´
M. Simonovic, R. Micura et al.
(N,N-diisopropyl)phosphoramidite[29,35] and following a standard synthesis
coupling cycle that lacked the detritylation step. For details see “RNA
synthesis”.
tended to methylated and/or phosphorylated peptidyl–
tRNA conjugates. Also, the serine analogue Abu(p), intro-
duced as a functional part of an adenosine-modified solid
support, could be used as an amino acid building block
(Fmoc-Abu(pAll2)) for incorporation at internal positions of
the peptidyl moiety.[22] Finally, our work included the devel-
opment of an efficient one-pot, three-strand tRNA ligation
protocol for tRNASec, which is generally applicable to other
tRNA species containing a long variable loop.
RNA synthesis: All oligonucleotides were synthesized on an ABI 392
Nucleic Acid Synthesizer following standard synthesis protocols. Detrity-
lation (120 s): dichloroacetic acid/1,2-dichloroethane (4:96); coupling
(120 s): phosphoramidites (0.1m in acetonitrile, 130 mL) were activated
with benzylthiotetrazole (0.3m in acetonitrile, 180 mL); capping (2ꢁ10 s,
Cap A/Cap B=1:1): Cap A: phenoxyacetic anhydride (0.2m in THF),
Cap B: N-methyl imidazole (0.2m), sym-collidine (0.2) in THF; oxidation
(20 s): I2 (0.2m) in THF/pyridine/H2O (35:10:5). Amidites, benzylthiote-
trazole, and capping solutions were dried over activated molecular sieves
(4 ꢂ) overnight.
We have prepared structurally complex tRNASec mimics
that carry modified amino acid moieties, which may, inter-
fere with discrete steps in the biosynthetic cycle of seleno-
cysteine. Because the aminoacyl moieties are attached to
tRNASec through a hydrolysis-resistant linkage, the new
mimics are attractive for high-resolution crystallographic
studies of the unexplored catalytic mechanism of SepSecS
and interactions between the active site of SepSecS and the
5’-end of Sep-tRNASec. Finally, a similar approach could be
utilized in studying other tRNA-dependent enzymes includ-
ing the ribosome.
Deprotection of 3’-aminoacylamino-RNA conjugates
Allyl deprotection: Conjugates synthesized on solid support 10 were
treated with a solution of N-methyl morpholine (37 mL, 0.34 mmol) and
acetic acid (37 mL, 65 mmol) in chloroform (1 mL). After addition of tet-
rakis(triphenylphosphine) palladium(0) (12 mg, 0.01 mmol), the suspen-
sion was agitated for 5 h at room temperature. Subsequently, the solid
support was collected on a Bꢃchner funnel, washed with chloroform and
dried under vacuum.
Acyl deprotection and cleavage from the solid support: For conjugates
synthesized on solid support 4, 5, 11, and 12 and for conjugates synthe-
sized on solid support 10 after allyl deprotection (see above), the beads
were transferred into an Eppendorf tube and equal volumes of methyla-
mine in ethanol (8m, 0.5 mL) and methylamine in H2O (40%, 0.5 mL)
were added. After 6 h shaking at room temperature the supernatant was
filtered and evaporated to dryness.
Experimental Section
2’-O-TOM deprotection: The obtained residue was treated with
TBAF·3H2O in THF (1m, 1 mL) overnight at room temperature. The re-
action was quenched by the addition of triethylammonium acetate
(TEAA) (1m, pH 7.4, 1 mL). After reducing the volume of the solution,
it was applied on a size-exclusion chromatography column (GE Health-
care, HiPre 26/10 Desalting, 2.6ꢁ10 cm, Sephadex G25). By eluating with
H2O, the conjugate-containing fractions were collected, evaporated to
dryness, and the residue was dissolved in H2O (1 mL). Analysis of the
crude products was performed by anion-exchange chromatography on a
Dionex DNAPac PA-100 column (4ꢁ250 mm) at 608C. Flow rate:
For the synthesis of the functionalized solid supports 4, 5, and 10 as well
as amino acid derivative 9 see the Supporting Information.
Na-Methylation on solid support
Fmoc deprotection: The solid supports 4, 5, or 10 (30 mg) were suspended
in acetonitrile for 1 min, followed by treatment with piperidine (20% sol-
ution in acetonitrile, 2 mL) for 10 min, and again for 5 min with fresh sol-
ution. Then, the beads were washed with acetonitrile and dried under
vacuum.
