Proc. Natl. Acad. Sci.
USA
Vol. 94, pp. 7126–7128, July 1997
Commentary
Natural and unnatural answers to evolutionary questions
Richard C. Conrad, Tonia L. Symensma, and Andrew D. Ellington
Department of Chemistry, Indiana University, Bloomington, IN 47405
The sequence, structure, and function of natural biopolymers
frequently appear to be optimally adapted to their metabolic
and physiological tasks. So much so that ‘‘scientific creation-
ists’’ frequently argue that natural biopolymers could not have
possibly arisen by chance, and that the conservation of histone
sequence is a sign of providential necessity rather than natural
chance. Evolutionary biologists reply that random mutation
and natural selection are more than capable of generating
highly functional macromolecules, but direct, experimental
validations of this stipulation sometimes have proven difficult
to find: it is one thing to hypothesize that a complex macro-
molecule such as tRNA could have arisen by chance, it is quite
another to prove it. Although the evolutionary basis of biology
has not been in doubt for more than a hundred years, it would
nonetheless be satisfying to actually witness the evolution of
complex macromolecules instead of merely inferring the
lengthy chain of mutation and selection that led to their
fixation. Experiments in artificial or directed evolution have
the potential not just to bolster evolutionary theory but to flesh
out the basis of this theory—the notion that function can be
acquired frequently enough by random chance to initiate and
sustain life. Stephen Gould (1) is fond of saying that if we could
rerun the tape of life, the biological answers that would be
found would be different. Although we cannot yet rerun the
full feature film, it is now possible to at least examine vignettes
for molecular evolution.
Although Sol Spiegelman, Manfred Eigen, and other pio-
neers (2, 3) initiated the study of directed molecular evolution
using Qb bacteriophage, in recent years such experiments have
become much more powerful due to the advent of facile
methods for DNA synthesis and nucleic acid amplification.
Now, functional nucleic acid binding species (aptamers) and
catalysts can be selected from partially or completely random
sequence pools (4–8). Such in vitro selection experiments can
provide insights into what possibilities may have been available
during the course of molecular evolution. For example, how
many different sequences or structures might be functionally
similar to tRNA, and thus, what is the probability that a
molecule such as tRNA might have arisen by chance?
The paper by Klug et al. (9) is an excellent example of the
application of in vitro selection methods to such evolutionary
questions. A nonstandard amino acid, selenocysteine, is spe-
cifically incorporated into formate dehydrogenase (fdhF) of
Escherichia coli during translation. An opal (UGA) codon in
the genes encoding these proteins can interact with a unique
aminoacylated tRNA (tRNASec). Obviously, this dual use of
the opal stop codon prompts questions such as what precludes
termination from occurring at selenocysteine insertion sites,
and what precludes other opal codons from coding for sel-
enocysteine? Previous work, primarily from the laboratory of
August Böck (10), has shown that there is a downstream
hairpin structure in the mRNA that directs this particular opal
codon to be used as a selenocysteine-incorporation site. The
hairpin in turn interacts with an EF-Tu variant known as SelB.
Whereas the N-terminal 70% of SelB is homologous to
© 1997 by The National Academy of Sciences 0027-8424y97y947126-3$2.00y0
PNAS is available online at http:yywww.pnas.org.
7126
elongation factor EF-Tu (11), the C-terminal region of the
protein contains the hairpin-binding activity (12). These find-
ings support a model in which the hairpin serves as the initial
docking site for SelB and its bound selenocysteyl-tRNASec
(Fig. 1). This model for selenocysteine incorporation appears
to be widely applicable and holds in all prokaryotes that have
been studied so far. For example, all known SelB-binding
hairpins fold into a similar stem–loop structure with a con-
sensus GGUC end loop. Studies using chemical protection
(13), nuclease and iodine-cleavage protection (14), and to-
eprinting analysis (15) had indicated that the end loop com-
prised the main recognition element. This was further verified
by demonstrating that mutations in this loop diminished or
abolished binding (16) and a 17-nucleotide minihelix com-
posed of the upper half of the hairpin was capable of binding
SelB without loss of specificity or affinity (12).
Klug et al. (9) have attempted to discern how likely or
optimal the evolution of the SelB-binding hairpin would have
been. A pool of fdhF hairpin variants was synthesized in which
each position contained 70% wild-type residues and 10% of
each non-wild-type residue; in addition, positions correspond-
ing to the selenocysteine-encoding opal codon were totally
randomized. The RNA pool was selected for its ability to bind
to SelB in vitro. The inclusion of wild-type hairpin as a specific
competitor helped to ensure that the best binding species were
extracted from the population. The resultant anti-SelB aptam-
ers fell into two groups. The first was composed of minor
variations of the wild-type sequence. The 16 nucleotides at the
top of the hairpin were completely conserved, with the excep-
tion of two adjacent base pair interactions close to the loop that
covaried between several Watson–Crick alternatives. None of
the wild-type-like aptamers had a UGA codon adjacent to the
stem, suggesting that the VGA codon does not directly interact
with SelB. The second group of aptamers contained widely
divergent sequences that nonetheless still could be folded to
form stem–loop structures. Most of the alternative stem–loops
bound at the same site on SelB as the original hairpin, but two
of the novel structures also appeared to interact with addi-
tional epitopes on SelB. Protection studies indicated that the
apical regions of both classes of aptamer hairpins were im-
portant for protein recognition.
