A flexizyme that selectively charges amino acids activated by a water-friendly leaving group

Article abstract of DOI:10.1016/j.bmcl.2009.03.114

We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water

Full text of DOI:10.1016/j.bmcl.2009.03.114

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3892–3894
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A flexizyme that selectively charges amino acids activated
by a water-friendly leaving group
Nobuyoshi Niwa a, , Yusuke Yamagishi a, , Hiroshi Murakami b, Hiroaki Suga a,b,
*
a Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
b Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-0894, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of
amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion
was designed to render the acyl-donor substrates better water solubility. Although the previously
reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexi-
zyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selec-
tivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to
prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids.
Received 11 February 2009
Revised 20 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Keywords:
Ribozyme
In vitro selection
Non-proteinogenic amino acid
Aminoacylation
Ó 2009 Elsevier Ltd. All rights reserved.
Engineering of the universal genetic code has given us a new
opportunity to express peptides or proteins bearing non-proteino-
genic amino acids.1–4 Recent developments in methodologies of
the genetic code reprogramming have enabled us to reassign multi-
ple codons to non-proteinogenic amino acids.5–21 By means of such
methodologies, libraries of non-standard peptides can be prepared
in the mRNA-encoding manner.14 We have engaged the develop-
ment of such a methodology integrated with flexizymes.10,22–24
Flexizymes are flexible tRNA acylation ribozymes and facilitate
the preparation of various non-proteinogenic aminoacyl-tRNAs,
which are otherwise complex and technically difficult processes.
Two flexizymes, referred to as eFx and dFx, have been devised
(Fig. 1); the eFx acylates tRNA with amino or hydroxy acids upon
activation with cyanomethyl ester (CME) group or p-chlorobenzyl
thioester (CBT), where its activity relies on the recognition of
aromatic sidechain or the CBT group in the substrate; on the
other hand, dFx acylates tRNA with those activated with 3,5-dinit-
robenzyl ester (DBE) group, where its activity fully relied on the
recognition of the DBE group. Since both flexizymes bind the
30-end sequence of tRNA via the 3-base pairing interaction
(RCCA-30, R = G, A and U are the preferable discriminator base at po-
sition 73, but even C is acceptable under prolonged incubation), any
tRNAs can be the acyl-acceptor for the flexizymes. Thus, by the
combination of these two flexizymes, virtually any desired acyl-
tRNAs can be readily prepared.
However, a shortcoming of the current flexizyme system is that
since the CBT or DBE-derived the acyl-donor substrates make them
more hydrophobic, some derivatives, particularly those with
hydrophobic sidechains, are occasionally difficult to dissolve in
the aqueous reaction buffer. Notably, eFx and dFx are able to retain
their catalytic activity in the buffer containing up to 20% and 40%
DMSO, respectively, but the addition of DMSO to the reaction buffer
Figure 1. Flexizymes and their cognate leaving groups in substrates. Chemical
structures of a benzyl ester and thioester leaving group. R represents amino acid
sidechains including non-proteinogenic ones. Each flexizyme recognizes the
specific leaving group and charges the acyl group onto the 30-hydroxyl group at
the tRNA 30-end. Abbreviations: LG, leaving group; CME, cyanomethyl ester; CBT,
p-chlorobenzyl thioester; eFx, enhanced flexizyme; DNB, 3,5-dinitrobenzyl ester;
dFx, dinitro flexizyme. U with asterisk denotes the absolutely conserved U-turn
base. Bold bases orchestrate to constitute the catalytic domain of each flexizyme.
* Corresponding author. Tel.: +81 03 5452 5495; fax: +81 3 5452 5496.
E-mail address: hsuga@rcast.u-tokyo.ac.jp (H. Suga).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.114

