2′-mercaptonucleotide interference reveals regions of close packing within folded RNA molecules

Article abstract of DOI:10.1021/ja035175y

The 2′-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2′-hydroxyl group, we synthesized 2′-mercaptonucleotides for use in nucle

Full text of DOI:10.1021/ja035175y

Published on Web 07/29/2003
2′-Mercaptonucleotide Interference Reveals Regions of Close
Packing within Folded RNA Molecules
Jason P. Schwans, Cecilia N. Cortez, Joe M. Olvera, and Joseph A. Piccirilli*
Contribution from the Howard Hughes Medical Institute, Departments of Biochemistry and
Molecular Biology, and Chemistry, The UniVersity of Chicago, Chicago, Illinois 60637
Received March 15, 2003; E-mail: jpicciri@midway.uchicago.edu
Abstract: The 2′-hydroxyl group makes essential contributions to RNA structure and function. As an
approach to assess the ability of a mercapto group to serve as a functional analogue for the 2′-hydroxyl
group, we synthesized 2′-mercaptonucleotides for use in nucleotide analogue interference mapping. To
correlate the observed interference effects with tertiary structure, we used the independently folding ∆C209
P4-P6 domain from the Tetrahymena group I intron. We generated populations of ∆C209 P4-P6 molecules
containing 2′-mercaptonucleotides located randomly throughout the domain and separated the folded
molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of
the RNA molecules revealed the sites at which 2′-mercaptonucleotides interfere with folding. These
interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites
that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2′-
mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed
regions within folded RNA molecules.
Introduction
ability of the mercapto group to serve as a functional analogue
for the 2′-hydroxyl group remains largely unexplored.7 Just what
Sulfur substitution endows nucleic acids with unique phys-
iochemical properties that empower biological chemists to
elucidate fundamental features of nucleic acid structure and
function. Virtually every oxygen atom on the nucleobases, sugar,
and phosphoryl group has succumbed to sulfur modification.1-3
Nevertheless, substitution of the 2′-hydroxyl group in RNA by
a mercapto (SH) group has received relatively little attention,
despite the potential of 2′-mercaptonucleosides as probes for
the role of the 2′-hydroxyl group in RNA.4 2′-Mercaptonucleo-
sides were first synthesized several decades ago, but most
investigations of their properties have focused on the nucleoside
or nucleotide level.5 Although strategies for their incorporation
into RNA oligonucleotides have become available recently,6 the
features of RNA structure and function could 2′-mercapto-
nucleotides expose and how effectively can they mimic natural
nucleotides in mediating tertiary interactions? To address these
questions, we synthesized 2′-mercapto-R-thiotriphosphate ana-
logues of adenosine, cytidine, guanosine, and uridine (Chart 1)
for use in nucleotide analogue interference mapping.8 To
evaluate the structural and energetic basis of 2′-mercapto-
nucleotide interference, we used as a model system the
structurally and biochemically well-defined P4-P6 independently
folding RNA domain.9 We obtained a “macromolecular profile”
(4) For examples of the role of the 2′-hydroxyl group in RNA structure and
function, see the following and references therein: (a) Saenger, W.
Principles of Nucleic Acid Structure. Springer AdVanced Text in Chemistry;
Springer-Verlag: New York, 1984. (b) Pyle, A. M.; Cech, T. R. Nature
1991, 350, 628. (c) Abramovitz, D. L.; Friedman, R. A.; Pyle, A. M. Science
1996, 271, 1410. (d) Egli, M.; Portmann, S.; Usman, N. Biochemistry 1996,
35, 8489. (e) Strobel, S. A.; Doudna, J. A. Trends Biochem. Sci. 1997, 22,
26. (f) Sjogren, A.-S.; Pettersson, E.; Sjoberg, B.-M.; Stromberg, R. Nucleic
Acids Res. 1997, 25, 648. (g) Batey, R. T.; Rambo, R. P.; Doudna, J. A.
Angew. Chem. 1999, 38, 2327. (h) Shan, S.; Yoshida, A.; Sun, S.; Piccirilli,
J. A.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12299. (i)
Gordon, P. M.; Sontheimer, E. J.; Piccirilli, J. A. Biochemistry 2000, 39,
12939. (j) Doherty, E. A.; Batey, R. T.; Masquida, B.; Doudna, J. A. Nat.
Struct. Biol. 2001, 8, 339.
(5) (a) Ryan, K. J.; Acton, E. M.; Goodman, L. J. Org. Chem. 1971, 36, 2646.
(b) Imazawa, M.; Ueda, T.; Ukita, T. Chem. Pharm. Bull. 1975, 23, 604.
(c) Patel, A. D.; Schrier, W. H.; Nagyvary, J. J. Org. Chem. 1980, 45,
4830. (d) Divakar, K. J.; Mottoh, A.; Reese, C. B.; Sanghvi; Y. S. J. Chem.
Soc., Perkin Trans. I 1990, 969. (e) Marriott, J. H.; Mottahedeh, M.; Reese,
C. B. Carbohydr. Res. 1991, 216, 257. (f) Dantzman, C. L.; Kiessling, L.
L. J. Am. Chem. Soc. 1996, 118, 11715.
(1) For examples of sulfur modification in the nucleobase, see the following
and references therein: (a) Sontheimer, E. J. Mol. Biol. Rep. 1994, 20, 35.
(b) Earnshaw, D. J.; Gait, M. J. Biopolymers 1998, 48, 39. (c) Yu, Y.-T.
Methods Enzymol. 2000, 318, 71. (d) Favre, A.; Saintome, C.; Fourrey,
J.-L.; Clivio, P.; Laugaa, P. J. Photochem. Photobiol., B 2000, 42, 109.
(2) For examples of sulfur modification in the sugar, see the following and
references therein: (a) Bellon, L.; Morvan, F.; Barascut, J. L.; Imbach, J.
L. Biochem. Biophys. Res. Commun. 1992, 184, 797. (b) Piccirilli, J. A.;
Vyle, J. S.; Caruthers, M. H.; Cech, T. R. Nature 1993, 361, 85. (c) Zhou,
D.-M.; Usman, N.; Wincott, F. E.; Matulic-Adamic, J.; Orita, M.; Zhang,
L.-H.; Komiyama, M.; Kumar, P. K. R.; Taira, K. J. Am. Chem. Soc. 1996,
118, 5862. (d) Kuimelis, R. G.; McLaughlin, L. W. Biochemistry 1996,
35, 5308. (e) Hancox, E. L.; Walker, R. T. Nuclosides Nucleotides 1996,
15, 135. (f) Weinstein, L. B.; Earnshaw, D. J.; Cosstick, R.; Cech, T. R. J.
