Native chemical ligation of hydrolysis-resistant 3′-peptidyl-tRNA mimics

Article abstract of DOI:10.1021/ja209053b

Hydrolysis-resistant 3′-peptidyl-RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is native chemical ligation of 3′-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3′-peptidyl-RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

Full text of DOI:10.1021/ja209053b

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Native Chemical Ligation of Hydrolysis-Resistant 30-PeptidylꢀtRNA Mimics
Anna-Skrollan Geiermann,† Norbert Polacek,‡ and Ronald Micura*,†
†Institute of Organic Chemistry and Center for Molecular Biosciences, University of Innsbruck, Austria
‡Innsbruck Biocenter, Division of Genomics and RNomics, Medical University of Innsbruck, Austria
S Supporting Information
b
ABSTRACT: Hydrolysis-resistant 30-peptidylꢀRNA con-
jugates that mimic tRNA termini represent a remarkable
synthetic challenge, particularly if they contain amino acids
with complex side-chain functionalities, such as arginines.
Here we demonstrate a novel approach that combines solid-
phase synthesis and bioconjugation to obtain these deriva-
tives with high efficiency and purity. The key step is native
chemical ligation of 30-cysteinyl-RNA fragments to highly
soluble peptide thioesters. The so-prepared 30-peptidylꢀ
RNA conjugates relate to resistance peptides that can render
the ribosome resistant to macrolide antibiotics by a yet
unknown ribosomal translation mechanism.
Figure 1. Novel strategy involving NCL for the synthesis of hydrolysis-
resistant 30-peptidylꢀtRNA mimics. The peptide shown was chosen
exemplarily and relates to a typical antibiotic resistance peptide.11
resistance peptides.11 When these peptides are translated, they can
render the ribosome resistant to macrolide antibiotics by a mechanism
yet to be explored. The prospect of an efficient synthetic access to
enable investigations that can shed light onto this process, particularly
in context with ribosomal X-ray structure determinations, prompted
us to target arginine-containing 30-peptidylꢀtRNA mimics.
Since many sequences of resistance peptides contain cysteines
beside arginines,11 retrosynthetic analysis suggested a convergent
pathway that involves native chemical ligation (NCL) as a
potential route (Figure 1). NCL was originally designed to link
unprotected peptide fragments under mild conditions.16 The
process involves a reaction between a weakly activated C-term-
inal thioester and an unprotected N-terminal cysteine residue.17
The thermodynamic strength of an amide bond over a thioester
bond is the driving force behind this reaction, which is made
possible through a proximity-driven S-to-N acyl migration.
To achieve our specific targets, one fragment must be an
RNAꢀpeptide conjugate providing the 30-terminal cysteine
moiety while the other fragment, the arginine-containing pep-
tide, must be functionalized as a thioester. On the basis of
our previously elaborated synthetic approach for generating
30-aminoacylamino-30-deoxyadenosine derivatives,14 we synthe-
sized the novel 30-cysteinylamino-30-deoxyadenosine-modified
solid supports 1 and 2 by analogy (Figure 2A).
