Interplay of structure, hydration and thermal stability in formacetal modified oligonucleotides: RNA may tolerate nonionic modifications better than DNA

Article abstract of DOI:10.1021/ja904926e

DNA and RNA oligonucleotides having formacetal internucleoside linkages between uridine and adenosine nucleosides have been prepared and studied using UV thermal melting, osmotic stress, and X-ray crystallography. Formacetal modifications have remarkably

Full text of DOI:10.1021/ja904926e

Published on Web 09/29/2009
Interplay of Structure, Hydration and Thermal Stability in
Formacetal Modified Oligonucleotides: RNA May Tolerate
Nonionic Modifications Better than DNA
Andrej Kolarovic,† Emma Schweizer,‡ Emily Greene,‡ Mark Gironda,‡
Pradeep S. Pallan,§ Martin Egli,§ and Eriks Rozners*,†,‡
Department of Chemistry, Binghamton UniVersity, State UniVersity of New York, Binghamton,
New York 13902, Department of Chemistry and Chemical Biology, Northeastern UniVersity,
Boston, Massachusetts 02115, and Department of Biochemistry, School of Medicine, Vanderbilt
UniVersity, NashVille, Tennessee 37232
Received June 16, 2009; E-mail: erozners@binghamton.edu
Abstract: DNA and RNA oligonucleotides having formacetal internucleoside linkages between uridine and
adenosine nucleosides have been prepared and studied using UV thermal melting, osmotic stress, and
X-ray crystallography. Formacetal modifications have remarkably different effects on double helical RNA
and DNAsthe formacetal stabilizes the RNA helix by +0.7 °C but destabilizes the DNA helix by -1.6 °C
per modification. The apparently hydrophobic formacetal has little effect on hydration of RNA but decreases
the hydration of DNA, which suggests that at least part of the difference in thermal stability may be related
to differences in hydration. A crystal structure of modified DNA shows that two isolated formacetal linkages
fit almost perfectly in an A-type helix (decamer). Taken together, the data suggest that RNA may tolerate
nonionic backbone modifications better than DNA. Overall, formacetal appears to be an excellent mimic of
phosphate linkage in RNA and an interesting modification for potential applications in fundamental studies
and RNA-based gene control strategies, such as RNA interference.
The potential of RNA interference (RNAi) to become a new
therapeutic approach stimulates interest in chemical modifica-
tions to optimize the efficacy of RNA-based drugs.1 Several
modifications, developed earlier to improve the properties of
antisense oligonucleotides, have also shown promising results
in RNAi.1,2 We are interested in developing RNA analogues
that have nonionic internucleoside linkages.3,4 Among such, we
have found that replacement of selected phosphates with amide
linkages (CH2-CO-NH-5′) does not change the overall
conformation and thermal stability of RNA.3 Recently, Iwase
et al.5 reported that introduction of two such amide linkages at
the overhanging uridines of an siRNA did not significantly
change the RNAi activity. The formacetal linkage 3′-O-CH2-O-
5′ is another interesting modification that has served as a
phosphate mimic in structural studies on a photolyase bound to
a DNA lesion6 and may have broader use in fundamental studies
and practical applications involving modified RNA.
Previous studies suggested that formacetal modification might
have different effect on DNA and RNA oligonucleotides. Van
Boom and co-workers found that formacetal modifications
strongly destabilized DNA duplexes by -1.4 to -2.4 °C per
modification.7 Gao and co-workers reported similar findings (-3
°C per modification); however, their preliminary NMR studies
suggested that formacetal linkage caused little structural per-
turbation of the DNA helix.8 Matteucci found that formacetals
in the DNA strand only slightly decreased the stability of
DNA-RNA heteroduplexes.9 In contrast, our preliminary
studies showed that formacetals were somewhat stabilizing in
all RNA duplexes (+0.2 to +0.8 °C per modification).4
However, each of these studies used different model sequences,
which makes it difficult to compare and rationalize the results.