Am. Chem. Soc. 1996, 118, 10341. (g) Thomson, J. B.; Patel, B. K.; Jimenez,
V.; Eckart, K.; Eckstein, F. J. Org. Chem. 1996, 61, 6273.
(3) For examples of phosphorothioate modifications, see the following and
references therein: (a) Eckstein, F. Angew. Chem. 1983, 95, 431. (b)
Eckstein, F. Annu. ReV. Biochem. 1985, 54, 367. (c) Ruffner, D. E.;
Uhlenbeck, O. C. Nucleic Acids Res. 1990, 18, 6025. (d) Verma, S.;
Eckstein, F. Annu. ReV. Biochem. 1998, 67, 99. (e) Shan, S.; Kravchuk, A.
V.; Piccirilli, J. A.; Herschlag, D. Biochemistry 2001, 40, 5161. (f) Eckstein,
F. Biochimie 2002, 84, 841. (g) Huppler, A.; Nikstad, L. J.; Allmann, A.
M.; Brow, D. A.; Butcher, S. E. Nat. Struct. Biol. 2002, 9, 431.
(6) (a) Hamm, M. L.; Piccirilli, J. A. J. Org. Chem. 1997, 62, 3415. (b) Raines,
K. R.; Gottlieb, P. A. RNA 1998, 4, 340.
(7) (a) Earnshaw D. J.; Hamm, M. L.; Piccirilli, J. A.; Karpeisky, A.;
Biegelman, L.; Ross, B. S.; Monoharan, M.; Gait, M. J. Biochemistry 2000,
39, 6410. (b) Hamm, M. L.; Schwans, J. P.; Piccirilli, J. A. J. Am. Chem.
Soc. 2000, 122, 4223. (c) Hamm, M. L.; Nikolic, D.; van Breemen, R. B.;
Piccirilli, J. A. J. Am. Chem. Soc. 2000, 122, 12069.
9
10012
J. AM. CHEM. SOC. 2003, 125, 10012-10018
10.1021/ja035175y CCC: $25.00 © 2003 American Chemical Society

2′-Mercaptonucleotide Interference within RNA Molecules
Chart 1. 2′-Mercaptonucleoside-R-thiotriphosphates
A R T I C L E S
mobility of the domain relative to a permanently unfolded
control molecule9a quantitatively describes the folding transition.
The apparent standard free-energy change associated with the
midpoint of the tertiary folding transition is given by ∆G°′ )
13
nRT ln[Mg2+
]
1/2
,
where n represents the experimentally
determined Hill coefficient and [Mg2+
]
gives the folding
1/2
midpoint, the magnesium concentration at which the P4-P6
molecules equally populate the folded and unfolded states. For
showing that 2′-mercaptonucleotides reveal regions of close
packing and structural rigidity within folded RNA molecules.
∆C209 P4-P6, n equals 4 and [Mg2+
]
1/2
equals 0.45 mM
MgCl2.9h
Results and Discussion
We allowed the population of ∆C209 P4-P6 to fold19 and
electrophoresed them through a nondenaturing polyacrylamide
gel.9d,f We excised those species with the same mobility as
folded native ∆C209 P4-P6. Exposure of these selected RNA
molecules to I2 induces cleavage at all of the phosphorothioate
linkages, revealing the location of 2′-mercaptonucleotides within
the RNA.16 Gaps in the resulting cleavage ladder as compared
to the phosphorothioate control reveal sites at which 2′-
mercaptonucleotides interfere with the tertiary folding transition
(Figure 1).8,9d,f To achieve maximal sensitivity to folding
interference without compromising signal, we conducted the gel
Raines and Gottlieb synthesized 2′-mercaptocytidine tri-
phosphate using POCl3 phosphorylation of 2′-deoxy-2′-(2-
nitrophenyldithio)cytidine followed by reaction with pyrophos-
phate and subsequent reduction of the disulfide.6b We could not
generate the corresponding R-thiotriphosphates using analogous
reaction conditions with PSCl3, however. Instead, we prepared
the R-thiotriphosphates from the more stable 2′-tert-butyldi-
sulfenyl10 nucleosides using a one-pot/two-step reaction se-
quence with pyridine as both solvent and activating agent.11
Subsequent reduction of the disulfide with DTT provided the
desired 2′-mercaptonucleoside R-thiotriphosphates. We chose
the ∆C209 P4-P6 domain12 from the Tetrahymena ribozyme
as the system with which to challenge 2′-mercaptonucleotides.9
P4-P6 represents one of the largest RNAs to date whose folding
has been characterized thermodynamically,13 kinetically,14 and
crystallographically.9b,c,h We incorporated the analogues ran-
domly throughout the ∆C209 P4-P6 domain by in vitro
transcription using the mutant Y639F T7 RNA polymerase.6b,15
I2 sequencing16 of the population revealed that the polymerase
incorporated the analogues evenly throughout the RNA transcript
at a level of approximately 1-5 substitutions per molecule (data
not shown).9b,g We established that the 2′-SH groups remain
intact following polyacrylamide gel electrophoresis (PAGE)
purification and isolation of the RNA.17,18
selection experiments at [Mg2+
]
1/2
(0.45 mM MgCl2). Under
these conditions, the gel mobility shift assay detects energetic
effects as small as 0.2 kcal/mol.20
2′-Mercaptonucleotide interference occurs at 24 of the 140
possible sites in the ∆C209 P4-P6 domain (Figure 2, red and
blue bars). Little interference occurs in simple duplex regions,
suggesting that the minor groove of A-form helical RNA readily
accommodates the bulky mercapto group. Instead, the interfer-
ences occur primarily in the single-stranded regions of ∆C209
P4-P6, spanning the entire secondary structure of the domain.