ydrolysis-resistant RNAꢀpeptide conjugates that mimic
H
acylated tRNA termini are highly requested compounds
for structural and functional studies of the ribosomal elongation
cycle.1 Such conjugates in their simplest form are represented by
puromycin derivatives.2,3 They were positioned as stable sub-
stratesinthe peptidyltransferasecenter(PTC) and gave riseto the
first detailed mechanistic insights for ribosomal peptide bond for-
mation based on high-resolution X-ray structures.3,4 In a very
recent example, synthetic 30-aminoacylꢀRNA conjugates signifi-
cantly contributed to our understanding of how the growing pep-
tide chain communicates within the PTC in ribosomal stalling
phenomena.5 Facile experimentalaccesstothis type of conjugate is
expected to stimulate further investigations and functional char-
acterizationofdifferentstatesof ribosomal translation, inparticular
ofpre- and postpeptidyl transferstates6 and tRNA hybrid states7 as
well as translation initiation,8 elongation,9 and termination.10 In
addition, such conjugates will enable the exploration of peptide-
mediated macrolide antibiotic resistance phenomena.11
The synthesis of 30-aminoacylꢀ and 30-peptidylꢀRNA con-
jugates represents a serious bottleneck for such studies, and there-
fore, ourresearchprogramis devotedtotheelaboration of straight-
forward routes to achieve these derivatives.12 A solid foundation for
their chemical synthesis has been presented that relies on the assembly
of both the peptide and the RNA on the same functionalized solid
support followed by cleavage and deprotection of the whole con-
jugate.13,14 This approach is convenient for most RNA and peptide
sequences, but it suffers from certain limitations concerning the side-
chain flexibility of the peptide moiety. For instance, partial oxidation of
theN-terminalmethionines of the resistance peptideꢀRNA targets
has been a drawback of the original pathway. Moreover, attempts to
obtain arginine-containing 30-peptidylꢀRNA conjugates by this
approach have failed to date.15 One important class of 30-peptidylꢀ
RNA conjugates that frequently contain arginines function as so-called
Starting from commercially available 30-amino-20,30-dideoxy-
adenosine, we obtained support 1 in four steps in good yields
[Figure 2A; also see the Supporting Information (SI)]. Thereby,
the cysteine thiol was masked as a tert-butyl disulfide group. This
functional moiety was stable during RNA assembly and, as the
sole protecting group, remained attached under the optimized
conditions for cleavage of the conjugate from the solid support
Received: September 26, 2011
Published: November 03, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ja209053b J. Am. Chem. Soc. 2011, 133, 19068–19071
|

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Figure 3. Characterization of 30-cysteinylꢀRNA and peptide thio-
esters. (A) Anion-exchange HPLC traces of (top) crude and (inset)
purified Cys-RNAs S1 and S2 and (bottom) negative-ion-mode
LCꢀESI-mass spectra. (B) RP HPLC traces of (top) crude and (inset)
purified thioesters P2 and P3 and (bottom) positive-ion-mode ESI mass
spectra. For detailed conditions, see the SI.
a major product under conditions typically applied for NCL reactions
[involving a high concentration of urea, tris(carboxyethyl)phosphine
for S-tBu removal in situ,21 and thiophenol to form more reactive
thioesters22 in sodium phosphate buffer at pH 7.5]. Yields were
∼65% for ligations with 30-cysteinylamino-20,30-dideoxyadenosine-
based thiol donors and ∼55% for 30-cysteinylamino-30-deoxyadeno-
sine donors, which may suggest that the 20-OH is slightly disfavorable
for the ligation reaction for either steric, conformational, or mechan-
istic reasons or a combination of these. Interestingly, we found by
liquid chromatographyꢀelectrospray ionization mass spectrometry
(LCꢀESI-MS) that the major peak in the analytical anion-exchange
HPLC chromatogram corresponds to the disulfide-bridged homo-
dimer of the desired 30-leucinylcysteinylꢀRNA ligation product
(Figure 5). The dimer was formed under the conditions of the
HPLC buffer system, which contained up to 0.5 M LiClO4.
Replacement of LiClO4 by NaCl led to a significant reduction in
dimer formation. However, we considered the dimers advantageous
because of their significantly slower migration and hence facile HPLC
purification from unreacted 30-cysteinylꢀRNA, S-tBu-protected start-
ing material, and potential byproducts. Moreover, subsequent reduc-
tion of the dimers to the corresponding thiol monomers with
dithiothreitol (DTT) proceeded cleanly and in quantitative yields.
Excess DTT could be efficiently removed by flash chromatography
on a short Sep-Pak cartridge filled with reversed-phase (RP) material.
Because of the ease of handling, this synthesis/purification strategy
was applied to all 30-peptidylꢀRNAs prepared here (Table 1). These
conjugates were purified and isolated as dimers by anion-exchange
chromatography, thus resulting in very high purities as evaluated by
LCꢀESI-MS (see the SI). We point out that we currently test mildly
oxidative reagents as additives to the HPLC buffers for further
optimization of the yields for dimer formation.
