Preparation of translationally competent tRNA by direct chemical acylation

Article abstract of DOI:10.1021/ol101408f

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.

Full text of DOI:10.1021/ol101408f

ORGANIC
LETTERS
2010
Vol. 12, No. 17
3776-3779
Preparation of Translationally
Competent tRNA by Direct Chemical
Acylation
Noah H. Duffy and Dennis A. Dougherty*
DiVision of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125
dadougherty@caltech.edu
Received June 17, 2010
ABSTRACT
Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical
acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA
mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor
tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.
Unnatural amino acid mutagenesis provides the researcher
with the ability to perform precise structure-function studies
with proteins, beyond that which can be performed with the
20 natural amino acids.1-3 This is usually achieved through
nonsense suppression, in which the site of interest is mutated
to a stop codon, and an aminoacyl-tRNA bearing the
appropriate anticodon delivers the unnatural amino acid
during translation. The aminoacyl-tRNA is critical to this
methodology, and several approaches to generating it have
been developed.2,4,5 Nature generates aminoacyl-tRNAs via
reactions of ATP-activated amino acids with the cognate
tRNA, mediated by the corresponding aminoacyl-tRNA
synthetase (aaRS).6 In some cases, the natural system can
be hijacked to charge an unnatural amino acid onto a tRNA
if the difference between the unnatural and cognate amino
acids is not large. Methods for generating orthogonal aaRS/
tRNA pairs that accept unnatural amino acids have been
shown to be a viable source of aminoacyl-tRNA. Alterna-
tively, amino acids may be appended to tRNA using chemical
synthesis. All strategies have particular advantages and
disadvantages, and the chemical acylation approach is the
focus of the present work.
The standard chemical aminoacylation strategy begins with
chemically synthesized dinucleotide pdCpA (dCA), which
is then acylated with the desired amino acid.4,7 The derived
aminoacyl-dCA is then enzymatically appended to tRNA via
T4 ligase. This method provides maximal flexibility, as
virtually any amino acid can be used. When combined with
an appropriate expression system, the chemically synthesized
aminoacyl-tRNA does not get recharged by an endogenous
aaRS (an intentional aspect of the experimental design), and
so the amionacyl-tRNA is a stoichiometric reagent. Since
(1) Dougherty, D. A. Curr. Opin. Chem. Biol. 2000, 4, 645
(2) Young, T. S.; Schultz, P. G. J. Biol. Chem. 2010, 285, 11039
(3) Link, A. J.; Tirrell, D. A. Methods 2005, 36, 291
(4) Nowak, M. W.; Gallivan, J. P.; Silverman, S. K.; Labarca, C. G.;
Dougherty, D. A.; Lester, H. A. Methods Enzymol. 1998, 293, 504
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(5) Ohuchi, M.; Murakami, H.; Suga, H. Curr. Opin. Chem. Biol. 2007,
11, 537
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(7) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc.
1991, 113, 2722.
(6) Arnez, J. G.; Moras, D. Trends Biochem. Sci. 1997, 22, 211.
10.1021/ol101408f  2010 American Chemical Society
Published on Web 08/05/2010

