367
Recent advances in the in vitro evolution of nucleic acids
Joshua A Bittker, Kevin J Phillips and David R Liu*
Molecular evolution allows chemists and biologists to generate
nucleic acids with tailor-made binding or catalytic activities.
Recent examples of nucleic acid evolution in vitro provide
insights into natural ribozyme evolution and also demonstrate
potential applications of evolved DNA and RNA molecules.
Efforts to expand the scope of nucleic acid evolution are also
underway, including the development of novel methods for
exploring nucleic acid sequence-space and the incorporation
of non-natural chemical functionality into nucleic acid libraries.
Addresses
Department of Chemistry and Chemical Biology, Harvard University,
12 Oxford Street, Cambridge, MA 02138, USA
*e-mail: [email protected]
Current Opinion in Chemical Biology 2002, 6:367–374
1367-5931/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 20 March 2002
Abbreviation
EGR-1 early growth response factor-1
Introduction
Although generating molecules with new functions is a
common goal of both chemists and nature, the approach
traditionally adopted by the chemist is fundamentally
different from that taken by nature. Nature encodes functional molecules (or the catalysts that generate them) with
a carrier of information that can be amplified and translated,
and therefore can use iterated cycles of diversification,
translation, selection, and amplification to evolve molecules with advantageous new functions. In contrast, the
chemist’s approach to generating new molecular function is
traditionally a linear process of synthesis, assaying or
screening, and elucidating structure–function relationships.
Chemists and biologists can, however, apply the power of
molecular evolution to a very select class of molecules —
those molecules that either can be amplified, or that can be
translated from other amplifiable molecules. Our ability to
amplify combinatorial libraries of DNA and RNA makes
nucleic acids attractive targets for the evolution of new
receptors and catalysts. Over the past 20 years, DNA and
RNA molecules capable of binding to a wide variety of
small-molecule and macromolecular targets or capable of
accelerating an assortment of chemical reactions have been
generated through iterated cycles of in vitro evolution
(Figure 1) [1,2]. Here, we review recent advances in nucleic
acid evolution. New examples of special interest provide
insights into natural nucleic acid evolution and demonstrate the potential applications of in vitro evolved DNA
and RNA. The continued development of novel methods
to explore the sequence space of nucleic acids and to
augment nucleic acid libraries with chemical functionality
will probably further enhance this powerful approach to
generating functional molecules.
Evolutionary insights from ribozymes
Several groups have used in vitro evolution to generate
nucleic acid catalysts of reactions thought to be essential to
a minimal life form [3]. Starting from a previously evolved
RNA ligase ribozyme, Bartel and co-workers [4••] recently
evolved an RNA-dependent RNA polymerase, a key
component of the hypothesized prebiotic ‘RNA world’ [5].
The evolved ribozyme demonstrates 97% sequence fidelity
when presented with RNA primer–template complexes
and equimolar concentrations of NTPs. Although not
highly processive, this ribozyme is able to perform multiple
turnovers, extending some primer–template pairs 11 bases
in 24 hours.
RNA and DNA molecules that promote other reactions
relevant to the prebiotic world, including pyrimidine
phosphoribosylation [6] and oligonucleotide adenosylation, [7] have also been recently generated by in vitro
evolution (Figure 2). These examples join the modern
ribosome [8] as nucleic acids that catalyze reactions at the
core of replicating molecular information and translating
information into functional molecules. Although these
studies demonstrate that nucleic acids are capable of
catalyzing some of the reactions thought to be essential for
primitive life, it is not known whether sequences evolved
in vitro resemble those that may have existed billions of
years ago, or even how closely the structure of the first
oligomeric catalysts resembled the structures of modern
nucleic acids [9].
The in vitro evolution of ribozymes and deoxyribozymes
has also provided insight into how naturally occurring RNA
catalysts may have evolved. Salehi-Ashtiani and Szostak
[10•] recently isolated from random RNA pools a number
of RNA-cleaving ribozymes that are surprisingly similar to
the naturally occurring hammerhead ribozyme motif. This
finding suggests that a convergent evolution pathway may
have led to the limited number of self-cleaving ribozymes
observed in nature. An in vitro evolved ribozyme was also
used to demonstrate that a new RNA fold can evolve from
an unrelated fold without requiring inactive intermediate
sequences [11]. Starting with a class III RNA ligase
ribozyme previously evolved in vitro and the natural
self-cleaving hepatitis delta virus ribozyme, Bartel and
co-workers generated variants of each ribozyme that
approached each other in sequence. At the intersection of
the two series of variants, a single sequence possessed the
ability to catalyze both the ligase and the self-cleavage
reactions, albeit weakly. These results suggest that natural
ribozymes with no functional or structural similarity can
evolve from a common ancestor.