Elevated Mg2+ concentration (2.0 mM Mg2+, Figure 2B)
attenuates most of the interferences, underscoring the need to
conduct these experiments near the midpoint of the folding
transition. This Mg2+-induced signal attenuation reveals an upper
limit for the energetic destabilization imposed by the mercapto
The P4-P6 domain undergoes a tertiary folding transition in
a Mg2+-dependent manner (eq 1).9 The folding transition renders
group (∆∆G°′ ) -nRT ln([0.45 mM Mg2+]/[2.0 mM Mg2+
) 3.5 kcal/mol).21
]
the domain more compact such that folded molecules (F‚nMg2+
)
migrate faster than unfolded molecules (U) during nondenaturing
PAGE.9,13
To explore the physical basis of 2′-mercaptonucleotide
interference, we obtained for comparison a 2′-deoxynucleotide
(2′-H) interference map, which allows us to classify each residue
in ∆C209 P4-P6 according to its 2′-SH/2′-H interference profile.
Deoxynucleotide interference functionally distinguishes residues
bearing dispensable 2′-hydroxyl groups from those bearing 2′-
U + nMg2+ h F‚nMg2+
(1)
The Mg2+ concentration dependence of the electrophoretic
(8) (a) Waring, R. B. Nucleic Acids Res. 1989, 17, 10281. (b) Christian, E. L.;
Yarus, M. J. Mol. Biol. 1992, 228, 743. (c) Strobel, S. A.; Shetty, K. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 2903. (d) Ortoleva-Donnelly, L.;
Szewczak, A. A.; Gutell, R. R.; Strobel, S. A. RNA 1998, 4, 498. (e) Strobel,
S. A. Curr. Opin. Struct. Biol. 1999, 9, 346. (f) Ryder, S. P.; Strobel, S. A.
Methods 1999, 1, 38. (g) Ryder, S. P.; Ortoleva-Donnelly, L.; Kosek, A.
B.; Strobel, S. A. Methods Enzymol. 2000, 317, 92 and references therein.
(9) (a) Murphy, F. L.; Cech, T. R. Biochemistry 1993, 32, 5291. (b) Cate, J.
H.; Gooding, A. R.; Podell, E.; Zhou, K.; Golden, B. L.; Kundrot, C. E.;
Cech, T. R.; Doudna, J. A. Science 1996, 273, 1687. (c) Cate, J. H.; Doudna,
J. A. Structure 1996, 4, 1221. (d) Cate, J. H.; Hanna, R. L.; Doudna, J. A.
Nat. Struct. Biol. 1997, 4, 553. (e) Szewczak, A. A.; Cech, T. R. RNA
1997, 3, 838. (f) Basu, S.; Strobel, S. A. RNA 1999, 5, 1399. (g) Juneau,
K.; Cech, T. R. RNA 1999, 5, 1119. (h) Juneau, K.; Podell, E.; Harrington,
D. J.; Cech, T. Structure 2001, 9, 221.
(15) Sousa, R.; Padilla, R. EMBO J. 1995, 14, 4609.
(16) Gish, G.; Eckstein, F. Science 1988, 240, 1520.
(17) Bonaventura, C.; Bonaventura, J.; Stevens, R.; Millington, D. Anal.
Biochem. 1994, 222, 44.
(18) The following data establish the integrity of the mercapto group: (1)
Following purification by DTT PAGE (see Experimental Section), a
synthetic oligonucleotide containing a single 2′-mercaptocytidine residue
reacted quantitatively with Thiolyte MQ, (2) transcription of pppGAUGGC
in the presence of 2′-mercaptouridine triphosphate and no UTP generates
a transcript that reacts quantitatively with Thiolyte MQ, and (3) incubation
with biotin-PEG3-maleimide followed by streptavidin shifted the electro-
phoretic mobility of the entire population of ∆C209 molecules (data not
shown).
(10) Pastuszak, J. J.; Chimiak, A. J. Org. Chem. 1981, 46, 1868.
(11) Fisher, B.; Chulkin, A.; Boyer, J. L.; Harden, K. T.; Gendron, F.-P.;
Beaudoin, A. R.; Chapal, J.; Hillaire-Buys, D.; Petit, P. J. Med. Chem.
1999, 42, 3636.
(19) Folding conditions: 50 °C for 10 min followed by slow cooling to room
temperature; Tris-Borate pH 8.3; 5% glycerol; MgCl2 as indicated. Under
these conditions, the gel mobility shift assay gives the same n value and
[Mg2+
]
as reported by Juneau et al.9h
1/2
(12) ∆C209 has the same sequence and global fold as the natural P4-P6 domain
(20) Site-specific substitution of A183 with 2′-deoxyadenosine destabilized P4-
P6 tertiary folding by 0.2 kcal/mol.13 The gel mobility shift assay detects
2′-deoxyadenosine interference at position 183.
but lacks cytidine 209.9g,h
(13) (a) Silverman, S. K.; Cech, T. R. Biochemistry 1999, 38, 8691. (b)
Silverman, S. K.; Cech, T. R. RNA 2001, 7, 161.
(21) We assume here that the individual mercaptan-modified RNA molecules
undergo a Mg2+-dependent, two-state folding transition with the same n
value as the unmodified ∆C209 P4-P6.
(14) Silverman, S. K.; Deras, M. L.; Woodson, S. A.; Scaringe, S. A.; Cech, T.
R. Biochemistry 2000, 39, 12465.
9
J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003 10013

A R T I C L E S
Schwans et al.
Figure 1. RNA folding interferences induced by nucleotide analogues.
Shown are representative sequencing ladders of ∆C209 P4-P6 populations
containing phosphorothioates at cytidine (CRS), 2′-deoxycytidine (dC), or
2′-mercaptocytidine (CSH) residues. Gaps in the I2-induced cleavage pattern
of the folded population reveal the sites of interference. The unselected
lane (Uns) gives the extent and location of analogue incorporation during
transcription and indicates an even distribution of the analogue throughout
the RNA. Arrows indicate sites of interference: 121, 127, 137, 154, 197,
222-23. Shorter electorophoresis times retain the smaller RNA fragments
and reveal interference at C109 (data not shown). As a control for degra-
dation, we also analyzed samples without I2 incubation (data not shown).
Figure 2. ∆C209 P4-P6 secondary structure with histogram indicating the
sites of 2′-H and 2′-SH interference at (A) 0.45 mM and (B) 2.0 mM MgCl2.