Figure 2. Fragment synthesis for NCL of 30-peptidylꢀRNA conjugates.
(A) Novel solid supports 1 and 2 for the solid-phase synthesis of 30-
cysteinylamino-20,30-dideoxy- and 30-cysteinylamino-30-deoxy-RNA
(for synthetic details, see the SI). (B) Preparation of peptide thioesters
with increased solubility in aqueous solution.
and deprotection (Figure 3A). Likewise, the corresponding 20-
deoxyadenosine solid support 2 was synthesized using tert-butyl
disulfide side-chain protection [seven steps starting from readily
available 9-(30-azido-30-deoxy-β-D-arabinofuranosyl)adenine;18
Figure 2A and the SI] and yielded the corresponding S-tBu-
masked 30-cysteinyl conjugates (Figure 3A and Table 1).
Concerning the required peptide thioester fragments, we first
prepared standard benzylthioesters using commercially available
preloaded sulfamylbutyryl resins, Fmoc chemistry for peptide assem-
bly, and N-tert-butyloxycarbonyl (Boc) protection of the N-terminal
methionine. Subsequent alkylation of the sulfamyl group followed by
thiolysis and acidic deprotection provided the corresponding peptide
benzylthioesters. However, the limited solubility of the synthesized
thioesters restricted their efficient application for NCL. Therefore, to
obtain thioesters with increased solubility, we synthesized amino-
modified benzylthioesters (ABTs), which were introduced by Suga
and co-workers19 in the context of flexizyme-catalyzed aminoacylation
reactions of tRNAs. We synthesized the required N-(2-aminoethyl)-
4-mercaptomethylbenzamide along the reported lines and prepared
side-chain-protected peptide acids with an N-terminal Boc protecting
group using Fmoc chemistry on a 2-chlorotrityl chloride resin
(Figure 2B).20 Carbodiimide-promoted esterification resulted in
the desired peptide 4-(N-(2-aminoethyl)carbamoyl)benzylthio-
esters, and indeed, these novel peptide thioester derivatives were
endowed with significantly increased solubility in aqueous buffer
solutions (Figure 3B and Table 1).
Having the 30-cysteinylꢀRNA and peptideꢀABT fragments in
hand, we attempted their NCL, which finally resulted in the work-
flow illustrated in Figure 4. Starting with a 22 nucleotide (nt) S-tBu-
protected 30-cysteinylꢀRNA minihelix and a 10-fold excess of
leucineꢀABT (Figure 5), we immediately observed conversion to
For NCL of our main targets carrying pentapeptide resistance
sequences, we further optimized the ligation conditions and found
that higher buffer concentrations of 1 M Tris HCl and a pH value
3
of 8.0 were advantageous for yields and reproducibility (Figure 6).