In all earlier studies formacetal linkages were inserted between
uridine or thymidine nucleosides only, which reduced the
synthetic effort but also limited the sequences that could be
studied.
† Northeastern University.
‡ Binghamton University.
§ Vanderbilt University.
(1) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug DiscoVery Today
2008, 13, 842–855. Corey, D. R. J. Clin. InVest. 2007, 117, 3615–
3622.
To rationalize the earlier results and to gain more insight in
structural and thermodynamic properties of formacetal-modified
(2) Rozners, E. Curr. Org. Chem. 2006, 10, 675–692. Manoharan, M.
Curr. Opin. Chem. Biol. 2004, 8, 570–579.
(3) Rozners, E.; Katkevica, D.; Bizdena, E.; Stro¨mberg, R. J. Am. Chem.
Soc. 2003, 125, 12125–12136.
(6) Mees, A.; Klar, T.; Gnau, P.; Hennecke, U.; Eker, A. P. M.; Carell,
T.; Essen, L. O. Science 2004, 306, 1789–1793.
(4) Rozners, E.; Stro¨mberg, R. J. Org. Chem. 1997, 62, 1846–1850.
Rozners, E.; Katkevica, D.; Stro¨mberg, R. ChemBioChem 2007, 8,
537–545.
(7) Quaedflieg, P. J. L. M.; Pikkemaat, J. A.; Van der Marel, G. A.; Kuyl-
Yeheskiely, E.; Altona, C.; Van Boom, J. H. Recl. TraV. Chim. Pays-
Bas 1993, 112, 15–21.
(5) Iwase, R.; Toyama, T.; Nishimori, K. Nucleosides, Nucleotides Nucleic
Acids 2007, 26, 1451–1454. Iwase, R.; Miyao, H.; Toyama, T.;
Nishimori, K. Nucleic Acids Symp. Ser. 2006, 175–176.
(8) Gao, X.; Brown, F. K.; Jeffs, P.; Bischofberger, N.; Lin, K. Y.; Pipe,
A. J.; Noble, S. A. Biochemistry 1992, 31, 6228–6236.
(9) Matteucci, M. Tetrahedron Lett. 1990, 31, 2385–2388.
9
14932 J. AM. CHEM. SOC. 2009, 131, 14932–14937
10.1021/ja904926e CCC: $40.75  2009 American Chemical Society

Formacetal-Modified Oligonucleotides
A R T I C L E S
Scheme 1. Synthesis of Formacetal Linked Dimersa
a Steps: (a) 1, 2, NIS, TfOH, THF, -40 to -25 °C, 1.5 h, 72%; (b) TBAF, THF, rt 18 h; (c) p-methoxytrityl chloride, pyridine, 35 °C, 1.5 h 39% (two
steps); (d) 2-chlorobenzoyl chloride, THF/pyridine, -78 °C, 1.5 h, then PCl3, imidazole, NEt3, THF/pyridine/CH2Cl2, -78 °C, 0.5 h, 73%; (e) TMSOTf,
ClCH2CH2Cl, rt, 40 min, 43%; (f) TBAF, THF, rt, 1 h, 50%; (g) p-dimethoxytrityl chloride, pyridine, rt, 20 h 75%; (h) NC(CH2)2OP(Cl)N(iPr)2, DIEA,
CH2Cl2, rt, 1 h 20 min, 50%.