Red bars represent sites that exhibit interference with both analogues. Blue
bars represent sites that exhibit 2′-SH interference uniquely. The green bar
represents a site that exhibits 2′-H interference uniquely. Gray bars represent
sites that exhibit phosphorothioate interference, which masks possible
interference from the 2′-modification. Interference values were calculated
as described by Ryder et al.8g
hydroxyl groups that provide a significant energetic contribution
to folding (Figure 2, red and green bars). All deoxynucleotide
interferences in P4-P6 occur at residues whose 2′-hydroxyl
groups engage in hydrogen bonds, as was inferred from the
crystal structure.9b,h Among these sites, only one exhibits no
2′-SH interference (Figure 2A, green bar): tetraloop residue
G150, whose 2′-OH donates a hydrogen bond to N7 of A152.
The absence of 2′-SH interference here indicates that a mercapto
group at this site functionally mimics the hydrogen bonding
interaction of the 2′-hydroxyl group. Although mercaptans form
considerably weaker hydrogen bonds than do hydroxyl groups,22
this interaction could provide a favorable thermodynamic
contribution to folding as mercaptans undergo desolvation with
considerably smaller energetic penalty than do hydroxyl
groups.23,24 The remaining sites of 2′-deoxynucleotide interfer-
ence (Figure 2, red bars) exhibit 2′-SH interference, indicating
that generally the 2′-mercapto group lacks the ability to supplant
these important 2′-hydroxyl groups. Apparently, the spatial and
geometric constraints imposed by the P4-P6 structure preclude
effective hydrogen bonding with the mercapto group. 2′-SH
interference at sites bearing dispensable 2′-hydroxyl groups
(Figure 2, blue bars) must arise as a consequence of the sulfur
atom rather than from disruption of a hydrogen bond. Interest-
ingly, these sites flank the 2′-H/2′-SH interferences (Figure 2
and see below), suggesting that the sulfur may interfere by
disrupting nearby tertiary interactions. When modeled into the
∆C209 P4-P6 structure, the sulfur atoms at all sites of 2′-SH
interference penetrate the van der Waals volumes of nearby
atoms (Figure 3). Presumably, the folded structure cannot
accommodate the large sulfur atom without energetic penalty
from short-range repulsions. Conversely, no such van der Waals
overlap occurs at the sites that exhibit no 2′-SH interference.
To provide a global view of the 2′-hydroxyl groups within
P4-P6, we modeled a sulfur atom at every residue in the space-
filling representation of the molecule (Figure 4A).9b,h Most of
(22) Crampton, M. R. In Chemistry of the -SH Group; Pati, S., Ed.; Wiley-
Interscience: New York, 1974; p 379.
(23) Fersht, A. Trends Biochem. Sci. 1987, 12, 301 and references therein.
(24) The thermodynamic contribution of these hydrogen bonds to the folded
state of the RNA reflects an energy balance between the intrinsic strength
of each hydrogen bond and the desolvation energy of the interacting
partners. 2′-SH interference therefore reflects an unfavorable hydrogen bond
exchange reaction between the hydrogen bonding partners and water in
going from the unfolded to the folded state.
9
10014 J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003

2′-Mercaptonucleotide Interference within RNA Molecules
A R T I C L E S
Conclusions
We have used the plethora of microenvironments offered by
a macromolecule of known structure to explore an atomic
modification. Steric interactions dominate the response of RNA
to 2′-mercaptonucleotide substitution, whereby the mercapto
group acts as a 35 Å3 sphere (a hydroxyl group occupies 13
Å3)25 that disrupts folding when the local environment lacks
the flexibility and space to accommodate it. Consequently, 2′-
mercaptonucleotides provide a method by which to expose
structurally rigid and densely packed regions of folded RNA
molecules.
Figure 3. A structural basis for 2′-mercaptonucleotide interference. (A)
Space-filling representation of residues C197 and G200 located in the hinge
region. C197 bears a dispensable 2′-hydroxyl group (dark red sphere) that
packs closely against the nucleobase G200. (B) A 2′-mercapto group at
C197 penetrates the van der Waals volume of nearby atoms.
Experimental Section
All reagents and anhydrous solvents were purchased from Aldrich;
other solvents were from Fisher. All reactions using air-sensitive or
moisture-sensitive reagents were carried out under an argon atmosphere.
1H and 31P NMR spectra were recorded on Bruker 500 or 400 MHz
1
NMR spectrometers. H chemical shifts are reported in ppm relative
to tetramethylsilane. 31P chemical shifts are reported relative to an
external standard of 85% aqueous H3PO4. Mass spectra were obtained
from the Department of Chemistry, University of California at
Riverside, using a VG-ZAB instrument or from the Department of
Chemistry, University of Chicago, using a HP ESI instrument. Merck
silica gel (9385 grade, 230-400 mesh, 60 Å, Aldrich) was used for
column chromatography. Silica gel on glass with fluorescent indicator
(Aldrich) was used for TLC.
Nucleotide modifying enzymes were from Amersham Pharmacia or
New England Biolabs. [γ-32P]-Adenosine triphosphate was obtained
from Perkin-Elmer Life Sciences. Nucleoside triphosphates were from
Amersham Pharmacia, and 2′-ribo- and deoxy-R-triphosphates were
from Glen Research. Biotin-PEG3-maleimide was purchased from
Pierce, and streptavidin was from Sigma. Denaturing polyacrylamide
gel electrophoresis (DPAGE) was carried out using 6-9% polyacryl-
amide (Fisher; acrylamide:bis-acrylamide, 29:1) with 7 M urea and
TBE (89 mM Tris, 89 mM boric acid; 2 mM EDTA). DTT gels (10
mM DTT/7 M urea/TBE) were cast and allowed to polymerize
overnight. Gel loading buffer contained 8 M urea (J. T. Baker), 50
mM EDTA (pH 8.0, Fisher), 0.02% bromophenol blue (EM Science),
0.02% xylene cyanol FF (Kodak), and 10 mM DTT.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-(t-butyl-
thio)adenosine. 6-N-Benzoyl-9-[3,5-O-(1,1,3,3-tetraisopropyldisilox-
ane-1,3-diyl)-2-O-trifluoromethanesulfonyl-â-D-arabinofuranosyl]-
adenine5e (838 mg, 1.13 mmol) was dissolved in DMSO. 2-Methyl-2-
propanethiol (0.3 mL, 2.26 mmol, 2 equiv) was added followed by
dropwise addition of 1,1,3,3-tetramethylguanidine (0.3 mL, 1.7 mmol,
1.5 equiv). After being stirred at room temperature for 2 h, the reaction
was diluted with cold CH2Cl2 (20 mL). The solution was washed with
saturated NaHCO3 and dried over MgSO4. Short column silica gel flash
chromatography using 0-2% EtOH in CH2Cl2 gave the product. The
material was dissolved in MeOH (25 mL) and cooled to 0 °C. NH3
was bubbled into the solution until saturated. The solution was then
allowed to warm to room temperature and stirred for 16 h. The solvent
was evaporated, and the residue was purified by silica gel chromatog-
raphy (0-2% EtOH in CH2Cl2) to give 3′,5′-O-(tetraisopropyldisilox-
ane-1,3-diyl)-2′-deoxy-2′-(tert-butylthio)adenosine (79% yield over the
two steps) as a white foam.1H NMR (CDCl3, 400 MHz): δ 8.33 (s,
1H), 8.06 (s, 1H), 6.09 (d, 1H), 5.80 (bs, 2H), 4.87 (t, 1H), 4.09 (m,
5H), 1.25 (s, 9H), 1.05 (m, 28H). HRMS (FAB+): calculated for
C26H48N5O4SSi2 (MH+) 582.2979; found 582.2989.