We also tested conditions recently published by the Seitz group
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Table 1. NCL of 30-Cysteinyloligonucleotides and Amino Acid/Peptide Thioesters to 30-PeptidylꢀtRNA Mimics
30-cysteinyloligonucleotidea
peptide thioesterb
conditionsc
product conjugated
yield (%)e
Mobs (Mcalc) (amu)f
GGGUGAUUUCGAUCACCC-
ACCdA-30ꢀNH-Cys(StBu)-NH2 (S1)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
GGGUGAUUUCGAUCACCC-
ACCA-30ꢀNH-Cys(StBu)-NH2 (S2)
p-CUCCGGAACGCGCC-
LeuꢀABT (P1)
A
RNA-ACCdA-30ꢀNH-CysLeu-NH2
(C1)
65
14352.0 (14351.1)
LeuꢀABT (P1)
A
B
B
B
B
B
B
RNA-ACCA-30ꢀNH-CysLeu-NH2
55
54
72
45
66
24
51
14364.8 (14365.1)
15122.0 (15122.5)
15302.2 (15303.3)
12840.6 (12839.1)
15074.0 (15073.8)
15216.2 (15216.2)
15156.1 (15159.2)
(C2)
MFFGꢀABT (P2)
MRVWꢀABT (P3)
MLLTꢀABT (P4)
MVLWꢀABT (P5)
MRVLꢀABT (P6)
MRVWꢀABT (P3)
RNA-ACCA-30ꢀNH-CGFFM-NH2
(C3) (clarythromycin)
RNA-ACCA-30ꢀNH-CWVRM-NH2
(C4) (telithromycin)
RNA-ACCA-30ꢀNH-CTLLM-NH2
(C5) (roxythromycin)
RNA-ACCA-30ꢀNH-CWLVM-NH2
(C6) (roxythromycin)
RNA-ACCA-30ꢀNH-CLVRM-NH2
(C7) (RU64399)
RNA-UCCA-30ꢀNH-CWVRM-NH2
UCCA-30ꢀNH-Cys(StBu)-NH2 (S3)
(C8) (telithromycin)
a Oligonucleotide sequence in the 50-to-30 direction. b Peptide sequence from the N-terminus to the C-terminus. c Conditions A: 0.25 mM Sn, 2.5 mM
Pn, 0.1 M sodium phosphate (pH 7.5), 0.1 M TCEP, 2% (v/v) thiophenol. Conditions B: 0.25 mM Sn, 8 mM Pn, 7 M urea, 1 M Tris HCl (pH 8.0), 0.1
3
M TCEP, 2% (v/v) thiophenol. d Isolated as the disulfide-bridged homodimer; the macrolide antibiotic to which resistance is conferred is noted in
parentheses. Peptide sequence from the C-terminus to the N-terminus. e Determined from areas in HPLC profiles. f Molecular weights M of disulfide-
bridged homodimers as obtained using LCꢀESI ion-trap MS.
Figure 4. Workflow elaborated for NCL of 30-cysteinylꢀRNA and
peptide thioesters to obtain high-quality 30-peptidylꢀRNA conjugates.
thatrely on the soleadditive ofsodium 2-mercaptoethanesulfonate
(MESNa)23 but could not further increase the yields.
According to the strategy presented here, we synthesized a
series of RNAꢀpeptide conjugates that mimic 30-acylated tRNA
termini carrying pentapeptides associated with macrolide anti-
biotic resistance (C3ꢀC8; Table 1). One of them, arginine-
containing C8, was further successfully ligated to the chemically
synthesized 56 nt 50 fragment of Escherichia coli tRNACys using
T4 RNA ligase12 to obtain the corresponding full-length tRNAꢀ
peptide conjugate (Figure 7 left).
Figure 5. NCL of 30-cysteinylꢀRNA S1 and leucine thioester P1 to obtain
the corresponding 30-dipeptidylꢀRNA conjugate. Substantial dimerization
of the ligation products was observed. Conditions: see Table 1.
P-site.24 The CMCT modifications cause a stop when the RNA is
reverse-transcribed, and this results in a specific band on a poly-
acrylamide gel when the reverse-transcription products are separated
by electrophoresis. The right panel of Figure 7 depicts an example of a
typical polyacrylamide gel of primer extension analysis of E. coli
ribosomes exposed to tRNACys-30ꢀNH-CWVRM-NH2, C80, and
the 18 nt RNA-30ꢀNH-Cys-NH2 (S30). From the protection pattern
of U2585, it is evident that tRNACys-30ꢀNH-CWVRM-NH2 pro-
tects the P-site uridine to a comparable extent as the reference
tRNATyr while the footprint is less for the short single-stranded RNA-
pentapeptide C80. In contrast, the same RNA with a single cysteine,
S30, provided no footprint, indicating no binding to the ribosomal
P-site under the used conditions.