Table 1. Thermal Melting and Osmotic Stress Results of Formacetal-Modified Oligonucleotidesa
-∆H
-∆H
-∆G310
ethylene
glycol ∆nW
glycerol
d
∆nW
acetamide
d
∆nW
c
no
sequence
Tm °C
∆S eu
d
kcal/molb
kcal/molc
kcal/molc
e
e
OL1
OL2
OL3
OL4
OL5
OL6
r(UpApUpApUpApUpApUpApUpA)
r(UfApUfApUfApUfApUfApUfA)
d(TpApTpApTpApTpApTpApTpA)
d(TfApTfApTfApTfApTfApTfA)
d(CpGpTpApTpApTpApTpApTpApCpG) 46.2 ( 0.4 30.6 ( 1.1 37.9 ( 2.0
d(CpGpTfApTfApTfApTfApTfApCpG) 30.4 ( 0.3 34.1 ( 1.0 32.8 ( 1.5
30.0 ( 0.3 82.4 ( 4.2 70.3 ( 4.0 206 ( 13 6.4 ( 0.1
38.3 ( 0.1 77.1 ( 0.7 82.2 ( 1.6 238 ( 5 8.4 ( 0.1
36 ( 6
37 ( 3
22 ( 7
-
15 ( 2
13 ( 2
40 ( 6
67 ( 6
34 ( 3 100 ( 2
22.9 ( 0.7 39.5 ( 3.3 41.4 ( 4.1 113 ( 14 6.2 ( 0.2
26 ( 8
-
19 ( 2
9 ( 3
38 ( 6
-
34 ( 3
19 ( 2
<10
-
-
-
93 ( 6
82 ( 5
-
9.2 ( 0.1
7.4 ( 0.1
a Oligonucleotide (2 µM) in 10 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and 300 mM NaCl. Results ( standard deviations. b From δR/
δ(Tm-1) vs Tm, ref 13. c From van’t Hoff analysis of melting curves. d ∆nW ) number of water molecules released upon melting. Results ( error
estimates. e Data from ref 13.
RNA and DNA we prepared formacetal (f) linked dinucleotides
r(UfA) and d(TfA). The self-complementary UA and TA motifs
allowed us to prepare a series of RNA and DNA oligonucle-
otides for thermodynamic (UV melting and osmotic stress) as
well as X-ray crystallographic studies. Herein, we show that
the formacetal modification has a surprisingly different effect
on double helical RNA compared to DNA. Although formacetal
stabilizes RNA helix by +0.7 °C per modification, it strongly
destabilizes DNA helix (-1.6 °C per modification). Interest-
ingly, the apparently hydrophobic formacetal has little effect
on hydration of RNA but decreases the hydration of DNA, as
judged by osmotic stress experiments. X-ray crystal structure
shows that two formacetal linkages do not distort the conforma-
tion of an A-type decamer. Our results suggest that the
formacetal linkage fits remarkably well in a double helical RNA
and may be an interesting modification to test in short interfering
RNAs (siRNAs). Taken together with our earlier studies on
amide3 and formacetal4 modified RNA, the current results also
suggest that RNA may tolerate nonionic backbone modifications
better than DNA.
and modifying literature procedures by us4 and van Boom and
co-workers,10 respectively. N-Iodosuccinimide and triflic acid
(TfOH)-mediated coupling11 of thioacetal 14 with adenosine
acceptor 212 gave r(UfA) dimer 3 (Scheme 1). Deprotection of
silyl groups, selective 5′-tritylation and one-pot 2′-benzoylation
and 3′-phosporylation following our previously reported pro-
cedures4 gave the phosphonate dimer 6. Analogous synthesis
of d(TfA) was not successful, apparently because of low stability
of deoxyadenosine 8 under the acidic (TfOH) coupling conditions.
However, trimethylsilyl triflate mediated coupling of dibutyl
phosphate 710 with deoxyadenosine acceptor 812 gave r(UfA)
dimer 9 in acceptable yield (Scheme 1). Deprotection of silyl
groups, selective 5′-tritylation and standard phosphoramidite
synthesis gave dimer 12. Using the dimers 6 and 12 in standard
H-phosphonate and phosphoramidite solid-phase synthesis
protocols we initially prepared two self-complementary model
sequences r(UfA)6 OL2 and d(TfA)6 OL4 (Table 1). The choice
of the sequences was based on our earlier UV thermal melting
(10) Quaedflieg, P. J. L. M.; Timmers, C. M.; van der Marel, G. A.; Kuyl-
Yeheskiely, E.; van Boom, J. H. Synthesis 1993, 627–633.