368
Combinatorial chemistry
Figure 1
In vitro evolution of nucleic acids. Typically,
randomized synthetic DNA libraries are either
selected directly for desired binding or
catalytic activities, or are transcribed into RNA
prior to selection. The DNA-encoding
molecules with desired activities are amplified
(and in some cases, intentionally diversified)
either by PCR or by reverse transcription
followed by PCR. The resulting enriched DNA
pool then re-enters the in vitro evolution cycle.
Constant
sequence
Constant sequence
(or T7 promoter)
Random sequence
Synthetic single-stranded
DNA pool
Ribozymes: transcribe into RNA
Deoxyribozymes: use directly
Single-stranded
DNA or RNA
pool
Selection
Active nucleic acid
receptors or catalysts
Ribozymes: transcribe
Deoxyribozymes: strand separate
Amplified doublestranded DNA
Ribozymes: reverse transcribe, then PCR (and diversify)
Deoxyribozymes: PCR (and diversify)
Current Opinion in Chemical Biology
Recent applications of evolved nucleic acids
The ability of in vitro evolution to tailor the specificity and
catalytic activity of nucleic acids has led to several recent
applications. RNA-cleaving deoxyribozymes such as the
10-23 motif [12] that contain both substrate base-pairing
regions and a catalytic core have been used to recognize
and destroy target RNA sequences with some generality.
For example, the 10-23 deoxyribozyme core was flanked
with sequences designed to complement the mRNA
encoding human early growth response factor-1 (EGR-1),
a transcription factor involved in the pathogenesis of
atherosclerosis [13]. Some of the resulting tailor-made
DNAzymes cleave EGR-1 mRNA in vitro, inhibit EGR-1
protein expression both in vitro and in pigs, and reduce
arterial wall thickening by 40% following coronary artery
stenting in pigs. Efficacy of the 10-23 DNAzyme in animals
was also demonstrated by using a nuclease-resistant,
phosphorothioate-modified DNAzyme analog to target
tumor necrosis factor alpha (TNF-α) mRNA, resulting in
decreased TNF-α expression and improved blood flow
following congestive heart failure in rats [14]. A library of
randomized flanking sequences surrounding the 10-23
core was recently evolved in vitro to target a variety of
RNA sequences present in the HIV-I gag mRNA [15].
When transfected into cells, the evolved deoxyribozymes
resulted in decreased HIV gene expression and inhibited
HIV replication in vitro.
Evolved nucleic acids have also recently been used as tools
in the analysis of metabolites and other small molecules.
Self-cleaving RNAs and RNA aptamer motifs have been
joined through a randomized linker and evolved in vitro to
generate allosterically activated ribozymes [16,17]. Several
allosteric switches that self-cleave only in the presence of
a specific ligand have been successfully created in this
manner, providing a means of detecting a wide range of
substrates. These results raise the possibility that arrays
of ribozymes can be used to analyze the metabolic profile
of organisms [18•].
New methods for exploring nucleic acid
sequence space
The development of new methods for evolving nucleic
acid receptors and catalysts will enhance their potency and
expand the scope of their applications. For example, a
metabolome-wide or proteome-wide detection array would
require a vast number of iterated selection and amplification
cycles to generate the constituent aptamers. The automation
of this process [19] allows in vitro evolution to take place on
an accelerated timescale. A prototype nucleic acid evolution
robot was developed that performs eight selections in parallel
and can complete up to eight sets of 12 selection rounds in
two days. RNA aptamers capable of binding to hen eggwhite lysozyme were evolved using this prototype and were
enriched an estimated 1012-fold from the initial random pool.