N. Niwa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3892–3894
3893
Figure 3. Sequence alignment and secondary structures of aFx and other flexzymes.
(a) Sequence alignment. Bases originating from the random regions were shown in
bold, and the conserved bass (U42) was shown with asterisk. (b) The secondary
structure of aFx compared with the catalytic domain in other flexizymes.
Figure 2. A new substrate design, synthesis, and solubility in the reaction buffer.
(a) Chemical structures of acyl-donor substrates. R represents amino acid sidechains
including non-proteinogenic ones. Abbreviations are as follows: LG, leaving group;
ABT, amino-modified benzyl thioester; aFx, amino flexizyme. (b) Synthesis of
aminoacyl-ABT. An example of Leu derivative is shown. Reagents and conditions:
(I) thiourea, H2O, 1 h, reflux, then10% NaOH, 1 h, reflux, quant.; (II) I2, EtOH, 0 °C, 1.5 h,
quant.; (III) N-hydroxysuccinimide (NHS), EDCꢀHCl, 1,4-dioxane, CH2Cl2, rt, 1 h,
quant.; (IV) mono-tBoc-ethylendiamine, H2O, 1,4-dioxane, 0 °C, 2 h, 91%; (V) DTT,
picked 5 representative clones and tested for self-aminoacylation;
and found that all are active toward Leu-ABT. Among them, clone
11 exhibited the desired 30-terminal specific activity in the highest
efficiency (Fig. S3). Therefore, we focused on this clone for further
studies. We referred this new flexizyme to as aFx as oppose to eFx
and dFx.
DMF, 95 °C, 1 h, 50%; (VI) tBoc- -leucine, EDCꢀHCl, DMAP, CH2Cl2, rt, 1 h, then HCl,
L
AcOEt, CH2Cl2, rt, 0.5 h, 30%. (c) Solubility of Leu-CBT and Leu-ABT. Each compound
was dissolved in 0.1 M Hepes-K (pH 7.5), 20% DMSO at the concentration as shown.
The sequence alignment and drawing the individual secondary
structure of aFx, eFx, dFx and the parental Fx revealed the differ-
ence in the composition of catalytic domain (Fig. 3a and b). The
only conserved base is U42; this base is known to form a unique
U-turn structure and plays a critical role in presenting the catalytic
domain to the 30-hydroxyl group at the tRNA 30-end.24 Because of
lacking our knowledge of the 3-dimensional structure of the cata-
lytic domain in the individual flexizyme except for Fx, it is yet un-
clear how the sequence variation is able to adapt to the respective
leaving group (Fig. 3b). However, even for Fx we have shown that
the catalytic domain is susceptible to mutagenesis,24 it seems that
the catalytic domain can be readily adapted to various benzyl leav-
ing groups by the sequence alterations. Nonetheless, the present
result indicates that the selection from the doped pool of flexizyme
against substrates with different characteristic leaving groups
readily generates new flexizyme variants.
hampers the intrinsic ability of the flexizymes. Therefore we have a
dilemma between poor solubility of the substrates and loss of flex-
izyme activity by the addition of DMSO. Here we report a new pair
of flexizyme and substrate activated by a benzyl thioester in which
p-position is derived to an amino group that is protonated in the
reaction buffer to give the ammonium salt. By using this pair, we
have demonstrated aminoacylation of tRNA with amino acids bear-
ing hydrophobic sidechains or the
the sidechain.
a-N-octanoyl modification on
In order to make the leaving group more water-soluble, we de-
rived a commercially available 4-chlorobenzoic acid to have a pri-
mary amino group and a thiol group for activation of the acyl group
(Fig. 2a). Synthesis of this leaving group and its derivatization of
amino acid was straightforward as shown in Figure 2b. We referred
this amino-derivatized benzyl thioester aminoacyl-donor to as
aminoacyl-ABT (Fig. 2a).
First, we compared the solubility of 10 mM Leu-CBT and Leu-
ABT in the flexizyme reaction buffer containing 20% (v/v) of DMSO.
Despite the solution of Leu-CBT with a cloudy suspension, Leu-ABT
gave a completely clear solution. The solution remained clear even
with 40 mM Leu-ABT (Fig. 2c). Thus, the ABT leaving group signif-
icantly improved the water solubility of the Leu substrate. How-
ever, the alteration of the leaving group turned out to be
detrimental for the acylation activity of the available flexizymes,
dFx and eFx, yielding less than 1% leucylation onto a microhelix
RNA (Fig. S1; regarding this analytical method see below). This
observation imposed us to perform in vitro selection of a new fle-
ixyzme capable of reacting the ABT-derived substrate.
The method of in vitro selection and the flexizyme pool contain-
ing three random regions (Fig. S2a) were the same as our previous
report10 except that the active sequences for self-aminoacylation
were enriched by the reaction with Leu-ABT followed by the selec-
Figure 4. Aminoacylation ability of aFx. (a) Selectivity of aFx to cognate Leu-ABT
against non-cognate substrates. Bands of I and II indicate aminoacyl-microhelix
RNA and microhelix RNA, respectively. Conditions: 0.1 M Hepes-K (pH 7.5), 50 mM
MgCl2, 20% DMSO, 20 lM microhelix RNA, 20 lM aFx, and 5 mM substrate on ice
tive biotinylation on the
a-amino group of the charged Leu. After
for 2 h; (b) Flexibility of aFx toward sidechain in the aminoacyl-ABT structures.
Conditions for Ile-ABT (lane 1): 0.1 M Hepes-K (pH 7.5) and 10 mM substrate on ice
for 24 h. Conditions for Val-ABT (lane 2): 0.1 M Bicine-K (pH 9.0) and 10 mM Val-
ABT on ice for 6 h. Reactions in lanes 3–5 were carried out by the same conditions
described in Figure 4a. (c) Chemical structure of Opa-LG. Opa, 2-amino-3-
(octanamido)propanoic acid; LG, leaving group. (d) Aminoaylation of Opa onto a
microhelix RNA using aFx or dFx against the cognate Opa-LG. The reaction
conditions were the same as those described in Figure 4a, lane 1.
six rounds of selection of active sequences by capturing on a strep-
tavidin-agarose resin (Fig. S2b), we observed an enrichment of the
pool. The 30-terminal specific activity of the pool round 6 was con-
firmed by the activity loss upon the periodate oxidation (Fig. S2c).
23 clones isolated from the pool were sequenced, and their align-
ment revealed that 19 clones fell in a single class (Fig. S2d). We