Figure 4. The spatial distribution of 2′-SH interferences in ∆C209 P4-P6.
(A) Space-filling representation of the ∆C209 P4-P6 structure with all 2′-
OH groups replaced by 2′-SH groups so as to highlight their locations in
the molecule. Most 2′-OH groups line the rim of the minor groove on the
solvent accessible surface. (B) Sites of 2′-mercaptonucleotide interference
at 0.45 mM MgCl2. Interferences cluster between the parallel stacked helices
in the densely packed regions involved in tertiary contacts. Atomic
coordinates are from PDB accession 1HR2.9h
the 2′-hydroxyl groups reside in the minor groove and other
solvent accessible regions. Figure 4B highlights the locations
at which 2′-mercaptonucleotides interfere with P4-P6 folding.
Strikingly, most regions of the structure experience no 2′-SH
interference. Instead, the interferences tightly cluster in the three
most densely packed regions of the molecule: the tetraloop/
receptor, the A-rich bulge/trihelical junction, and the hinge
region (J5/5a). The residues in these regions experience
significant surface area burial, engage in hydrogen bonding
interactions via their 2′-hydroxyl groups, and exhibit strong
deviations from A-form helical geometry.9b,h
2′-Deoxy-2′-(t-butylthio)adenosine. 3′,5′-O-(Tetraisopropyldisilox-
ane-1,3-diyl)-2′-deoxy-2′-(tert-butylthio)adenosine (131.5 mg, 0.226
mmol) was dissolved in THF (5 mL). TBAF‚3H2O (71 mg, 0.271
(25) Ammon, H. Struct. Chem. 2001, 12, 205.
9
J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003 10015

A R T I C L E S
Schwans et al.
mmol) was added, and the solution turned slightly yellow. Within 15
min, TLC indicated the disappearance of the starting material. The
solvent was evaporated, and the residue was dissolved in 5% MeOH/
CHCl3. Silica gel column chromatography (5-10% MeOH in CHCl3)
(1:99, 25 mL) and extracted with CH2Cl2 (2 × 250 mL). The organic
phase was combined and washed with ice-cold saturated aqueous
NaHCO3 and brine and was dried over MgSO4. Column chromatog-
raphy of the residue (0-5% MeOH in CH2Cl2) gave 9-[3,5-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-2-O-trifluoromethanesulfonyl-â-D-
1
eluted the product as a white solid in 60% yield. H NMR (CD3OD,
1
500 MHz): δ 8.25 (s, 1H), 8.19 (s, 1H), 5.87 (d, 1H), 4.33 (d, 1H),
4.23 (bs, 1H), 4.09 (q, 1H), 3.95-3.76 (dd, 2H), 0.98 (s, 9H). HRMS
(FAB+): calculated for C25H22N5O3S (MH+) 340.1443; found 340.1445.
2′-Deoxy-2′-(2-nitrophenyldithio)adenosine. 2′-Deoxy-2′-(tert-
butylthio)adenosine (259 mg, 0.763 mmol) was dissolved in glacial
acetic acid (7 mL). 2-Nitrobenzenesulfenyl chloride (173 mg, 0.916
mmol, 1.2 equiv) was added, and the solution was stirred at room
temperature for 16 h. The solution was concentrated, and the residue
was purified by column chromatography (0-5% MeOH in CHCl3) to
give 2′-deoxy-2′-(2-nitrophenyldithio)adenosine (88% yield) as a yellow
arabinofuranosyl]guanosine as a white solid in 51% yield. H NMR
(CDCl3, 500 MHz): δ 7.69 (s, 1H), 6.19 (d, 2H), 5.34 (t, 1H), 4.85
(m, 1H), 4.04 (m, 2H), 3.85 (m, 1H), 1.07 (s, 9H), 0.99 (m, 28H).
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-(t-butyl-
thio)guanosine. 9-[3, 5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-
2-O-trifluoromethanesulfonyl-â-D-arabinofuranosyl]guanosine (500 mg,
0.76 mmol) was dissolved in DMSO (20 mL). 2-Methyl-2-propanethiol
(137 mg, 1.52 mmol) and 1,1,3,3-tetramethylguanidine (175 mg, 1.52
mmol) were added, and the mixture was stirred for 24 h at room
temperature. Saturated NaHCO3 was added, and a precipitate formed.
The precipitate was filtered, washed, and dried under vacuum. Column
chromatography (0-5% EtOH in CHCl3) gave 3′,5′-O-(tetraisopropyl-
disiloxane-1,3-diyl)-2′-deoxy-2′-(tert-butylthio)guanosine (360 mg, 79%)
as a white solid. 1H NMR (CDCl3, 500 MHz): δ 8.08 (s, 1H), 5.88 (s,
1H), 4.65 (t, 1H), 3.85-4.17 (m, 4H), 1.18 (s, 9H), 0.98 (m, 28H).