Furthermore, by employing ribosome chemical probing experi-
ments, we demonstrated that this peptidylꢀtRNA and the corre-
sponding tRNA 30 fragment, the 18 nt RNA-30ꢀNH-CWVRM-NH2
(C80), bind to their expected binding sites in the peptidyl transferase
center (PTC) of the ribosome. The location of interacting conjugates
can be identified through analysis of diagnostic nucleobase protections
in the 23S rRNA. P-site-bound tRNA protects characteristic nucleo-
bases from chemical modifications: in particular, modification of U2585
with 1-cyclohexyl-3-(2-(N-methylmorpholinoethyl)carbodiimide
p-toluenesulfonate (CMCT) is sensitive for tRNA binding to the
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’ ACKNOWLEDGMENT
Jasmin Morandell (Polacek lab) is thanked for contributions to the
ribosomal binding assay. Funding by the Austrian Science Foundation
FWF (P21641, I317 to R.M., Y315 to N.P.) and the Ministry of
Science and Research (GenAU III “Noncoding RNAs”; Projects
P0726-012-012 to R.M., D110420-012-012 to N.P.) is acknowledged.
’ REFERENCES
(1) Wilson, D. N.; Nierhaus, K. H. Angew. Chem., Int. Ed. 2003, 42, 3464.
(2) Darken, M. A. Pharmacol. Rev. 1964, 16, 223.
(3) Okuda, K.; Seila, A.; Strobel, S. A. Tetrahedron 2004, 60, 12101.
(4) (a) Nissen, P.; Hansen, J.; Ban, N.; Moore, P. B.; Steitz, T. A. Science
2000, 289, 920. (b) Hansen, J. L.; Ippolito, J. A.; Ban, N.; Nissen, P.; Moore,
P. B.; Steitz, T. A. Mol. Cell 2005, 20, 437. (c) Lang, K.; Erlacher, M.; Wilson,
D. N.; Micura, R.; Polacek, N. Chem. Biol. 2008, 15, 485. (d) Hiller, D. A.;
Singh, V.; Zhong, M.; Strobel, S. A. Nature 2011, 476, 236.
(5) Ramu, H.; Vꢀazquez-Laslop, N.; Klepacki, D.; Dai, Q.; Piccirilli, J.;
Micura, R.; Mankin, A. S. Mol. Cell 2011, 41, 321.
(6) (a) Schmeing, T. M.; Seila, A. C.; Hansen, J. L.; Freeborn, B.;
Soukup, J. K.; Scaringe, S. A.; Strobel, S. A.; Moore, P. B.; Steitz, T. A.
Nat. Struct. Biol. 2002, 9, 225. (b) Zhang, W.; Dunkle, J. A.; Cate, J. H.
Science 2009, 325, 1014.
Figure 6. NCL of a 30-peptidylꢀtRNA mimic with an arginine-contain-
ing resistance peptide (MRVLC). Conditions: see Table 1.
(7) (a) Fischer, N.; Konevega, A. L.; Wintermeyer, W.; Rodnina,
M. V.; Stark, H. Nature 2010, 466, 329. (b) Munro, J. B.; Altman, R. B.;
O’Connor, N.; Blanchard, S. C. Mol. Cell 2007, 25, 505.
(8) Walker, S. E.; Shoji, S.; Pan, D.; Cooperman, B. S.; Fredrick, K.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9192.
(9) (a) Clementi, N.; Chirkova, A.; Puffer, B.; Micura, R.; Polacek, N.
Nat. Chem. Biol. 2010, 6, 344. (b) Gao, Y. G.; Selmer, M.; Dunham, C. M.;
Weixlbaumer, A.; Kelley, A. C.; Ramakrishnan, V. Science 2009, 326, 694.
(10) (a) Weixlbaumer, A.; Jin, H.; Neubauer, C.; Voorhees, R. M.;
Petry, S.; Kelley, A. C.; Ramakrishnan, V. Science 2008, 322, 953.
(b) Amort, M.; Wotzel, B.; Bakowska-Zywicka, K.; Erlacher, M. D.;
Micura, R.; Polacek, N. Nucleic Acids Res. 2007, 35, 5130.
