Synthesis and Biophysical Characterization of tRNALys,3 Anticodon Stem-loop RNAs Containing the mcm5s2U Nucleoside

Article abstract of DOI:10.1021/ol006605h

equation presented Phosphoramidite reagents of the naturally occurring modified nucleosides mcm⁵s²U and mcm⁵U were synthesized and along with pseudouridine were incorporated into 17-nucleotide lysine tRNA anticodon stem-lo

Full text of DOI:10.1021/ol006605h

ORGANIC
LETTERS
2000
Vol. 2, No. 24
3865-3868
Synthesis and Biophysical
Characterization of tRNALys,3 Anticodon
Stem-Loop RNAs Containing the
mcm5s2U Nucleoside
Ashok C. Bajji and Darrell R. Davis*
Department of Medicinal Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112
daVis@adenosine.pharm.utah.edu
Received September 16, 2000
ABSTRACT
Phosphoramidite reagents of the naturally occurring modified nucleosides mcm5s2U and mcm5U were synthesized and along with pseudouridine
were incorporated into 17-nucleotide lysine tRNA anticodon stem-loop domains. Standard RNA phosphoramidite coupling chemistry allowed
us to systematically investigate the thermodynamic effects of nucleoside modification and to correlate thermodynamic trends with qualitative
structure effects seen by NMR spectroscopy.
Transfer RNAs are unique among natural RNAs in that a
very high percentage of the nucleosides are chemically
modified.1,2 Despite the wide distribution of modified
nucleosides, with few exceptions, the functional role of many
of the modified nucleosides is unknown. Reviews published
in recent years describe the known biochemical roles of the
modified nucleosides,3-5 but structural studies have been
limited by the availability of suitably modified RNAs.
Natural and unnatural modified nucleosides have also been
used as tools to study RNA structure-function relationships.
Current methods and a summary of applications have been
recently reviewed.6,7 Among the uridine modifications,
2-thiouridine (s2U) and the C-5 modified 2-thiouridines are
found predominantly at the wobble position of tRNAs.1 For
s2U, it has been shown that sulfur substantially stabilizes
the 3′-endo sugar conformation at the nucleoside and
dinucleotide level.3,8-10 It has also been shown that 2-thio,5-
methyluridine (s2T) stabilizes tRNAs isolated from thermo-
philic bacteria.11-13
A “modified wobble hypothesis” has been proposed to
explain the role of 2-thiouridine and the 5-modified 2-thio-
uridines during translation.14 The modified nucleoside 5-me-
(7) Ryder, S. P.; Ortoleva-Donnelly, L.; Kosek, A. B.; Strobel, S. A.
Methods Enzymol. 2000, 317, 92-109.
(8) Agris, P.; Sierzputowska-Gracz, H.; Smith, W.; Malkiewicz, A.;
Sochacka, E.; Nawrot, B. J. Am. Chem. Soc. 1992, 114, 2652-2656.
(9) Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz, A.; Kuo, K.;
Gehrke, C.; Agris, P. J. Am. Chem. Soc. 1987, 109, 7171-7177.
(10) Smith, W. S.; Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz,
A.; Agris, P. F. J. Am. Chem. Soc. 1992, 114, 7989-7997.
(11) Watanabe, K.; Yokoyama, S.; Hansske, F.; Kasai, H.; Miyazawa,
T. Biochem. Biophys. Res. Commun. 1979, 91, 671-677.
(12) Davanloo, P.; Sprinzl, M.; Watanabe, k.; Albani, M.; Kersten, H.
Nucleic Acids Res. 1979, 6, 1571-1581.
(1) Sprinzl, M.; Horn, C.; Brown, M.; Ioudovitch, A.; Steinberg, S.
Nucleic Acids Res. 1998, 26, 148-153.
(2) Limbach, P. A.; Crain, P. F.; McCloskey, J. A. Nucleic Acids Res.
1994, 22, 2183-2196.
(3) Yokoyama, S.; Nishimura, S. In tRNA: Structure, Biosynthesis and
Function; Soll, D., RajBhandary, U., Eds.; ASM Press: Washington, DC,
1995; pp 207-224.
(4) Agris, P. F. Prog. Nucleic Acids Res. Mol. Biol. 1996, 53, 74-129.
(5) Bjo¨rk, G. R. In Transfer RNA in Protein Synthesis; Hatfield, D. L.,
Lee, B. J., Pirtle, R. M., Eds.; CRC Press: Ann Arbor, 1992; pp 23-85.
(6) Beigleman, L.; Matulic-Adamic, M.; Karpeisky, A.; Haeberli, P.;
Sweedler, D. Methods Enzymol. 2000, 317, 39-65.
(13) Yamamoto, Y.; Yokoyama, S.; Miyazawa, T.; Watanabe, K.;
Higuchi, S. FEBS 1983, 157, 95-99.
(14) Agris, P. Biochimie 1991, 73, 1345-1349.
10.1021/ol006605h CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/03/2000

