Supplementary Note
New designs for aminoacyl-donors
Our previous investigations1 with Fx have revealed that it is able to accept three
distinct types of leaving groups: cyanomethyl esters (CME), thioesters, and adenosine
monophosphate. This suggests that the leaving group is not involved in recognition of the
substrate for Fx. Moreover, Fx accepts aminoacyl-donors with not only amino groups
(including ammonium as well as N-acylated forms) but also hydroxy groups (D.
Kourouklis, H.S., unpublished data), and therefore the amino group is also not a critical
element for recognition. These observations have suggested that the essential recognition
elements of Fx toward the acyl-donor substrate might reside on the methylene–aromatic
moiety of the sidechain and the carbonyl group of the ester.
With this hypothesis in mind, we redesigned the substrate into one that can be
widely recognized by Fx, or its mutants if necessary, without dependence upon the
methylene–aromatic group in the sidechain. One possible solution was to alter the
recognition element from the sidechain to a generic group in the substrate, such as the
leaving group, and thereby Fx is able to react with substrates bearing various types of
sidechains. We thus considered a substrate that has a benzyl ester as a leaving group (Fig.
1c). When such a substrate was compared structurally with the original substrate
(Phe-CME), the sidechain and amino group in the new substrate was positioned similar to
the CME leaving group in Phe-CME. In addition, based on the knowledge that the CME
group could be altered to other leaving groups, the substitution of CME with a benzyl
ester would not eliminate the reactivity toward Fx. More importantly, its reactive center
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could be superimposed on that of Phe-CME, and Fx might then be able to retain
reactivity toward this substrate.
In vitro selection of Fx mutants
In order to rapidly and successfully isolate the desired Flexizymes, we set two
seeds for the selection design. The first one was set in the RNA pool design. Only the
bases involved in amino acid recognition were randomized without or with a single
random base insertion (Supplementary Figure 2 online); there were a total of 10 or 11
nucleotides with a complexity of approximately one to four million (= 410 to 411)].
Moreover, a microhelix RNA, an analog of tRNA that is also a good substrate for Fx, was
chosen as the acyl-acceptor domain. To this end, each RNA pool, of which the random
domain was connected to the microhelix via a 10-mer poly-adenosine linker, was
constructed and the resulting two pools were mixed as a single set of pool. The second
seed was set in the substrate, where -(N-biotinyl)amino--(S)-hydroxybutanoic acid
DBE (Hbi-DBE, Supplementary Figure 2 online) was used as a substrate. The
-hydroxy acid ester, instead of a protonated -amino acid ester, was chosen to aim at
increasing the potential for selecting Flexizymes with broader specificity for acid esters
with -substituted heteroatoms. In parallel to the above selection, we also carried out in
vitro selection using a similar RNA pool containing Phe-CME, with the aim of enhancing
the original activity of Fx (Supplementary Figure 3 online).
After cloning of the enriched pool (Supplementary Figures 2 and 3 online) and
testing each clone for trans-activity toward tRNAAsnCUA, we selected the single best
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catalyst from each selection, named dFx (d: dinitro) and eFx (e: enhanced) as shown in
Fig. 1a and 1b, respectively. Both Flexizymes contained several base mutations from the
original Fx; dFx had two mutations (G20U and G21C) and one mutation-insertion
(U34CA), while eFx contained four mutations (A19G, G21C, U32G and G36A). The dFx
was able to charge Hbi using Hbi-DBE onto tRNAAsnCUA with a 10% yield whereas Fx
was nearly inactive (Supplementary Figure 1 online, lanes 9 and 7). The most striking
observation for dFx was that when Aly-DBE was used Aly-tRNAAsnCUA was obtained in
17% yield, whereas Fx produced less than a 3% yield (Supplementary Figure 1 online,
lanes 8 and 10). Destruction of the 3'-terminal diol of tRNA by NaIO4-oxidation
completely eliminated the activity, indicating that aminoacylation occurred at the
3'-terminal hydroxyl group (Supplementary Figure 1 online, lanes 12 and 14).
Reaction toward a wide collection of DBE and CBT substrates
Based
on
our
past
experience,
an
approximate
20%
yield
of
aminoacyl-tRNAAsnCUA was the threshold for obtaining over 20% suppression efficiency
using suppressor tRNAAsnCUA under our translation conditions. The efficiencies observed
in most reactions for natural amino acids met this requirement but unfortunately some
amino acids did not behave as anticipated. Such amino acids fell into two groups based
on the characteristics of their sidechains. The members of the first group are Cys, Asn,
and Asp, in which sidechains bear nucleophiles (Fig. 1d, lanes 10, 11 and 13; see also
Supplementary Table 1 online). We suspected that the ester bond of these
aminoacyl-tRNAs was hydrolyzed during the selective biotinylation of the -amino
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group performed for the conventional streptavidin-dependent gel-shift assays. In addition,
the secondary amino group on Pro (lane 7) might be inefficient for the biotinylation. To
investigate whether these are the reasons for the modest yields, we performed the
aminoacylation of the microhelix RNA, the same RNA that was used for the selection,
and analyzed the products directly on acid PAGE (aPAGE) without the biotinylation
process (Fig. 1e). Because of its short length, this aminoacyl-acceptor with homogenous
3'-ends could be obtained following PAGE purification. This allowed us to assess more
accurately the aminoacylation abilities of dFx for 3'-end aminoacylation compared to in
vitro transcription of tRNAs, bearing heterogeneous 3'-ends due to difficulty in their
purification. Indeed, this microhelix–aPAGE analytical method generally resulted in
aminoacylation yields which were approximately 2-fold higher than when tRNA was
used (Fig. 1e, lanes 26 and 32 for Leu and Lys, and Supplementary Table 1 online,
compare their yields with aPAGE). Upon analyzing the dFx-catalyzed aminoacylation of
microhelix RNA, we found that all amino acids in the first group (Cys, Asn, Asp, and
Pro) produced yields of more than 20% (Fig. 1e, lanes 27–31, and Supplementary Table
1 online labeled with aPAGE), and thus these amino acids also met the threshold
requirement.