(11) He, G. X.; Bischofberger, N. Tetrahedron Lett. 1997, 38, 945–948.
(12) Zhu, X. F.; Williams, H. J.; Scott, A. I. J. Chem. Soc., Perkin Trans.
1 2000, 2305–2306.
Results
Synthesis of Formacetal-Modified Oligonucleotides. Synthesis
of formacetal-modified RNA and DNA was done by adopting
9
J. AM. CHEM. SOC. VOL. 131, NO. 41, 2009 14933

A R T I C L E S
Kolarovic et al.
and osmotic stress study,13 which had characterized the unmodi-
fied RNA (OL1) and DNA (OL3) controls.
Table 2. Selected Crystal and Refinement Data
Space group
Tetragonal P41212
UV Thermal Melting and Osmotic Stress. To gain insight
into the effect of formacetal modification on thermal stability
and hydration of DNA and RNA, we studied the model
sequences OL2 and OL4 using the UV thermal melting and
osmotic stress method as previously reported.13,14 Substitution
of 12 out of 22 phosphates with formacetals increased the
thermal stability of the formacetal-modified RNA duplex OL2
(Table 1, +0.7 °C per modification). Most remarkably, the
relatiVely hydrophobic formacetal linkages did not decrease the
hydration of RNA, as judged by little change in ∆nW, the number
of water molecules released upon melting. In fact, in acetamide,
the formacetal duplex OL2 was more hydrated than the
unmodified control OL1. In contrast, the DNA OL4 was
strongly destabilized by the formacetal modification (at least
-1 °C per modification). Because OL4 was not suitable for
osmotic stress due to the low thermal stability, we prepared
OL6 with five formacetal linkages between the central T and
A nucleosides and two terminal unmodified CG base pairs.
Compared to the unmodified control OL5, replacement of 10
out of 26 phosphates with formacetals resulted in strong
destabilization of DNA (Table 1, -1.6 °C per modification).
Most remarkably, the glycerol and acetamide series showed
substantial loss of hydration upon formacetal modification of
DNA.
Crystal Structure of Formacetal-Modified DNA. To gain
more insight into structural features of formacetal-modified
oligonucleotides we synthesized and attempted crystallization
of a series of self-complementary DNA sequences having the
TfA dimer inserted at a central position. The DNA decamer
with sequence GCGTATACGC does not form crystals suitable
for high-resolution diffraction studies. Accordingly, our attempts
to solve crystal structures of GCGTATACGC having a for-
macetal modification so far have not been successful. However,
replacement of a single 2′-deoxynucleotide at various positions
of GCGTATACGC with a ribonucleotide or a 2′-O-modified
nucleotide analogue typically yields well diffracting crystals.15
Following such a design, we succeeded in solving the structure
of d(GCGTfAUMeACGC) with a formacetal linkage between
residues T4 and A5 and 2′-O-methyl-uridine (UMe) at position
6 (OL7; residues in the strand are numbered 1-10). The crystals
diffracted to 1.75 Å resolution and were of space group P41212,
(a ) b ) 33.20 Å; c ) 68.51 Å), with one strand per asymmetric
unit. Selected crystal data and refinement parameters are
summarized in Table 2 and an example of the quality of the
final electron density is depicted in Figure 1.
Unit cell constants [Å]
Resolution [Å]
No. of unique reflections
Completeness (1.78-1.75 Å) [%]
R-merge (1.78-1.75 Å) [%]
R-work [%]
R-free [%]
No. of DNA atoms
No. of waters
a ) b ) 33.20, c ) 68.51
1.75
4,006
95.2 (72.4)
5.8 (18.7)
22.8
28.2
201
25
0.022
3.0
Rms deviations bonds [Å]
Rms deviations angles [deg]
Average B-factor [Å2]
35.2
-77°, 174°, 65°, 76°, -153°, -72° (fA5) and -72°, 168°, 57°,
78°, -144°, -77° (UMe6), respectively (R, ꢀ, γ, δ, ꢀ, ꢁ). Overall,
the crystal structure demonstrates that formacetal fits almost
perfectly within the A-type duplex. This is consistent with earlier
studies by us4 and others.8
The backbone around formacetal linkages is relatively dry.