thylaminomethyl-2-thiouridine (mnm5s2U) has been shown
to be important for reading frame maintenance in Escherichia
coli,15 and we have recently reported the structure of the
tRNA anticodon domain containing mnm5s2U.16 There is less
known about the eukaryotic modified nucleoside 5-car-
bomethoxymethyl-2-thiouridine (mcm5s2U) than its close
analogue mnm5s2U. An understanding of the biophysical and
structural effects of mcm5s2U is particularly important
because this nucleoside appears to be directly involved in
RNA and protein recognition during HIV-1 reverse tran-
scription initiation.17,18 The present study describes the
synthesis of protected nucleoside phosphoramidites of
mcm5s2U and 5-carbomethoxymethyluridine (mcm5U) and
describes their incorporation, along with pseudouridine, into
mixture of 2′- and 3′-TBS derivatives, which after chro-
matographic separation, isomerization of the 3′-TBS, and
subsequent chromatographic separation (repeated twice)
yielded the pure 2′-isomers 3a and 3b in 69% and 83%
yields, respectively. The 5′-DMT and 2′-TBS nucleosides
3a and 3b were then reacted with 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite for 3 h in the presence of
diisopropylethylamine with DMAP catalyst to afford phos-
phoramidites 4a and 4b in 75% and 88% yields, respectively.
The modified tRNALys,3 anticodon domain in Figure 1 was
synthesized using commercial PAC-protected phosphora-
the anticodon stem loop (ASL) domain of human tRNALys,3
.
The mcm5s2U and mcm5U nucleosides19 were prepared
in good yields by the coupling of the bis-silyl derivative of
5-carbomethoxymethyl-2-thiouracil and 5-carbomethox-
yuracil with 1-O-acetyl-2,3,5-tri-O-benzoyl-â-D-ribofuranose
following the procedure of Vorbruggen.20 The nucleoside
sugar protection and phosphoramidite synthesis follows a
standard literature protocol for RNA oligonucleotide chem-
istry (Scheme 1).21 The 5′-hydroxyl groups of 1a and 1b
Scheme 1
Figure 1. Secondary structure of the 17-nucleotide anticodon stem-
loop domain of human tRNALys,3. Structures of the modified
nucleosides at positions 34 and 39 (tRNA numbering) are shown.
The native human tRNA also has the hypermodified ms2t6A
nucleoside at position 37.
midites (Glen Research) and standard coupling chemistry21
with t-BuOOH oxidation.22-24 Trityl assays indicated >98%
coupling for all the phosphoramidites; this was verified by
HPLC and MALDI/MS of the crude deprotected RNA. The
deprotection was carried out by treating column-bound RNA
with 10% DBU in methanol for 12 h at 25 °C.25 After DBU/
methanol treatment, the RNA solution was dried and then
treated with Et3N‚3HF26 to remove the TBS ethers. The fully
deprotected RNA was then purified by HPLC,27 and MALDI-
MS was used to verify that the purified oligonucleotides had
the correct molecular weight. The presence of the correct
modified nucleosides was further verified by digesting the
RNA to mononucleosides and then analyzing the digest by
combined LC/MS28,29 (Supporting Information). The 1H
NMR spectrum in Figure 2 shows strong peaks at 13.20,
12.30, and 11.60 from the central base pairs of the stem.
were protected as their dimethoxytrityl ethers using DMT-
Cl to give compounds 2a and 2b in 80% and 68% yields,
respectively. Treatment of 2a and 2b with TBS-Cl gave a
(22) Jager, A.; Engels, J. Tetrahedron Lett. 1984, 25, 1437-1440.
(23) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986,
27, 4191-4194.
(15) Brierley, I.; Meredith, M. R.; Bloys, A. J.; Hagervall, T. G. J. Mol.
Biol. 1997, 270, 360-373.
(16) Sundaram, M.; Durant, P. C.; Davis, D. R. Biochemistry 2000, 39,
12575-12584.
(24) Kumar, R. K.; Davis, D. R. J. Org. Chem. 1995, 60, 7726-7727.
(25) Shah, K.; Wu, H.; Rana, T. M. Bioconjugate Chem. 1994, 5, 508-
512.
(17) Isel, C.; Ehresmann, C.; Keith, G.; Ehresmann, B.; Marquet, R. J.
Mol. Biol. 1995, 247, 236-250.
(26) Gasparutto, D.; Livache, T.; Bazin, H.; Dupla, A.; Guy, A.; Khordin,
A.; Molko, D.; Roget, A.; Teoule, R. Nucleic Acids Res. 1992, 20, 5159-
5166.
(27) Sproat, B.; Colonna, F.; Mulla, B.; Tsou, D.; Andrus, A.; Hampel,
A.; Vinayak, R. Nucleosides Nucleotides 1995, 14, 255-273.
(28) Crain, P. F. Methods Enzymol. 1990, 193, 782-790.
(29) Pomerantz, S. C.; McCloskey, J. A. Methods Enzymol. 1990, 193,
796-824.
(18) Isel, C.; Westhof, E.; Massire, C.; Le Grice, S. F. J.; Ehresmann,
B.; Ehresmann, C.; Marquet, R. EMBO J. 1999, 18, 1038-1048.
(19) Fissekis, J. D.; Sweet, F. Biochemistry 1970, 9, 3136-3142.
(20) Vorbruggen, H.; Strehlke, P. Chem. Ber. 1973, 106, 3039-3061.
(21) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N. Nucleic
Acids Res. 1995, 23, 2677-2684.
3866
Org. Lett., Vol. 2, No. 24, 2000