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N. Niwa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3892–3894
To verify the ability of aFx for trans-activity, we prepared the
istry for Professor Carlos F. Barbas. This work was supported by
grants of Japan Society for the promotion of Science Grants-in-
Aid for Scientific Research (S) (16101007) to H.S., Grants-in-Aid
for JSPS Fellows (7734) to Y.Y., and a research and development
projects of the Industrial Science and Technology Program in the
New Energy and Industrial Technology Development Organization
(NEDO).
aFx domain alone by in vitro transcription from the correspond-
ing DNA template, and examined the aminoacylation onto a
microhelix RNA using denaturing acid PAGE (poly-acrylamide
gel electrophoresis), a previously reported method for the con-
ventional and reliable activity assay for trans-acting flexizymes.10
The trans-acting aFx was able to charge Leu using Leu-ABT onto
the microhelix RNA in 82% yield (Fig. 4a, lane 1). Interestingly,
it was virtually inactive toward Leu-CBT and Leu-DNB (Fig. 4a,
lanes 2 and 3), indicating that aFx exhibited high specificity to
the ABT leaving group.
Supplementary data
Supplementary data (synthetic procedures, a method of in vitro
selection, and analysis for aminoacylation were reported in supple-
mentary data) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2009.03.114.
To investigate the tolerance of aFx toward amino acid side-
chains and
a-chirality, we synthesized five additional amino acids,
Ile, Val, Met, Pro, and
D-Leu, activated by ABT (Fig. 4b). The CBT or
DNB derivatives of these amino acids were known to have modest
or poor solubility at 5 or 10 mM in the reaction buffer unless
including above 20% (v/v) DMSO. The ABT derivatives of these ami-
no acids were, however, efficiently charged onto the microhelix
RNA in high yields (Fig. 4b). This result shows that aFx is able to
charge the ABT-derived amino acids independent from their side-
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Lastly, we performed aminoacylation using a non-proteinogenic
amino acid bearing a fatty acid, 2-amino-3-(octanamido)propanoic
acid (Opa, Fig. 4c). We found that eFx was able to charge Opa-CBT
onto the microhelix RNA in 26% yield (Fig. 4d, lane 1), whereas aFx
was able to charge Opa-ABT in 48% yield (Fig. 4d, lane 2). Clearly,
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In conclusion, we have devised a new pair of flexizyme and sub-
strate activating group for tRNA aminoacylation, referred to as aFx
and ABT. aFx selectively charges ABT-activated amino acids onto
tRNA independent from the sidechain kinds. Importantly, the
ABT leaving group makes hydrophobic amino acid substrates more
water-friendly than CBT and DBE. We now have three pairs of flex-
izyme and acyl-donor, allowing us to choose the most appropriate
pair of flexizyme and acyl-donor depending upon the substrate
properties for the genetic code reprogramming.
Acknowledgments
This paper is dedicated to the recognition of the 2009 Tetrahe-
dron Young Investigator Award in Bioorganic and Medicinal Chem-