-Hind),0.06 pmol pT7L-21(ABsa-Xmn),
15 pmol of mutagenicoligodeoxynucleotide,40
mMtris-HCI(pH 7.2), and 8 mMMgSO4in 12-pl
volume,whichwas heated to 100?Cfor3 minutes,
then incubatedat 300Cfor30 minutes,25?Cfor30
minutes, 4?C for 30 minutes, and 0?C for 10
minutes.The annealing productwas made fully
double-strandedwith the Klenowfragmentof E.
coli DNApolymeraseI (Boehringer)and T4 DNA
ligase (USB)and then used to transformcompetent DH5a-F'cells, whichwere grownon ampicillin-containingplates. Colonieswere screened by
the colony hybridizationmethod with (5'-32p)labeled mutagenic oligodeoxynucleotideas a
probe [M. Grunsteinand D. S. Hogness, Proc.
al., J. Mol.Biol.215, 345 (1990);A. A. Beaudry
Natl. Acad. Sci. U.S.A. 72, 3961 (1975)]. DNA was
and G. F. Joyce, Biochemistry29, 6534 (1990).
prepared from positive colonies (35) and seG. F. Joyce and T. Inoue,NucleicAcids Res. 17,
quenced throughoutthe ribozyme gene (36).
711 (1989).
RNAwas prepared by in vitro transcriptionas
The (3'-32P)-labeledDNAsubstratewas prepared
described in the legend to Fig. 2.
withterminaldeoxynucleotidetransferase.Reac40. D. L. Robertsonand G. F. Joyce, unpublished
tionconditions:4 F.Md(GGCCCTCT-A3(TA3)3),
1
results.
F.M[a&-32P]dATP
1 mMCoCI2,1 mM
(3 p.Ci/pmol),
41. J. F. Milligan,D. R. Groebe, G. W.Witherell,0. C.
DTT,50 mMpotassiumcacodylate (pH7.2), and
terminaltransferase(BRL)at 2.7 U/pl, incubated
Uhlenbeck, Nucleic Acids Res. 15, 8783 (1987).
at 37?Cfor 30 minutes.The productcorrespond42. G. S. Eadie,J. Biol.Chem.146, 85 (1942);B. H.J.
ing to additionof a single dA residuewas purified
Hofstee, ibid.199, 357 (1952). Because of limited
by electrophoresisin a 20 percent polyacrylamproductyield, we were forced to workat a subide-8 Mureagel and subsequentaffinitychromastrateconcentration[S] 2 K,,; thus Kmshould be
tographyon Nensorb (DuPont).
regardedas only an estimate of the truevalue.
T. Inoue, F. X. Sullivan,T. R. Cech, J. Mol.Biol.
43. We thankD. Decker and D. Rumschitzkifor ad189, 143 (1986).
vice and N. Lehmanand L. Orgel for comments
DNA'sused to transcribethe populationof RNA's
on the manuscript.Supported by NASA grant
at each generation(legend to Fig. 1) were ampliNAGW-1671and NIHgrantAl30882.
fied in a second PCR reactionwith primers5'CCMAGCTTGATCTCGAGTACTCCAMAACTMA-12 February1992; accepted 15 June 1992
SCIENCE * VOL. 257 * 31 JULY 1992
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All use subject to JSTOR Terms and Conditions
641