the synthesis of the aminoacyl-tRNA is arduous, the quantity
of protein that can be produced using the chemical aminoa-
cylation strategy is limited. For the most part, the methodol-
ogy has been exploited to study ion channels and neurore-
ceptors,wheretheextraordinarysensitivityofelectrophysiology
overcomes the quantity limitation.1
Scheme 2. Synthesis of Amino Acid Ethyl Phosphates
Recently, Tzvetkova and Kluger showed selective acyla-
tion of the 3′ end of RNAs through the use of La3+ salts and
amino acids activated as ethyl phosphate esters (Scheme 1).8
Scheme 1.
La3+-Mediated Acylation of the 3′ End of tRNA
by lyophilization. The resulting foamy materials were used
directly without further purification.
We will refer to this biomimetic strategy as the La3+/EP
approach. If direct aminoacylation of full-length tRNA can
produce translationally competent tRNAs, the synthesis of
dCA, one of the most laborious aspects of the chemical
aminoacylation strategy, would become unnecessary. As
such, we have evaluated the La3+/EP strategy, focusing on
two questions: (1) can we optimize the direct acylation of
full-length suppressor tRNAs; and (2) are the resulting
aminoacyl-tRNAs translationally competent in protein syn-
thesis? In particular, our application of nonsense suppression
to studies of neuroreceptors and ion channels requires
expression in a vertebrate cell, the Xenopus laeVis oocyte.4
This adds additional challenges to the methodology, com-
pared to in vitro protein synthesis strategies.
To address these questions, we prepared a representative
collection of amino acids as derivatives 1-4 (Scheme 2), in
which the carboxylate is activated as an ethyl phosphate and
the amino group is protected with NVOC. This protecting
group has been previously established to be compatible with
the Xenopus oocyte expression systemsit is removed pho-
tochemically before use in translation.4 The amino acid ethyl
phosphates (aaEP) 1-4 were prepared using established
procedures,9 by coupling to bis(tetraethylammonium)ethyl
phosphate using DCC (Scheme 2). As a consequence of our
selected protecting groups, addition of THF as a cosolvent
to CH2Cl2 was required to alleviate solubility issues. Protec-
tion of the phenolic OH on tyrosine was not necessary under
these reaction conditions. Tetraethylammonium salts of
aaEPs 1-4 were obtained by extraction into water, followed
In their report, Tzvetkova and Kluger detected acylation
of RNA by careful selection of the amino acidsspecifically,
a fluorinated amino acid (19F NMR detection) or a dansylated
amino acid (fluorescence detection). Our aaEPs were not
selected with these detection methods in mind, and therefore,
we needed a more general method of detecting acylation.
Previously, our laboratory developed a MALDI-TOF mass
spectrometry method for detecting enzymatic ligation of a
dCA-amino acid to a tRNA transcript lacking the 3′-terminal
pCpA.10 We anticipated that the amino acids 1-4 would
shift the MALDI signal sufficiently that the acylated tRNA
could be detected even if conversion was not complete.
Applying the reported La3+-mediated acylation conditions
to uridine and cytidine with activated amino acids such as
1-4 gave 2′/3′ esters in low overall conversion (∼5-10%
as measured by LC/MS). Increasing the equivalents of the
aaEPs and La3+ to a large excess (e20 equiv) with respect
to the nucleoside improved the conversion (∼25-50%, data
not shown). Using large excesses of aaEPs introduced
solubility issues that could be alleviated by use of DMSO
as a cosolvent. These modified conditions were then applied
to a full-length tRNA (Figure 1). Very large excesses of
aaEPs and La3+ with respect to the tRNA (>1000 equiv) were
required to detect any acylation in the MALDI mass spectra.
Key to the successful application of MALDI was treatment
of the reaction mixture with the well-known lanthanide
chelator diethylenetriamine pentaacetic acid (DTPA).11 While
the standard tRNA precipitation protocols removed most of
(8) Tzvetkova, S.; Kluger, R. J. Am. Chem. Soc. 2007, 129, 15848.
(9) Kluger, R.; Li, X.; Loo, R. W. Can. J. Chem. 1996, 74, 2395.
(10) Petersson, E. J.; Shahgholi, M.; Lester, H. A.; Dougherty, D. A.
RNA 2002, 8, 542.
Org. Lett., Vol. 12, No. 17, 2010
3777