We next extended our test to two sets of nonstandard amino acids
(Supplementary Figure 5 online). The first set included those having altered sidechains
(Cit, Aly, Bly, and Iph) and D-configurations (Dle). The second set included those that
alter the ordinary -peptide bond to N-methyl-peptide (Mle), -peptide (Bal), or -ester
(Hle and Hph) bonds, upon incorporation into proteins. As expected, all nonnatural amino
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acids in the first set could be efficiently charged by dFx or eFx onto tRNA (Fig. 1d, lanes
18–23 and Supplementary Table 1 online). Due to the difficulty of selective
biotinylation on the secondary amino group in Mle (see Fig. 1d, lane 21) and the
-hydroxy group in Hle and Hph, the microhelix–aPAGE method was utilized to observe
directly the acylated-RNA. Again these acyl-donors were charged efficiently (Fig. 1e,
lanes 33, 34 and 37). One exception was Bal for which the yield was only 18% when the
generic dFx system was used, probably because the change in position of the protonated
amino (i.e. ammonium) group from  to  resulted in less activation of the carbonyl
group. However, when the corresponding CBT was used the yield increased to 40% (Fig.
1f, lane 47).
Amber and programmed frame-shift suppression for site-specific protein
mutagenesis
To verify whether the acyl-tRNAs prepared by dFx and eFx function in
translation, we demonstrated site-specific mutagenesis of a model protein, green
fluorescent protein (GFP), using amber suppression in an E. coli high-performance
cell-free translation system (RTS-100). The same expression system9 has been previously
utilized for evaluating acyl-tRNAAsnCUA (where a few mutations are introduced into the
wildtype tRNAAsnCUA in order to increase the orthogonality of endogenous ARSs; see
Supplementary Figure 6a, lane 3) prepared by the original Fx. Therefore, this system is
very reliable for testing the function of acyl-tRNAAsnCUA prepared by the new
Flexizymes.
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Initially, the tRNAs charged with the 20 natural amino acids were individually
tested for suppression of the amber codon at position 151 on the GFP mRNA. As
expected, most amino acids were incorporated with over 20% suppression efficiencies (a
dozen of the amino acids showed near or above 50% efficiencies, Supplementary
Figure 6a, lanes 5–23). One exception was Gly, which was not incorporated into GFP at
all (lanes 4 and 24). Because it was confirmed that Gly was attached to the 3'-end of
tRNA like the other amino acids, the complete lack of incorporation was unanticipated.
We thus suspected that unknown mechanisms were operating in RTS-100 to prevent the
use of Gly-tRNAAsnCUA for elongation, e.g. its selective hydrolysis. To verify this, we
used a reconstituted cell-free translation apparatus, the PURE system (see also ref. 13 in
main text). Because the PURE system contains minimal numbers of recombinant protein
enzymes and factors that are essential for translation, the contaminants responsible for the
lack of incorporation of Gly may not be present. Indeed, Gly was incorporated into GFP
in the PURE system (Supplementary Figure 6b), suggesting that there could be
unknown factors present in RTS-100 which prevented amber suppression by Gly.
Next, we tested tRNAAsnCUA charged with nonnatural amino acids that were
prepared by dFx or eFx (Supplementary Figure 6c). These were divided into two
groups; (1) derivatives of -amino acids, consisting of Cit, Aly, Bly, and Iph (lanes
25–27 and 31, respectively), and (2) more exotic nonnatural amino acids, consisting of
Mle, Dle, and Bal (lanes 28–30). In the first group, the suppression efficiency of Cit, Aly,
and Iph exceeded 50% as expected. Bly was also incorporated but its efficiency was
lower than the others, probably because the biotinyl group hindered the interaction with a
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component(s) of the translation machinery, such as EF-Tu or/and the ribosome peptidyl
transfer center. On the other hand, the nonnatural amino acids in the second group were
not incorporated into GFP using the RTS-100 (lanes 28–30) or PURE systems (data not
shown). Our results suggested that these nonnatural amino acids are poor substrates for
the E. coli cell-free translation systems tested in this study.
The Fx system is also able to charge -hydroxy acids onto tRNA as with amino
acids. Therefore, three hydroxy acids, Hph, Hle, and Hbi, were chosen for the
incorporation test into GFP (Supplementary Figure 6d, lanes 36–38). To also
demonstrate the versatility of the Fx system toward tRNA bearing different anticodons,
we performed programmed frame-shift suppression at position 178 using tRNAAsnACCC
charged with the hydroxy acids. Both Hph and Hle were incorporated into GFP
efficiently. In contrast, Hbi was incorporated poorly, most likely for the same reasons as
the inefficient incorporation of Bly.
References
1.
Saito, H., Kourouklis, D. & Suga, H. EMBO J. 20, 1797-806 (2001).
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