Several water molecules are located in the vicinity of the
formacetal moiety but the distances from individual atoms
exceed 3.5 Å (Figure 4A). Hydrogen bonds between water
molecules and residues T4 and T5 are only established to base
atoms (not shown). The phosphate groups of flanking residues
are well hydrated (Figure 4B). However, at the current resolution
of the crystal structure, hydration patterns around the duplex
can only be visualized to a limited extent. A more complete
account of the effects of the formacetal moiety on the local water
structure will have to await the determination of crystal
structures at higher resolution.
Circular Dichroism of Formacetal-Modified Oligo-nucle-
otides. To gain insight into overall helix conformation of
formacetal-modified oligonucleotides in solution we studied the
circular dichroism (CD) spectra of RNA OL1 and OL2 and
DNA OL5, OL6, and OL7. All DNA oligonucleotides gave
CD spectra (Figure 5A) typical for B-form helical conforma-
tions: Positive bands at 260-290 nm, strong negative bands
around 250 nm and positive bands at 220-225 nm accompanied
In the crystal, the duplex adopts an A-form conformation
(Figure 2), with the paired strands exhibiting an identical
geometry due to the dyad symmetry.
In the formacetal moiety all torsion angles adopt a conforma-
tion that is consistent with a standard A-form backbone (Figure
3; R sc-, ꢀ ap, γ sc+, δ sc+, ꢀ ap, ꢁ sc-). The conformation of
the sugars in T4, fA5 and UMe6 is C3′-endo and their backbone
torsion angles are -60°, 165°, 49°, 80°, -155°, -71° (T4),
(13) Rozners, E.; Moulder, J. Nucleic Acids Res. 2004, 32, 248–254.
(14) Spink, C. H.; Chaires, J. B. Biochemistry 1999, 38, 496–508.
(15) (a) Egli, M.; Usman, N.; Rich, A. Biochemistry 1993, 32, 3221–3237.
(b) Egli, M. AdVances in Enzyme Regulation; Weber, G., Ed.; Elsevier
Science Ltd.: Oxford, UK, 1998; Vol. 38, pp 181-203. (c) Egli, M.;
Minasov, G.; Tereshko, V.; Pallan, P. S.; Teplova, M.; Inamati, G. B.;
Lesnik, E. A.; Owens, S. R.; Ross, B. S.; Prakash, T. P.; Manoharan,
M. Biochemistry 2005, 44, 9045–9057. (d) Egli, M.; Pallan, P. S. Annu.
ReV. Biophys. Biomol. Struct. 2007, 36, 281–305.
Figure 1. Quality of the electron density. Final Fourier sum 2Fo - Fc
electron density (1σ threshold) around the T4fA5 dimer is shown as a green
meshwork and water molecules are shown as magenta spheres.
9
14934 J. AM. CHEM. SOC. VOL. 131, NO. 41, 2009

Formacetal-Modified Oligonucleotides
A R T I C L E S
of either typical A or B helices and displayed a set of unique
bands - negative around 285 and 260 nm and positive around
215 nm. This result was surprising because our previous studies
had shown that the CD spectra of formacetal-modified RNA
were virtually identical to CD spectra of unmodified RNA.4
However, the RNAs in our earlier studies contained less
formacetal modifications (up to six formacetals out of 24
linkages) compared to OL2 (12 formacetals out of 22 linkages).