be used in unnatural amino acid mutagenesis, we sought a
functional test. Nonsense suppression has been extensively
employed to probe the nicotinic acetylcholine receptor
(nAChR) expressed in Xenopus oocytes, involving hundreds
of experiments with scores of unnatural amino acids.1,14 The
nAChR is a membrane-bound, ligand-gated ion channel that
can be functionally interrogated by whole cell electrophysi-
ology. Briefly, aminoacyl-tRNA and mRNA coding for the
nAChR and containing a strategically positioned stop codon
are microinjected into Xenopus oocytes. Following an
incubation time of 24-48 h, expressed nAChRs are probed
by application of acetylcholine (ACh) and measuring the
resulting current. Channel function is typically expressed as
EC50, the concentration of ACh required to achieve the half-
maximal response. As a negative control, injection of
unacylated suppressor tRNA produces no measurable current
as a result of the introduction of the stop codon for nonsense
suppression.
Initial studies focused on so-called wild-type recovery
experiments, in which the amino acid appended to the
suppressor tRNA is the amino acid conventionally found at
the site where the stop codon was inserted. This experiment
should produce wild-type receptor, with behaviors indistin-
guishable from conventionally expressed receptor. As shown
in Table 1, using three different amino acids (Leu, Tyr, and
Trp), two different tRNAs (THG7315 and TQOpS′16), and
two variants of the nAChR (the neuronal (R4)2(ꢀ2)3 and the
muscle-type (R1)2ꢀ1δγ), wild-type receptors were prepared.
This provides strong evidence that the La3+/EP methodology
produces translationally competent aminoacyl-tRNAs. To
confirm compatibility with unnatural amino acids, 5-fluo-
rotryptophan (F1-Trp) was incorporated at the cation-π
binding site of the muscle-type receptor (Trp149 of the R
subunit).17 The 4-fold shift in EC50 produced in previous
studies was recapitulated (Figure 2). These results clearly
establish that the aminoacyl-tRNA derived from the La3+/
EP method produced ion channels whose functional re-
sponses match those of channels produced by the dCA
method.
Figure 1. MALDI mass spectra of THG73 tRNA before (top) and
after (below) exposure to La3+-mediated acylation conditions using
leucine derivative 1. Observed masses shown, theoretical masses
in parentheses. A new peak indicative of monoacylation appears,
along with unacylated tRNA. Additional peak (*) at 24 438 m/z
present from transcription of tRNA, which remains unaffected by
the acylation conditions. Yield of acylation estimated to be 25%.
the excess La3+, enough remained (∼500 µM),12 presumably
strongly associated with the tRNA, to disrupt the MALDI.
DTPA application followed by size exclusion chromatog-
raphy resulted in clean samples (e100 nM).12,13 In addition,
it seems likely that injection of large amounts of La3+ into
the Xenopus oocyte would be detrimental to receptor
synthesis and/or function, further justifying the DTPA
treatment.
Even though a large excess of aaEP was used, products
arising from multiple acylations were not detected in the
MALDI mass spectra; only unacylated and monoacyled
tRNA were seen (Figure 1). Our previous work has shown
that the aminoacyl-tRNA ionizes differently than the free
tRNA, and therefore, exact quantitation of acylation via
MALDI is not possible. Therefore, reference mixtures with
known ratios of tRNA/aminoacyl-tRNA were prepared and
evaluated by MALDI. Mass spectra of reaction products then
allowed us to estimate conversion of tRNA to aminoacyl-
tRNA to be 5-25%.
A notable difference between the dCA and La3+/EP
experiments relates to protein yield. The whole cell maximal
currents measured for dCA-derived aminoacyl-tRNAs were
generally higher than those from La3+/EP-derived tRNAs
(Table 1). We suspected that this reflects the decreased
quantity of aminoacyl-tRNA being injected with the La3+/
EP method because of the incomplete acylation. Indeed,
when we correct for the amount of acylated tRNA injected,
the yields of receptor from La3+/EP-derived tRNAs are
comparable to dCA-derived (Table 1). It is worth reiterating
that control experiments show that THG73 and TQOpS′ are
The monoacylation detected in the MALDI mass spectra
was consistent with, but does not prove, the notion that only
the 3′ end of the tRNA was acylated. As further evidence,
as well as to determine if these La3+/EP-derived tRNAs could
(14) Dougherty, D. A. Chem. ReV. 2008, 108, 1642
.
(15) Saks, M. E.; Sampson, J. R.; Nowak, M. W.; Kearney, P. C.; Du,
F. Y.; Abelson, J. N.; Lester, H. A.; Dougherty, D. A. J. Biol. Chem. 1996,
271, 23169.
(16) (a) Rodriguez, E. A.; Lester, H. A.; Dougherty, D. A. RNA 2007,
13, 1703. (b) Rodriguez, E. A.; Lester, H. A.; Dougherty, D. A. RNA 2007,
13, 1715.
(11) (a) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem.
ReV. 1999, 99, 2293. (b) Parker, D.; Dickins, R. S.; Puschmann, H.;
Crossland, C.; Howard, J. A. K. Chem. ReV. 2002, 102, 1977.
(12) As measured by IPC-MS.
(17) (a) Xiu, X. A.; Puskar, N. L.; Shanata, J. A. P.; Lester, H. A.;
Dougherty, D. A. Nature 2009, 458, 534. (b) Zhong, W.; Gallivan, J. P.;
Zhang, Y.; Li, L.; Lester, H. A.; Dougherty, D. A. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12088.
(13) Typical micoinjection volume of 50 nL exposes oocytes to e5 fmol
of La3+
.
3778
Org. Lett., Vol. 12, No. 17, 2010