Recent advances in the in vitro evolution of nucleic acids Bittker, Phillips and Liu
369
Figure 2
(a)
S
O
–O P O
–O
S
O
HO
O
NH
O
–O P O
–O
O– O–
O P O P O–
O
OH O
+
NH
N
H
Nucleotidesynthesizing
ribozyme
N
O
O
+
HO
PPi
OH
(b)
NH2
N
O
–O P O
O
N
O
–O P O
–O
N
OH
N
O
N
N
O
NH
N
O
N
N
N
Capase
deoxyribozyme
NH2
+
O
HO
ATP
O P O
O–
O
DNA
O
NH
+
PPi
HO
O
O
DNA
(c)
NH2
RNA template
RNA template
HO
H2N
O
RNA
O
O P O
–O
N
NH
N
O
N
N
O
N
HO
H2N
–O
O P O
O
OH
O
O
O
–O P O P O P O
–O –O
O–
HO
O
N
N
NH
N
N
NH2
O
N
NH
O
O P O
O–
H2N
N
O
RNA
O
O
HO
O
N
N
N
HO
O
N
O
H2N
O
O
RNA polymerase
ribozyme
OH
O P
O
–O
N
N
O
HO
O—RNA
NH2
+
OH
O
O
NH
N
N
N
O
O—RNA
-O
O P O
O
O
PPi
OH
Current Opinion in Chemical Biology
Reactions relevant to prebiotic chemistry that are catalyzed by nucleic
acids evolved in the laboratory. (a) Pyrimidine phosphoribosylation is
an important step in nucleotide synthesis. (b) Oligonucleotides can be
activated for other reactions such as ligation by 5′-adenosylation.
(c) An essential step towards self-replication is an RNA that catalyzes
RNA polymerization.
The final group of sequences bound to lysozyme with affinities
(Kd <50 nM) comparable with those of protein-binding
aptamers generated by traditional in vitro selection methods.
the sequence space of a nucleic acid in a purposeful manner
but rather simply enriches active molecules from the starting
pool of sequences. In contrast, diversifying an evolving nucleic
acid library with error-prone PCR between some rounds of
selection explores sequence space in a modest but potentially
beneficial manner, as point mutations that may enhance
desired binding or catalytic properties arise at an accelerated
rate and can compete with unmutated sequences. Although
enhanced point mutation rates can access more active neighbors in sequence space, the overall solutions found will still be
similar to the sequences in the initial pool (Figure 3).
Existing methods for the in vitro evolution of nucleic acids
include pure SELEX (iterated cycles of selection and amplification without intentional additional mutagenesis between
rounds) [1], SELEX with cassette mutagenesis [20], and
SELEX with error-prone PCR [21,22] in which point mutations are introduced at a frequency of roughly 1–10% per base
per PCR reaction. In the purest form, SELEX does not explore
370
Combinatorial chemistry
Figure 3
Constant
sequence
Homologous region
Constant
sequence
Parental sequences
SELEX
Enriches active parental sequences
Error-prone PCR
Parental-like sequences with point mutations
Homologous
recombination
Sequences with crossovers at homologous regions
Crossovers at any site
Non-homologous
random recombination
Repetition of sequences
Reordering of sequences
Elimination of sequences
Current Opinion in Chemical Biology
Diversification methods for nucleic acid evolution. Starting with parental
sequences, SELEX enriches active parental sequences. Error-prone
PCR yields parental sequences with point mutations. Homologous
recombination methods, such as DNA shuffling, allow crossovers
between sequences only in regions of homology. Non-homologous
random recombination would theoretically allow many changes, including
random crossovers between any sequences, repetition, reordering, or
elimination of sequence information, and any combination of these.
Protein evolution over the past seven years has been
revolutionized by methods of exploring protein sequence
space that allow homologous recombination to take place
between genes encoding related but distinct proteins in a
process known as DNA shuffling [23,24]. Evidence points
to the potential usefulness of recombination during nucleic
acid evolution as well. Chimeric [25] and allosteric
ribozymes [16,26] use recombination by design, connecting
known active DNA or RNA motifs with possible linkers
before screening or selecting active recombined hybrids.
Methods to randomly recombine any regions of two or
more nucleic acids during in vitro evolution may provide
benefits not offered by SELEX or error-prone PCR
methods. For example, recombination may allow binding
affinities or catalytic activities to be enhanced through
repetition of active sequences allowing polyvalent
interactions [27] with ligands or substrates, or by combining
nucleic acid motifs that each bind to a distinct moiety of
the target molecule. Randomly recombining nucleic acid
aptamers or catalysts may therefore provide access to areas
of sequence space not readily available in an initial random
pool, but which contain interesting chemical solutions
derived from multiple parental sequences with weak or
partial activities (Figure 3).
A method to controllably induce random recombination
during the in vitro evolution of nucleic acid aptamers or
catalysts has not yet been reported. Unintentional
recombination during SELEX can take place by template
switching during PCR [28] or reverse transcription [29].