MS (ESI): calculated for C26H48N5O5SSi2 (MH+) 598.28; found
598.2.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-(2-nitrophenyldi-
thio)guanosine. 3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-
2′-(tert-butylthio)guanosine (70 mg, 0.12 mmol) was dissolved in glacial
acetic acid (5 mL). 2-Nitrobenzenesulfenyl chloride (68 mg, 0.36 mmol,
1.2 equiv) was added, and the reaction was stirred at room temperature
for 24 h. The acetic acid was removed by evaporation. The resulting
residue was purified by column chromatography (0-5% MeOH in
CHCl3) to give 2′-deoxy-2′-(2-nitrophenyldithio)guanosine (79 mg,
73%) as a yellow solid. 1H NMR (CDCl3, 500 MHz): δ 8.06 (m, 3H),
7.68 (bs, 2H), 7.52 (m, 2H), 7.25 (m, 2H), 5.97 (s, 1H), 4.78 (m, 1H),
3.76-4.10 (m, 4H), 1.01 (m, 28 H). MS (ESI): calculated for
C28H43N6O7S2Si2 (MH+) 695.21; found 695.1.
2′-Deoxy-2′-(2-nitrophenyldithio)guanosine. 9-[3,5-O-(1,1,3,3-Tetra-
isopropyldisiloxane-1,3-diyl)-2′-(2-nitrophenyldithio)-â-D-arabinofuran-
osyl]guanosine (180 mg, 0.26 mmol) was dissolved in THF (10 mL).
TBAF (1 M in THF, 0.52 mL) was added; TLC indicated that the
reaction was complete within 3 min. The solvent was evaporated, and
the residue was purified by column chromatography (0-10% MeOH
in CHCl3) to give 2′-deoxy-2′-(2-nitrophenyldithio)guanosine (80 mg,
68%) as a yellow solid. 1H NMR (CD3OD, 400 MHz): δ 8.05 (d, 1H,
J ) 7.1 Hz), 7.94 (d, 1H, J ) 8.1 Hz), 7.77 (s, 1H), 7.50 (t, 1H, J )
7.1 Hz), 7.27 (t, 1H, J ) 7.1 Hz), 6.10 (d, 1H, J ) 9.5 Hz), 4.51 (d,
1H, J ) 5.2 Hz), 4.44 (m, 1H), 4.07 (m, 1H), 3.68 (m, 2H). MS (ESI):
calculated for C16H15N6O6S2 (MH-) 452.05; found 451.0.
1
solid. H NMR (CD3OD, 500 MHz): δ 8.35 (s, 1H), 8.18 (s, 1H),
8.03 (t, 2H), 7.87 (s, 1H), 7.56 (t, 1H), 7.31 (t, 1H), 6.38 (d, 1H), 4.58
(d, 1H), 4.34 (m, 1H), 4.14 (m, 1H), 3.72 (m, 2H). MS (ESI): calculated
for C16H17N6O5S2 (MH+) 437.06; found 437.0.
2′-Deoxy-2′-(t-butyldithio)adenosine. 2′-Deoxy-2′-(2-nitrophenyl-
dithio)adenosine (47.5 mg, 0.109 mmol) was dissolved in 10% MeOH/
CHCl3 (5 mL). In a separate flask, 2-methyl-2-propanethiol (80 µL,
0.71 mmol), triethylamine (0.17 mL), and methanol (1.5 mL) were
combined. The solution was transferred to the reaction flask and stirred
for 3 h at room temperature. The reaction was concentrated, and the
residue was purified by column chromatography (0-10% MeOH in
CHCl3) to give 2′-deoxy-2′-(tert-butyldithio)adenosine as a white solid
1
in 89% yield. H NMR (CD3OD, 400 MHz): δ 8.29 (s, 1H), 8.14 (s,
1H), 6.15 (d, 1H), 4.55 (d, 1H), 4.26 (m, 1H), 4.17 (bs, 1H), 3.76-
3.72 dd, 2H). MS (ESI): calculated for C14H22N5O3S2 (MH+) 372.12;
found 372.0.
2′-Deoxy-2′-(t-butyldithio)cytidine. 2′-Deoxy-2′-(2-nitrophenyldithio)-
cytidine)5d (70 mg, 0.170 mmol) was dissolved in 10% MeOH/CHCl3
(10 mL). In a separate flask, 2-methyl-2-propanethiol (115 µL, 1.02
mmol), triethylamine (277 µL), and methanol (2.8 mL) were combined.
The solution was transferred to the reaction flask and stirred for 3 h at
room temperature. The reaction was concentrated, and the residue was
purified by column chromatography (0-10% MeOH in CHCl3) to give
the product as a white solid in 90% yield. 1H NMR (CDCl3, 400
MHz): δ 7.77 (d, 1H, J ) 7.5 Hz), 6.06 (d, 1H, J ) 9.0 Hz), 5.81 (d,
1H, J ) 7.4 Hz), 4.30 (m, 1H), 3.89 (m, 1H), 3.61 (m, 3H), 1.14 (s,
9H). MS (ESI): calculated for C13H22N3O4S2 (MH+) 348.10; found
348.1.
2′-Deoxy-2′-(t-butyldithio)uridine. 2′-Deoxy-2′-(2-nitrophenyldithio)-
uridine5d (94 mg, 0.243 mmol) was dissolved in 10% MeOH/CHCl3
(12.5 mL). In a separate flask, 2-methyl-2-propanethiol (164 µL, 1.46
mmol), triethylamine (375 µL), and methanol (3.8 mL) were combined.
The solution was transferred to the reaction flask and stirred for 3 h at
room temperature. The reaction was concentrated, and the residue was
purified by column chromatography (0-10% MeOH in CHCl3) to give
2′-Deoxy-2′-(t-butyldithio)guanosine. 2′-Deoxy-2′-(2-nitrophenyl-
dithio)guanosine (60 mg, 0.130 mmol) was dissolved in 10% methanol/
CHCl3 (20 mL). In a separate flask, 2-methyl-2-propanethiol (24 µL,
0.26 mmol), triethylamine (70 µL), and methanol (3.0 mL) were
combined. The solution was transferred to the reaction flask and stirred
for 3 h at room temperature. The reaction was concentrated, and the
resulting mixture was purified by column chromatography (0-10%
MeOH in CHCl3) to give 2′-deoxy-2′-(tert-butyldithio)guanosine (40
1
2′-deoxy-2′-(tert-butyldithio)uridine as a white solid (90% yield). H
NMR (CDCl3, 400 MHz): δ 7.96 (d, 1H, J ) 8.1 Hz), 5.85 (d, 1H, J
) 9.8 Hz), 5.77 (d, 1H, J ) 8.1 Hz), 4.26 (d, 1H, J ) 4.6 Hz), 4.03
(m, 1H), 3.78 (m, 2H), 3.63 (q, 1H), 1.35 (s, 9H). HRMS (FAB+):
calculated for C13H21N2O5S2 (MH+) 349.0879; found 349.0878.