Table 1. Thermodynamic Effects of Nucleoside Modification in the Anticodon of Lysine tRNAa
tRNA ASLb
Mg (mM)
Tm (°C)
∆H° (kcal/mol)
∆S° (eu)
∆G° (kcal/mol)
∆Tm (°C)
∆∆G° (kcal/mol)
tRNALys,3
0
0
0
10
0
10
0
5
10
0
5
10
50.0 ( 0.1
55.3 ( 0.2
49.4 ( 0.2
55.8 ( 0.1
51.6 ( 0.3
55.6 ( 0.2
54.6 ( 0.1
60.2 ( 0.1
64.2 ( 0.1
55.3 ( 0.2
59.9 ( 0.3
60.4 ( 0.2
-44 ( 1
-42 ( 1
-43 ( 1
-43 ( 2
-37 ( 1
-44 ( 2
-42.8 ( 0.5
-43 ( 1
-36 ( 2
-43 ( 2
-45 ( 1
-42.2 ( 0.3
-136 ( 2
-127 ( 2
-132 ( 1
-131 ( 1
-114 ( 1
-133.8 ( 0.5
-131 ( 2
-130 ( 1
-106 ( 1
-132 ( 2
-136 ( 2
-127 ( 1
-1.8 ( 0.1
-2.3 ( 0.1
-1.6 ( 0.2
-2.4 ( 0.2
-1.7 ( 0.1
-2.4 ( 0.2
-2.3 ( 0.1
-3.2 ( 0.1
-2.8 ( 0.1
-2.4 ( 0.3
-3.2 ( 0.1
-2.9 ( 0.1
0
0
tRNALys,3
ψ
+5.3c
-0.6c
+6.4d
+1.6c
+4.0d
+4.6c
+5.6d
+9.6d
+5.3c
+4.6d
+5.1d
-0.5c
+0.2c
-0.8d
+0.1c
-0.7d
-0.5c
-0.9d
-0.6d
-0.6c
-0.8d
-0.5d
tRNALys,3 U9
tRNALys,3 U9
tRNALys,3 U7
tRNALys,3 U7
tRNALys,3 U9,ψ
tRNALys,3 U9,ψ
tRNALys,3 U9,ψ
tRNALys,3 U7,ψ
tRNALys,3 U7,ψ
tRNALys,3 U7,ψ
a Oligonucleotide stability was measured on samples containing 10 µM RNA dissolved in 10 mM sodium phosphate, pH 7.0, containing, 50 mM KCl and
50 mM NaCl. To the parent buffer was added Mg2+ in the concentration indicated. Reported values are the average of three independent measurements.b The
tRNA ASLs have the sequence shown in Figure 1 and the modifications indicated. tRNALys,3 U7 for example has the U7 (mcm5U) modification at position
34 and unmodified ribonucleosides at the other positions. c The modification effect is compared to unmodified tRNALys,3
of 5 or 10 mM MgCl2 compared to the measurements on the same oligonucleotide at 0 mM MgCl2.
.
d ∆Tm and ∆∆G° depict the effect
The far downfield shifted resonance is for the N3-H proton
of ψ39, which forms a Watson-Crick base pair with
A31.30,31 The resonance at 10.50 is the ψ39N1-H proton.
The peaks at 13.50 and 10.50 are absent in the NMR spectra
for tRNALys,3-mcm5s2U34 and tRNALys,3-mcm5U34 which do
not have the ψ39 modification (not shown). The 31P spectrum
(Figure 2b) shows a downfield-shifted phosphorus assigned
To quantitate the modification effects, temperature-de-
pendent UV spectroscopy was used to measure the melting
transition (Tm) and to derive thermodynamic parameters for
the tRNALys,3 ASL oligonucleotides. The Tm’s were measured
at pH 7.0 for tRNALys,3 ASLs containing different combina-
tions of three naturally occurring modified nucleosides
mcm5s2U34, mcm5U34, and ψ39. The modification effects
were compared with data obtained previously on unmodified
tRNALys.3 and tRNALys,3-ψ39 (Table 1).
A comparison of unmodified tRNALys,3, tRNALys,3-ψ39,
tRNALys,3-mcm5s2U34, and tRNALys,3-mcm5s2U34,ψ39 shows
that the wobble modification has a minimal effect on the
overall thermodynamic stability. This indicates that although
the mcm5s2U modification stabilizes the U-turn as seen in
the E. coli tRNA, any overall gain in base stacking within
the loop has a minimal global thermodynamic effect. In
contrast, the stabilization in base stacking upon ψ39 modi-
fication is substantial (∼5 °C Tm increase) as has been seen
in other tRNA ASLs.16,30,35 Furthermore, a comparison of
mcm5U34- and mcm5s2U34-modified RNAs serves to em-
phasize that the sulfur has little effect on thermodynamic
stability. However, the 31P NMR spectra clearly show that
sulfur modification in combination with the side chain is
necessary to affect the RNA backbone conformational change
reflected by the downfield-shifted 33p34 resonance. The
backbone conformation is also stabilized by Mg2+ as shown
by a further downfield shift in the 31P NMR resonance when
going from 0 to 10 mM MgCl2 (not shown).
Figure 2. NMR spectra of the human tRNALys,3 ASL in Figure 1,
containing mcm5s2U34, ψ39, and unmodified nucleosides at the
remaining sequence locations. NMR spectra were acquired at 500
MHz on a 2 mM RNA sample in 10 mM phosphate buffer, pH
1
7.4, containing 50 mM NaCl, 50 mM KCl, at 5 °C: (a) H NMR
spectrum of the downfield region; (b) 31P NMR spectrum.
A noticeable difference is seen in the effect Mg2+ has on
the Tm’s for tRNALys,3-mcm5U34,ψ39 and tRNALys,3-mcm5
s2U34,ψ39. The sulfur-modified tRNALys,3 ASL is the only
case where we have seen a substantial increase in Tm upon
to the U33-p-mcm5s2U34 phosphate.16,32-34 There was no
downfield-shifted phosphorus observed for tRNALys,3-mcm5-
U34,ψ39, indicating that the 2-thio modification has a similar
effect on the anticodon structure of the human tRNALys,3 as
(32) Jucker, F. M.; Pardi, A. RNA 1995, 1, 219-222.
(33) Legault, P.; Pardi, A. J. Magn. Reson. B 1994, 103, 82-86.
(34) Schweisguth, D. C.; Moore, P. B. J. Mol. Biol. 1997, 267, 505-
519.
that found for mnm5s2U34 in the E. coli tRNALys 16
.
(30) Durant, P. C.; Davis, D. R. J. Mol. Biol. 1999, 285, 115-131.
(31) Sundaram, M.; Crain, P. F.; Davis, D. R. J. Org. Chem. 2000, 65,
5609-5614.
(35) Yarian, C. S.; Basti, M. M.; Cain, R. J.; Ansari, G.; Guenther, R.
H.; Sochacka, E.; Gzerwinska, G.; Malkiewicz, A.; Agris, P. F. Nucleic
Acids Res. 1999, 27, 3543-3549.
Org. Lett., Vol. 2, No. 24, 2000
3867