Like DNA shuffling methods, however, these sources of
recombination require levels of sequence homology that
are rarely present in immature libraries of DNA or RNA
derived from random sequences and therefore may only
Recent advances in the in vitro evolution of nucleic acids Bittker, Phillips and Liu
benefit fairly mature pools containing similar solutions. A
small number of newer methods allow non-homologous
recombination of protein libraries [30–33] but carry
significant limitations on the nature of crossovers or are
labor-intensive and have yet to be applied to nucleic acid
evolution. The development and application of nonhomologous random recombination methods to DNA or RNA
evolution may enhance the power of nucleic acid evolution
considerably, although it remains to be seen if the benefits
of recombination among nucleic acid libraries are as
dramatic as the corresponding benefits of recombination
among proteins.
Evolution of nucleic acids with expanded
chemical functionality
In addition to developing new methods of exploring nucleic
acid sequence space, the scope of nucleic acid evolution
can also be expanded by equipping DNA or RNA with
functional groups not available to natural nucleic acids.
This approach relies on the ability of DNA and RNA polymerases to accept, both as templates and as triphosphates,
modified nucleotides that possess functional groups
attached to the non-Watson–Crick face of the bases while
preserving the sequence fidelity of polymerization. Several
examples of in vitro evolution using modified nucleotides
have been reported and are described below.
In the first example, thrombin-binding aptamers were
evolved from a random pool of DNA containing 5-pentynyldeoxyuridine, demonstrating that nucleotides bearing
expanded functionality could be successfully used
during in vitro selections [34]. Eaton and co-workers [35]
later evolved the first nucleic acid catalyst to catalyze
carbon–carbon bond formation by selecting Diels–Alder
catalysts from a pool of modified RNA oligonucleotides
containing pyridyl groups appended to the C5 of uridine.
The catalytic activities of the evolved RNAs were dependent
on the presence of Cu2+ and on the modified base,
implying that coordination of Cu2+ by the pyridyl moiety
may play a key role either in the structure of the RNA or in
the direct Lewis-acid-mediated catalysis of the Diels–Alder
reaction. The first example of nucleic evolution using two
modified nucleotides simultaneously was also reported
recently [36•]. Perrin and co-workers successfully evolved
an RNAse mimic that contained both imidazole-modified
deoxyadenosines and amino-modified deoxyuridines, thus
introducing into nucleic acids two functionalities present in
the active site of some protein-based RNA-cleaving
enzymes. Another DNA-based RNAse mimic containing
imidazole-functionalized deoxyuridines was recently
evolved to cleave RNA in the presence of Zn2+ [37•].
To date, researchers have used polymerase enzymes to
incorporate more than 20 functionalized nucleotides into
DNA or RNA (Figure 4). Extensive modifications at C5
of deoxyuridine triphosphates are tolerated by several
thermostable DNA polymerases [38]. Recent studies on
C5-modified deoxyuridines have provided insights into
371
the effects of the functional group linkers on the efficiency
of polymerase-mediated incorporation [39,40]. In addition,
Williams and co-workers [41] have recently shown that
modifications at C7 of 7-deazadeoxyadenosine are well
tolerated by Taq DNA polymerase. Analogs of 7-deazadeoxyadenosine bearing alkyl, alkenyl, and alkynyl groups at C7
were incorporated into DNA with similar efficiency to that
of unmodified dATP, suggesting a high tolerance for
structural modifications at this position. Famulok and
co-workers [42•] achieved the simultaneous incorporation
of four functionalized dNTPs into a 79-mer DNA
oligo-nucleotide. The resulting modified DNA strand was
successfully transcribed and sequenced, demonstrating
that fidelity was maintained when the modified nucleotides
were incorporated against a natural template and also when
the functionalized nucleotides served as the template.
RNA nucleotide functionalization has focused on the C5
position of uridine. The feasibility of incorporating C5-functionalized thiol- and amine-containing uridine analogs into
RNA oligonucleotides was recently demonstrated [43].
These nucleotides were successfully incorporated using T7
RNA polymerase, albeit with somewhat reduced efficiency
relative to that of natural NTPs. Although researchers have
yet to demonstrate that a reaction catalyzed by a modified
nucleic acid cannot be catalyzed by an unmodified nucleic
acid subjected to identical in vitro evolution protocols, these
recent findings suggest a greatly increased use of functionalized nucleotides in future nucleic acid evolution efforts.