The finding that alternative sequences and structures can
bind protein targets is far from unique. For example, in one of
the first applications of in vitro selection methods to a protein
target, a translational operator that bound T4 DNA polymer-
ase was randomized, binding sequences were selected, and
both the wild-type and an alternative stem–loop were recov-
ered (8). The unnatural variant bound as well as the wild-type,
indicating that nature apparently had found one of two equiv-
alent ‘‘best answers.’’ Nonetheless, the three-dimensional
structures of the two different RNAs were virtually identical
(17, 18). Similarly, a region of 16S ribosomal RNA bound by
protein S8 was completely randomized and binding variants
were selected (19). Three families of related sequences were
found: one family was similar to the natural consensus se-
quence whereas the other two families deviated significantly
from this sequence. However, comparison of these families
suggested that they could all form a base triplex with a
 Commentary: Conrad et al.
FIG. 1. Schematic model of site-specific selenocysteine incorporation in E. coli formate dehydrogenases by alternative usage of the UGA stop
codon. A SelB-GTP-selenocysteyl-tRNASec ternary complex (GTP not shown) docks to the apical region of the mRNA hairpin located immediately
downstream of the opal codon. The N-terminal and central domains of SelB are homologous to EF-Tu and bind the selenocysteyl-tRNASec and
GTP. The C-terminal region of SelB is responsible for recognition of the mRNA hairpin. SelB thus facilitates the incorporation of selenocysteine
into the A site of the ribosome and into the nascent fDHF protein.
looped-out A residue. As a final example, Olke Uhlenbeck and
coworkers (20) partially randomized tRNAPhe and selected
variants that could fulfill a number of independent, functional
requirements, including the ability to (i) bind to phenylalanyl-
tRNA synthetase, (ii) be aminoacylated, and (iii) bind in
aminoacylated form to EF-Tu. Again, the selected variants
showed much greater sequence diversity than had previously
been observed in nature.
Although unnatural RNA sequences may bind as well as
natural sequences, it is unclear whether they can serve as
functional replacements for the natural sequences. It is pos-
sible that natural evolution never arrived at these alternative
solutions because they were incompatible with other functions
of the RNA. For example, although variants of tRNA might be
aminoacylated and carried by EF-Tu, there is no guarantee
that they would fit into the A or P sites of the ribosome. In this
respect, the paper by Klug et al. (9) is particularly instructive.
Several of the anti-SelB aptamers were cloned into a portion
of the fdhF mRNA in place of the wild-type hairpin structure;
care was taken to retain spacing requirements that previously
had been observed to be important for hairpin function. The
chimeras were then assayed for their ability to readthrough the
opal codon by facilitating the insertion of selenocysteine. An
aptamer from their first group, in which the apical region was
identical to the wild type except for one base pair, was found
to substitute for the wild-type stem–loop with 87% efficiency.
However, none of the aptamers from the second group were
able to efficiently substitute despite the fact that they formed
hairpin structures of similar size and contained at least some
residues that were similar to those of the wild type. One of the
aptamers was able to partially substitute for the wild-type
hairpin (17% of wild-type activity) when only its apical region
was switched. The authors concluded that some aspect of the
wild-type sequence and structure beyond SelB binding was
essential for selenocysteine insertion. These results can be
directly compared with a study in which anti-Rev aptamers
were inserted into the Rev-responsive element in place of the
wild-type Rev-binding element and assayed for the ability to
facilitate Rev-dependent mRNA transport (21). As was the
case with the studies performed by Klug et al. (9), one family
of aptamers closely resembled the wild-type sequence, whereas
the other diverged significantly. Tellingly, all could be modeled
to resemble the wild-type, three-dimensional shape (22). How-
Proc. Natl. Acad. Sci. USA 94 (1997) 7127
ever, in contrast to the results obtained by Klug et al. (9), both
families of aptamers could efficiently replace the function of
the wild-type element. Moreover, when the aptamers were
expressed in trans they could inhibit interaction of the Rev
protein with the wild-type element and concomitantly inhibit
viral replication (23). Thus, it would appear that in some
instances wild-type sequences are functionally optimal, and in
other cases wild-type sequences can be readily replaced by
alternative sequences. The structural corollary to this conclu-
sion may be that some RNA-binding sequences, such as the
Rev-binding element, are like DNA enhancers and can func-
tion in an architecture-independent manner, whereas other
RNA-binding sequences, such as the hairpin that binds SelB,
function in an architecture-dependent manner. Given the
model shown in Fig. 1, it is not surprising that the latter system
may require precise placement of the SelB:aminoacyl tRNA
complex relative to the opal codon, or that the act of binding
the hairpin may create a conformational shift in SelB, causing
it to more effectively place the selenocysteyl-tRNASec in the
ribosome.
However, there is an important caveat to this conclusion. In
both experiments, the observation and quantitation of function
may be highly dependent on assay conditions. Although the
anti-Rev aptamers bound from 3- to 10-fold better than Rev in
vitro, they initially appeared to facilitate mRNA transport no
better than the wild-type Rev-binding element in vivo. It was
hypothesized that this apparent discrepancy might be due to
saturation of both wild-type and aptamer sequences with Rev.
Accordingly, when the same constructs were expressed in the
presence of subsaturating concentrations of Rev, the aptamers
showed improved mRNA transport activity. Similarly, al-
though anti-SelB aptamers that diverged from the wild-type
hairpin sequence and structure do not appear to support
selenocysteine incorporation, it should be noted that the
effects of aptamer sequence and structure on overall transla-
tional efficiency were never measured. However, this is prob-
ably not a major consideration, as all the aptamers form stems
with roughly equivalent stability, and it has been shown that
lengthening the bottom stem by three base pairs has minimal
effect on translational readthrough (much of it probably due
to the greater distance between the opal codon and the hairpin
apex) (16). Thus, depending on assay conditions and interpre-
tations, unnatural sequences may function worse than, as well
7128 Commentary: Conrad et al. Proc. Natl. Acad. Sci. USA 94 (1997)

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