Discussion
The present UV thermal melting study confirms the earlier
literature data that formacetal modification has different effect
on double helical DNA and RNA (Table 1). In the present study,
formacetal modification strongly destabilizes double helical
DNA by -1.6 °C per modification, which is consistent with
the results of van Boom and co-workers7 (-1.4 to -2.4 °C per
modification) and Gao et al.8 (-3 °C per modification). In
contrast, formacetal modification stabilizes double helical RNA
by +0.7 °C per modification, which is consistent with our earlier
studies4 (+0.2 to +0.8 °C per modification).
Taken together with our earlier results on amide modified
RNA,3 the present data suggest that RNA may tolerate nonionic
backbone modifications better than DNA. If this phenomenon
holds true for other modifications, there are two implications
important for design of RNA analogues, for example such as,
modified siRNAs. First, it is conceivable that the phosphate
backbone may be in general an excellent site to introduce
nonionic modifications in RNA. Second, many nonionic back-
bone modifications have been introduced in DNA and found to
destabilize DNA-RNA heteroduplexes.17 One may conclude
that such modifications are not useful for applications involving
RNA as well. However, our formacetal data suggest that
modifications that are even strongly destabilizing in DNA may
actually be stabilizing in RNA. Therefore, the data obtained on
modified DNA17 should not be used to predict the biophysical
properties of the same modification in RNA without additional
experimental support. It is possible that other nonionic backbone
modifications that may not look promising in DNA, may in fact
be well tolerated in RNA.
Figure 2. Crystal structure of the duplex [d(GCGTfAUMeACGC)]2 (OL7).
The formacetal carbon is highlighted with a sphere and the hash symbol
indicates a symmetry-related strand. The view is across the major (top)
and minor grooves (bottom).
Osmotic stress results show that formacetal modification
causes little change in RNA’s hydration. This result is somewhat
unexpected because more than half (12 out of 22) of the polar
phosphates are replaced by the relatively hydrophobic formac-
etals in OL2. As could have been expected, formacetal
modification causes significant loss of hydration in DNA OL6.
These results suggest an intriguing possibility that at least part
of the observed differences in thermal stability of formacetal-
modified OL2 and OL6 may be caused by favorable hydration
of formacetal-modified RNA and unfavorable hydration of
formacetal-modified DNA. However, direct comparison is
complicated by the fact that the CD spectra suggest that
formacetal-modified RNA OL2 may not be in the expected
A-form helix. This result is unexpected because in earlier studies
formacetal modifications caused virtually no change in CD
spectra of RNA. Unfortunately, all attempts to crystallize OL2
were not successful. Work on structural aspects of formacetal
linkages in RNA by crystallography and solution phase NMR
is currently in progress in our laboratories and should clarify
the unexpected CD result.
Figure 3. Overlay of the T4fA5 and UMe6pA7 (green bonds) dinucleotides.
Torsion angles of the formacetal linkage are depicted in black. Values of
the ꢀ and ꢁ torsion angles in the 5′-flanking T4 residue are highlighted in
purple.
with weaker bands around 210 nm (negative).16 Consistent with
our previous findings in the RNA series,4 the CD spectra of
formacetal-modified DNA OL6 were similar to the unmodified
control OL5.
The unmodified RNA OL1 showed CD spectrum typical for
an A-form helical conformation with a strong positive band at
250-260 nm (Figure 5B).16 However, the CD spectrum of the
formacetal-modified RNA OL2 was different from the spectra
The crystal structure (Figures 2 and 3) demonstrates that
formacetal (two substitutions per decamer OL7) fits almost
(16) Sosnick, T. R.; Fang, X.; Shelton, V. M. Methods Enzymol. 2000,
317, 393–409. Gray, D. M.; Ratliff, R. L.; Vaughan, M. R. Methods
Enzymol. 1992, 211, 389–406.
(17) Freier, S. M.; Altmann, K. H. Nucleic Acids Res. 1997, 25, 4429–
4443.
9
J. AM. CHEM. SOC. VOL. 131, NO. 41, 2009 14935

A R T I C L E S
Kolarovic et al.