Thus far, the efforts on expanding the functionality accessible to nucleic acids during in vitro evolution have focused
on incorporating protein-like functionality into nucleic
acid oligonucleotides. Imidazoles, phenols, and primary
amines have been incorporated using polymerase enzymes
into RNA or DNA as surrogates for histidine, tyrosine and
lysine side chains. Given the more limited diversity of
monomers and the greater conformational restrictions of
nucleic acids compared with those of proteins [44], however,
attempts to mimic the functional abilities of proteins by
adding protein-like groups to nucleotide bases may prove
to be challenging.
Rather than attempting to mimic with modified nucleic
acids what nature already does best with proteins, playing
to the unique strengths of nucleic acids as targets for
in vitro evolution may prove to be more fruitful. Nonnatural functionality can be introduced into nucleic acid
libraries simply by functionalizing synthetic nucleotides at
positions known to be tolerated by polymerase enzymes
and adding the corresponding triphosphates into PCR or
transcription reactions. In contrast, incorporating nonnatural functionality into proteins requires engineering or
subverting complex components of the protein biosynthetic
machinery for each new amino acid added [45–48]. The
incorporation into nucleic acids of functionality beyond
that present in proteins is therefore a highly attractive
prospect. Although nucleic acids may not be able to match
372
Combinatorial chemistry
Figure 4
NH
N
H
NH2
NH2
NH2
O
H
N
N
R1 = H, R2=
O
N
H
R3 =
R3
N
HN
H
N
NH
N
O
R2
O
HO
O
O
N
H
CO2H
N
H
NH2
N
OH
O
HO
R1
O
HN
O
N
NH2
R1 = OH, R2=
O
SH
NH2
R4
N
H
N
H
N
O
N
H
N
N
H
N
NH2
N
N
HO
N
O
HO
R4=
NH2
CO2H
NH2
H
NH2
N
O
HO
N
N
HO
H
N
O
HO
NH2
HO
N
N
N
N
H
O
HO
H
HN
N
N
NH
O
HO
NH2
O
NH2
H
Current Opinion in Chemical Biology
Modified nucleotides reported to serve as substrates from DNA or RNA polymerase enzymes.
the catalytic potency of proteins in certain situations, the
potential to incorporate ad hoc functionality into DNA or
RNA libraries may allow nucleic acids to address problems
that are difficult to solve with proteins.
For example, equipping nucleic acids with metal-binding
ligands such as phosphines may result in novel transition
metal coordination complexes that can be evolved in vitro
using direct selections for bond-forming or bond-cleaving
catalysis followed by PCR amplification and diversification.
In addition, the conformational rigidity of nucleic acids
together with their lack of hydrophobic moieties may be
responsible for their limited abilities to bind small molecules [18•,44]. Incorporating into nucleic acids hydrophobic
groups attached to bases through flexible linkers may
increase substantially their ability to bind small molecules
with high affinity and specificity. Directing the power of
in vitro evolution to select, rather than screen, libraries of up
to 1015 metal-binding and small-molecule-binding nucleic
acids for those capable of stereoselectively catalyzing
chemical reactions not found in nature is one of many
tantalizing possibilities in this area.
Recent advances in the in vitro evolution of nucleic acids Bittker, Phillips and Liu
Conclusion
Recent examples of generating novel DNA or RNA
molecules by in vitro evolution have provided new insights
into the possible origins of catalytic activities essential to
early forms of life. In addition, the application of evolved
nucleic acids as tailor-made sensors or catalysts continues
to progress towards goals of industrial and therapeutic
significance. The development of new methods for exploring nucleic acid sequence space and for augmenting the
functionality available to nucleic acids during their in vitro
evolution may enhance considerably the chemical properties
of receptors and catalysts generated by these methods.
Ultimately, these efforts to expand the scope of nucleic
acid evolution may allow us to address longstanding questions including ‘What are the most efficient methods of searching
molecule space for desired activites?’, and ‘How do the chemical
properties of building blocks affect the functional potential of an
evolvable molecule?’. These questions lie at the heart of
understanding and applying nature’s powerful approach to
generating molecular function.
Acknowledgements
We thank Gerald Joyce, Ronald Breaker and David Bartel for helpful
discussions. KJP is supported by a National Defense Science and
Engineering Graduate (NDSEG) graduate research fellowship and by
Searle Scholars Program grant #00-C-101. JAB is supported by a Howard
Hughes Medical Institute (HHMI) predoctoral fellowship and by a
National Science Foundation (NSF) CAREER award #MCB-0094128.
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