increasing the Mg2+ concentration from 5 to 10 mM. This
effect is significant and may indicate that the conformational
change caused by sulfur modification provides a discrete
millimolar KD binding site for Mg2+ which stabilizes the
tRNALys,3 anticodon structure.
We have described an efficient approach for the synthesis
of the mcm5s2U and mcm5U nucleoside phosphoramidites.
The synthetic methodology allowed for facile integration of
these nucleosides into tRNALys,3 model oligonucleotides, and
we established deprotection conditions enabling us to obtain
quantities of modified RNA necessary for biophysical and
structural investigation. The presence of mcm5s2U at the
tRNA wobble position, along with ψ at position 39,
significantly alters the local structure and the thermodynamic
stability of these model oligonucleotides. The versatile
synthetic protocol will enable us to pursue high-resolution
structural studies and to investigate the biochemical effects
of nucleoside modification in tRNALys,3. These synthetic tools
will aid in our investigations to understand the established
role of nucleoside modification in the HIV-1 transcription
initiation complex.
Acknowledgment. This research was supported by the
NIH (AI/GM55508). NMR instrumentation was obtained in
part with funds from the NIH and the NSF (RR06262,
RR13030, DBI-0002806). MS and oligonucleotide facilities
are supported by NIH Grant CA42014.
Supporting Information Available: HPLC traces, MAL-
DI-MS spectra, and LC/MS nucleoside analysis for modified
oligonucleotides. Experimental procedures and 1H NMR data
for nucleosides and phosphoramidites. This material is
available free of charge via the Internet at http://pubs.acs.org.
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