9-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2-O-tri-
fluoromethanesulfonyl-â-D-arabinofuranosyl]guanosine. 9-[3,5-O-
(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2′-â-D-arabinofuranosyl]gua-
nosine26 (1.2 g, 2.28 mmol) and 4-(dimethylamino)pyridine (1.2 g, 13.2
mmol) were dissolved in CH2Cl2 (10 mL). The reaction mixture was
cooled to 0 °C, and trifluoromethanesulfonyl chloride (0.46 g, 2.7 mmol)
was added dropwise. The yellow solution was stirred for 10 min. The
reaction mixture was partitioned between ice-cooled acetic acid/water
1
mg, 80% yield) as a white solid. H NMR (CD3OD, 400 MHz): δ
7.94 (s, 1H), 6.02 (d, 1H, J ) 9.2 Hz), 4.50 (d, 1H, J ) 5.1 Hz), 4.16
(m, 1H), 4.14 (s, 1H) 3.73-3.84 (dd, 2H), 1.15 (s, 9H). MS (ESI):
calculated for C14H20N5O4S2 (MH-) 386.10; found 386.0.
General Method for the Synthesis of r-Thiotriphosphates. A
suspension of the nucleoside (25 mg) and pyridine (3 mL) was heated
with an air gun for several minutes until a clear solution was obtained.
Following cooling of the solution to room temperature, proton sponge
(20 mg, 2 equiv) was added. The solution was further cooled to 0 °C
and stirred for 10 min. Thiophosphoryl chloride (10 µL, 2 equiv) was
added dropwise, and the solution was stirred at 0 °C for 15 min. Tri-
n-butylammonium pyrophosphate (500 mg), tri-n-butylamine (0.25 mL),
(26) Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.; Zounes, M.
C.; Cummins, L. L.; Gonzalez, C.; Cook, P. D. J. Med. Chem. 1993, 36,
831.
9
10016 J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003

2′-Mercaptonucleotide Interference within RNA Molecules
A R T I C L E S
and DMF (2 mL) were combined in a separate flask and transferred
under anhydrous conditions to the reaction flask via cannula. The
reaction was stirred at 0 °C for 2 min. Triethylammonium bicarbonate
(20 mL, 0.2 M) and proton sponge (20 mg) were then added, and the
solution was stirred for 1 h at room temperature. The reaction mixture
was purified by ion-exchange chromatography (Sephadex DEAE-A25)
at room temperature using a gradient of triethylammonium bicarbonate
(0.1-1.0 M). Fractions containing the R-thiotriphosphate were visual-
ized by TLC, pooled, and dried. The material was purified further by
reverse phase HPLC on a Beckman ODS Ultrasphere C18 reverse-phase
column (10 × 250 mm) using a gradient of 0.1 M triethylammonium
acetate (solvent A) and acetonitrile (solvent B). The following linear
gradient program was used: 0-12% solvent B over 25 min to 40%
solvent B over 40 min.
at room temperature for 2 h, and extracted with phenol/chloroform/
isoamyl alcohol (25:24:1) followed by extraction with chloroform. The
samples were resuspended in water and treated with 20 mM HEPES
pH 7.4, 1.0 M NaCl, 5 mM EDTA, and 15 µg of streptavidin. The
reactions were incubated at room temperature for 20 min and quenched
with an equal volume of 90% formamide, 1X TBE (89 mM Tris; 89
mM boric acid; 2 mM EDTA). Nonradioactive samples were run on
1% agarose gels and visualized by ethidium bromide staining. Radio-
active samples were run on DPAGE. Modification by biotin/streptavidin
resulted in a slower migrating band or smear as compared to that of
the unmodified transcripts.
Nondenaturing Gel Electrophoresis. Nondenaturing gels were
electrophoresed in a manner similar to that of Silverman and Cech.13a
Gels were cast with 8% acrylamide, TB (89 mM Tris; 89 mM boric
acid; pH 8.3), 2.0 or 0.45 mM MgCl2, 0.05 mM EDTA, and 10 mM
DTT and allowed to polymerize overnight. Electrophoresis buffer
contained the same components as the gel. Gels were electrophoresed
at room temperature at 150 V for 5 h. The temperature of the gel buffer
was checked periodically. Fluctuation in temperature was not observed
under the electrophoresis conditions.
2′-Deoxy-2′-(t-butylthio)adenosine-r-thiotriphosphate. 31P NMR
(D2O, 400 MHz): δ 44.2 m, -8.9 d, -22.8-23.1 m. HRMS (FAB):
calculated for C14H23N5O11P3S3 (M-) 625.9775; found 625.9770.
-
2′-Deoxy-2′-(t-butylthio)cytidine-r-thiotriphosphate. 31P NMR
(D2O, 400 MHz): δ 41.1 m, -13 dd, -26.2 m. HRMS (FAB):
calculated for C13H23N3O12P3S3 (M-) 601.9662; found 601.9623.
-
2′-Deoxy-2′-(t-butylthio)guanosine-r-thiotriphosphate. 31P NMR
(D2O, 500 MHz): δ 44 m, -10.2 dd, -23 m. MS (ESI): calculated
Folding Reactions. Transcripts were treated with calf alkaline
intestinal phosphatase (Amersham Pharmacia) and 5′-end-labeled with
[γ-32P]-ATP (Perkin-Elmer Life Sciences) and T4 polynucleotide kinase
(New England Biolabs) according to the manufacturer’s instructions.
The samples were purified by DTT PAGE (10 mM DTT), and the
integrity of the mercapto group was verified by gel shift assay. Folding
reactions contained 5′-radiolabeled RNA, 2.0 or 0.45 mM MgCl2, TB
(pH 8.3), 10 mM DTT, and 5% glycerol. The samples were heated at
50 °C for 5 min and allowed to cool at room temperature for 10 min
before loading on the native gel. The bands were visualized by
autoradiography, excised, and eluted into TE (10 mM Tris‚HCl pH
7.5, 1 mM EDTA) at 4 °C overnight. The samples were ethanol
precipitated and resuspended in 10 µL of 50 mM HEPES (pH 8.0),
containing 100 mM iodoacetamide. Iodoacetamide modification prior
to sequencing reduces smearing during electrophoresis, thereby improv-
ing resolution, and prevents modification of the mercapto group during
I2-induced cleavage. The iodoacetamide reaction was allowed to proceed
for 15 min. The reactions were then quenched and purified by DPAGE.