(11) (a) Vimberg, V.; Xiong, L.; Bailey, M.; Tenson, T.; Mankin,
A. S. Mol. Microbiol. 2004, 54, 376. (b) Tenson, T.; Mankin, A. S. Peptides
2001, 22, 1661.
(12) (a) Steger, J.; Graber, D.; Moroder, H.; Geiermann, A.-S.;
Aigner, M.; Micura, R. Angew. Chem., Int. Ed. 2010, 49, 7470. (b) Graber,
D.; Moroder, H.; Steger, J.; Trappl, K.; Polacek, N.; Micura, R. Nucleic
Acids Res. 2010, 38, 6796.
(13) Terenzi, S.; Biaza, E.; Nguyen-Trung, N. Q.; Strazewski, P.
Angew. Chem., Int. Ed. 2003, 42, 2909.
(14) Moroder, H.; Steger, J.; Graber, D.; Fauster, K.; Trappl, K.;
Marquez, V.; Polacek, N.; Wilson, D. N.; Micura, R. Angew. Chem., Int.
Ed. 2009, 48, 4056.
Figure 7. (left) Enzymatic ligation of the full-length tRNACys-30ꢀNH-
CWVRM-NH2 and (right) ribosomal binding assay using chemical probing
(by CMCT) and primer extension analysis of E. coli 23S rRNA. The footprint
at U2585 indicates efficient tRNAꢀpeptide conjugate binding to the ribosomal
P-site of the peptidyl transferase center (PTC). For details, see the main text.
Taken together, these results indicate that our approach creates
efficient access to hydrolysis-resistant, biologically active 30-peptidylꢀ
tRNA mimics. With the use of native chemical ligation, greater side-
chain flexibility is achieved in comparison with the previously intro-
duced route that relied exclusively on solid-phase synthesis.14 Using
this novel approach, we have obtained five resistance peptideꢀRNA
conjugates that are awaited for structural and functional ribosomal
studies to shed light on that specific antibiotic resistance phenomenon.
(15) Steger, J.; Micura, R. Bioorg. Med. Chem. 2011, 19, 5167.
(16) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776. (b) Kent, S. B. Chem. Soc. Rev. 2009, 38, 338.
(17) (a) Shang, S.; Tan, Z.; Dong, S.; Danishefsky, S. J. J. Am. Chem.
Soc. 2011, 133, 10784. (b) Rohde, H.; Schmalisch, J.; Harpaz, Z.;
Diezmann, F.; Seitz, O. ChemBioChem 2011, 12, 1396. (c) Raz, R.;
Rademann, J. Org. Lett. 2011, 13, 1606. (d) McGrath, N. A.; Raines, R. T.
Acc. Chem. Res. 2011, 44, 752. (e) Premdjee, B.; Adams, A. L.;
Macmillan, D. Bioorg. Med. Chem. Lett. 2011, 21, 4973 .
(18) Chen, Y. C. J.; Hansske, F.; Janda, K. D.; Robins, M. J. J. Org.
Chem. 1991, 56, 3410.
(19) Niwa, N.; Yamagishi, Y.; Murakami, H.; Suga, H. Bioorg. Med.
Chem. Lett. 2009, 19, 3892.
(20) Goto, Y.; Suga, H. J. Am. Chem. Soc. 2009, 131, 5040.
(21) Stetsenko, D. A.; Gait, M. J. J. Org. Chem. 2000, 65, 4900.
(22) (a) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006,
128, 6640. (b) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent,
S. B. H. J. Am. Chem. Soc. 1997, 119, 4325.
’ ASSOCIATED CONTENT
S
Supporting Information. Procedures and characteriza-
b
tion data. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
(23) Diezmann, F.; Eberhard, H.; Seitz, O. Biopolymers 2010, 94, 397.
(24) Moazed, D.; Noller, H. F. Cell 1989, 57, 585.
Corresponding Author
ronald.micura@uibk.ac.at
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