Table 1. Data for Suppression Experiments Using La3+/EP or dCA Methods
Leu (1)
Tyr (2)
Trp (3)
F1-Trp (4)
receptor
mutation(s)
tRNA
acylation method
oocytes (N)
EC50 (µM)
R42ꢀ23
RL9′A:ꢀ119TGA
TQOpS′
R12ꢀ12δγ
R1Y190TAG
THG73
R12ꢀ12δγ
R1W149TAG
THG73
R12ꢀ12δγ
R1W149TAG
THG73
La3+/EP
dCA
18
La3+/EP
26
dCA
16
La3+/EP
52
dCA
15
La3+/EP
36
dCA
27
19
0.55 ( 0.02
2 ( 2
0.47 ( 0.01 52 ( 2
44 ( 2
9 ( 8
55 ( 2
8 ( 8
47 ( 1
15 ( 11 3 ( 3
230 ( 18
200 ( 9
11 ( 5
0.6 ( 0.4
mean Imax (µA)
4 ( 2
0.8 ( 0.5
1 ( 1
mean Imax/ngtRNA (µA/ng)a 0.5 ( 0.3
0.6 ( 0.4
1.0 ( 0.5 2 ( 2
3 ( 2 2 ( 2
a Imax values normalized to ng of acylated tRNA injected into oocytes. Acylated tRNA estimated as fraction of total tRNA injected based on percent
conversion measured by MALDI-MS. The La3+/EP method yielded 5-25% of total tRNA acylated; the dCA method yielded 100% of total tRNA acylated.
not appreciably acylated by an endogenous aaRS, and
injection of unacylated tRNA yields no meaningful signal.
Injection of a mixture of acylated and unacylated tRNA from
the La3+/EP method therefore simply produces an effective
dilution of acylated tRNA.
In summary, we have shown that direct acylation of tRNA
is a viable alternative to the more labor-instensive dCA
methodology. Although acylation yields are not high, in
appropriately chosen systems, the unacylated tRNA does not
interfere with ion channel characterization. The low acylation
yield gives rise to a comparable reduction in protein
expression, suggesting the approach will only be viable for
systems that express well in the Xenopus oocyte or other
cell of choice. Importantly, ion channels derived from
aminoacyl-tRNA prepared by the La3+/EP method are
functionally indistinguishable from channels derived from
the dCA method, establishing the viability of the approach
for whole-cell expression of proteins. Further work will seek
to improve protein yield by improving acylation efficiency
or alternatively by separation and concentration of the
aminoacyl-tRNA.
Acknowledgment. We thank Dr. Scott Virgil (Caltech)
for assistance with LC/MS, Dr. Russell Jacobs (Caltech) for
suggesting DTPA, Dr. Ronald Kluger (University of Toronto)
and students for helpful suggestions. This work was sup-
ported by the NIH (NS 34407).
Supporting Information Available: Experimental details
for tRNA acylation, ooctye protein expression, electrophysi-
ology, and synthesis of compounds 1-4. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Dose-response curves of mouse muscle nAChR,
[R1(W149TAG)]2ꢀ1δγ when injected with suppressor tRNA.
Source of tRNA indicated.
OL101408F
Org. Lett., Vol. 12, No. 17, 2010
3779