The bands were visualized by autoradiography, excised, and eluted into
TE at 4 °C for 4-6 h. The samples were ethanol precipitated and
washed with 70% ethanol.
I2 Sequencing. The samples were resuspended in 10 µL of gel
loading solution and split into two fractions. One fraction was treated
with 1/10 volume 100 mM I2 in ethanol. The samples were heated to
90 °C for 1.5 min and loaded on a 9% DPAGE gel. The samples were
electrophoresed for varying amounts of time so as to achieve the best
resolution of the residues near the ends of the transcripts.
Interference Quantitation. Interference values were assigned
essentially as described in Ryder et al.8g Peak intensities were
quantitated by PhosphorImager analysis at each residue for the selected
and unselected lanes. Values were normalized against total intensity
for each lane to account for loading differences. Interference was
calculated at each position according to the equation:
for C14H23N5O12P3S3 (M-) 641.97; found 641.9.
-
2′-Deoxy-2′-(t-butylthio)uridine-r-thiotriphosphate. 31P NMR (D2O,
400 MHz): δ 46.1 m, -8.0 dd, -22.4 m. HRMS (FAB): calculated
for C13H22N2O13P3S3 (M-) 602.9502; found 602.9526.
-
Transcription Reactions. ∆C209 P4-P6 was transcribed from EarI
digested ∆C209 plasmid (gift from K. Juneau and T. Cech). Transcrip-
tion reactions contained 40 mM tris(hydroxylmethyl)aminomethane
hydrochloride (Tris-HCl, pH 7.5), 4 mM spermidine, 10 mM dithio-
threitol (DTT), 15 mM MgCl2, 0.05% Triton X-100, 1.0 mM NTPs,
0.05 µg/µL DNA template, and 0.08 µg/µL Y639F T7 RNA poly-
merase. In separate reactions 2′-mercapto-R-thiotriphosphates were
added at the following concentrations: 0.4 mM ASHRS, 0.5 mM GSHRS,
0.4 mM CSHRS, and 0.4 mM USHRS. All transcription reactions were
incubated for 2 h at 37 °C. The NSHTPRS concentrations were chosen
to achieve a level of approximately 5% analogue incorporation,
determined by comparing I2-induced cleavage intensities to a NTPRS
standard.8b,f,g Transcripts were purified by denaturing PAGE (10 mM
DTT in gel and buffer). Samples were eluted, and ethanol was
precipitated, washed with 70% ethanol, and resuspended in 1X TE with
10 mM DTT.
Biotin/Streptavidin Gel Shift Assay Verifying 2′-SH Integrity.
To ensure that the mercapto group remains intact following transcription
and isolation of the RNA, the transcripts were treated with a mercaptan
modifying reagent. Modification was detected by observing a difference
in gel mobility due to the increased steric bulk introduced by the
modification reagent (biotin/streptavidin). Maleimide-PEG3-biotin con-
tains a maleimide group linked to biotin that reacts predominantly with
free -SH groups at pH 8.0. Selective alkylation of the mercapto group
covalently attaches the biotin moiety to the transcript. Modification
with maleimide-PEG3-biotin alone resulted in a gel shift of short RNAs
(6-12 nt; data not shown); however, due to the length of P4-P6 (∼160
nt), a gel shift was not observed. Subsequent reaction of the biotinylated
species with streptavidin resulted in a gel shift, where the single band
of the unmodified material became a smear with lower mobility. The
smear is likely due to the variability in the number of residues
containing the 2′-mercapto analogue in a single transcript (ap-
proximately 1-5 substitutions per transcript). Because the 2′-SH
transcripts also contained a phosphorothioate linkage, transcriptions with
2′-OH R-thiotriphosphates (also containing a phosphorothioate linkage)
were included as controls to ensure that modification was specific to
the 2′-mercapto group.
NRS 0.45 or 2.0 mM Mg2+ selected lane
2′-H or 2′-SH 0.45 or 2.0 mM Mg2+ selected lane
interference )
NRS unselected lane
2′-H or 2′-SH unselected lane
The data were normalized to give a value of 1 where no interference
occurs.8g Each interference value was determined from at least three
independent experiments.
Computer Modeling. Space-filling models of the ∆C209 P4-P6
domain (PDB accession: 1HR2)9h were generated using Chem3D
version 5.0 (CambridgeSoft Corp.). 2′-Mercapto substitution was
modeled by replacing the 2′-oxygen with 2′-sulfur. The bond length
Cold RNA (∼10 pmol) or radiolabeled RNA (∼20 pmol) was
modified with maleimide-PEG3-biotin (10 mM) in 50 mM HEPES pH
8.0, 300 mM NaCl, and 1 mM EDTA. The reactions were incubated
9
J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003 10017

A R T I C L E S
Schwans et al.
was increased to 1.819 Å, the average C-S bond length from gas-
phase molecules.27 Close contacts were calculated and tabulated for
each residue using Chem3D and the modified models. Sites at which
the increased steric bulk of the 2′-SH substitution could not be
accommodated without overlapping of nearest neighbor van der Waals
radii were designated as sites of steric clash. This designation excluded
sites at which the steric clash arose from a neighboring atom within
the same residue, such as the 3′-oxygen.
Juneau and Professor Tom Cech for the ∆C209 P4-P6 plasmid,
and Professor Michelle Hamm and members of the Piccirilli
lab for comments on the manuscript. J.A.P. is an associate
investigator of the Howard Hughes Medical Institute.
Supporting Information Available: Representative non-
denaturing gel and enlarged space-filling space models for
Figure 4 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. The authors thank Professor Rui Sousa
for the gift of the Y639F T7 RNA polymerase plasmid, Kara
(27) Liede, D. R., Ed. CRC Handbook of Chemistry and Physics, 73rd ed.; CRC
Press: Boca Raton, FL, 1992; pp 9-15-41.
JA035175Y
9
10018 J. AM. CHEM. SOC. VOL. 125, NO. 33, 2003