NBS activation: The deprotected supports were suspended in a solution
of 2-nitrobenzenesulfonyl chloride (150 mg, 0.68 mmol) and sym-collidine
(98 mL, 0.72 mmol) in CH2Cl2 (1 mL) and agitated for 2 h at room tem-
perature. The beads were washed with CH2Cl2 and dried.
1 mLminÀ1
; eluent A: 25 mm Tris·HCl (pH 8.0), 6m urea; eluent B:
25 mm Tris·HCl (pH 8.0), 0.5m NaClO4, 6m urea; gradient: 0–60% B in
A within 45 min or 0–40% B in A within 30 min for short sequences up
to fifteen nucleotides, UV detection at l=260 nm.
N-Methylation: The beads were swelled in DMF and subsequently treat-
ed with a solution of MTBD (10 mL, 0.07 mmol) and methyl-4-nitroben-
zene sulfonate (18 mg, 0.08 mmol) in DMF (1 mL) for 30 min. The solu-
tion was discharged and the solid support was washed with DMF for
direct use in the next step.
Purification of 3’-aminoacylamino-RNA conjugates: The crude RNA
products were purified on a semipreparative Dionex DNAPac PA-100
column (9ꢁ250 mm) at 608C with flow rate of 2 mLminÀ1 (for eluents
see above). Fractions containing the conjugate were loaded on a C18
SepPak Plus cartridge (Waters/Millipore), washed with 0.1–0.15m
(Et3NH)+HCO3À, H2O, and eluted with H2O/CH3CN (1:1). Conjugate-
containing fractions were evaporated to dryness and dissolved in H2O
(1 mL). The quality of the purified conjugate was analyzed by analytical
anion-exchange chromatography (for conditions see above). The molecu-
lar weight of all synthesized RNAs was confirmed by LC-ESI mass spec-
trometry (see the Supporting Information). Yields were determined by
UV photometrical analysis of conjugate solutions.
NBS cleavage: The beads were incubated with a solution of 2-mercaptoe-
thanol (18 mL, 0.26 mmol) and 1,5-diazabicycloACTHNUTRGENUG[N 5.4.0]undec-5-ene (DBU)
(16 mL, 0.11 mmol) in DMF (1 mL) for 30 min. During that time, the sol-
ution turned yellow. The beads were washed and a second treatment with
2-mercaptoethanol (9 mL, 0.13 mmol) and DBU (8 mL, 0.06 mmol) in
DMF (1 mL) for 1 min showed no color reaction and confirmed that
cleavage was complete. The beads were washed with DMF and acetoni-
trile, and finally dried under vacuum.
Enzymatic ligation of tRNASec: Equimolar amounts of three chemically
Fmoc protection: Sodium carbonate (100 mg) was dissolved in H2O/diox-
ane (1:1, 2 mL), followed by addition of the beads. After 15 min, Fmoc
N-hydroxysuccinimide ester (3 mg, 0.08 mmol) was added and the sus-
pension was agitated overnight at room temperature. The beads were
washed with water, acetonitrile, and CH2Cl2 and dried under vacuum.
synthesized tRNA fragments (see Figure 2 for sequence, Figure 4 for 5’-
phosphate modification, and Table 2) were dissolved in water (2= of the
3
final total reaction volume, final RNA concentration of 40 mm for each
strand). One tenth of the final reaction volume of 10ꢁ ligation buffer
(10 mm adenosintriphosphate (ATP), 500 mm 4-(2-hydroxyethyl)-1-piper-
azineehtanesulfonic acid (HEPES)-NaOH, 100 mm MgCl2, and 100 mm
dithiothreitol (DTT)) was added. The solution was heated to 908C for
5 min and then allowed to cool to room temperature within 3 h. Then,
bovine serum albumin (BSA) stock solution (1 mgmLÀ1) was added (Fer-
mentas). The reaction mixture was vortexed and centrifuged before it
was treated with T4 RNA ligase (0.4 UmLÀ1, Fermentas, 10 UmLÀ1 in
storage buffer). Final concentrations: 40 mm for each RNA strand, 1 mm
ATP, 50 mm HEPES-NaOH (pH 8.0), 10 mm MgCl2, 10 mm DTT,
O-Phosphorylation on solid support
TBDMS deprotection: After swelling of the solid support 4, 5, 11, or 12
(30 mg) in THF (1 mL) for 10 min, it was treated with a solution of
TBAF·3H2O (1.0m) and acetic acid (0.5m) in THF (1 mL) for 30 min at
room temperature. Then, the beads were washed with THF and CH2Cl2
and dried.
O-Phosphorylation: Phosphorylation was performed in an automated
manner on an oligonucleotide synthesizer by using bis(2-cyanoethyl)-
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