Figure 4. Comparison of water molecules associated with the backbones of (A) formacetal linkage in T4fA5 and (B) phosphate linkage in A7pC8. Contacts
between water molecules associated with the formacetal and phosphate moiety in the structures of the chemically modified and native decamers, respectively,
are shown with thin lines and distances in Å.
Supporting Information). Although OL7 exists as a B-form helix
in solution (Figure 5A), it crystallizes in the A-form (Figure
2). This is likely due to an intrinsic tendency of this decamer
to relatively easily flip into the A-form as well as to dehydration
and packing interactions in the crystal. Moreover, the decamer
sequence here features a 2′-O-methyl-uridine residue and
incorporation of a single ribonucleotide into a DNA oligonucle-
otide was previously demonstrated to convert the B- to the
A-form in the solid state.15a,18
The crystal structure of OL7 confirms our current and
previous4 thermal melting results that the formacetal linkages
are well tolerated in A-form helix. Thus, the overall conforma-
tion of the decamer duplex is little perturbed by substitution of
two phosphate linkages with formacetals. The bridging C-O
bonds in formacetal are somewhat shorter (∼1.4 Å) than the
P-O bonds in phosphate (∼1.6 Å). However, the O-C-O
angle (∼110°) is wider than the analogous O-P-O angle
(102-104°). The wider angle compensates for the shorter bonds
in formacetal and, thus, the distance between the 3′-O and the
5′-O of the next nucleoside changes only from ∼2.5 Å in
phosphate to ∼2.3 Å in formacetal. The backbone of nucleic
acid is flexible enough to accommodate such a relatively small
change. Taken together, our data suggest that isolated formacetal
linkages are remarkably good mimics of the internucleoside
phosphate linkages in RNA, but not necessarily in DNA.
Conclusions
The present study is to our knowledge the first attempt at
direct comparison of nonionic backbone modification in RNA
and DNA. Our results suggest that RNA may tolerate nonionic
backbone modifications better than DNA. Therefore, conclusions
based on thermal stability of modified DNA17 should be used
with caution when designing chemically modified RNA ana-
logues. Taken together the excellent thermal stability and the
fact that two formacetal linkages do not disturb the A-form
decamer duplex suggest that formacetals may be excellent
mimics of phosphate for certain biochemical studies of RNA.
Figure 5. CD spectra of oligonucleotides.
For example, RNA fragments having single formacetal modi-
fication as a non-cleavable phosphate analogue may be useful
perfectly within an A-form helix, the conformation adopted by
double-stranded RNA. This is consistent with earlier studies
by us4 and others8 and is further confirmed by the modeled
structure of a DNA duplex with several formacetal linkages per
strand that exhibits an overall A-form (see Figure S2 in
as substrates for cocrystallization with RNA-processing en-
zymes. Similar application have been demonstrated in DNA.6
(18) Ban, C.; Ramakrishnan, B.; Sundaralingam, M. J. Mol. Biol. 1994,
236, 275–285.
9
14936 J. AM. CHEM. SOC. VOL. 131, NO. 41, 2009

Formacetal-Modified Oligonucleotides
A R T I C L E S
concentrations were provided by Drs. Spink and Chaires. The slope
of the plot of reciprocal temperature (in K) of melting vs the
logarithm of water activity (lnaw) at different concentrations (0, 5,
10, 15, and 20%) of small cosolutes gave the value of d(Tm-1)/
d(lnaw). The final ∆nW were obtained by linear fitting using
KaleidaGraph software (Version 3.51) with a confidence level
usually better than 98%. The experimental uncertainties in the final
∆nW were calculated as previously described by us.13
Our studies also suggest that formacetal may be an interesting
modification to test in siRNAs, where the charge reduction may
help cellular uptake and biodistribution. However, the structural
consequences of extensive formacetal modification of RNA need
to be further investigated.
Experimental Section
Crystallization and Data Collection. Crystals of 5′-GCGT-
fAUMeACGC-3′ (UMe ) 2′-OMe-U) were grown by the hanging-
drop vapor diffusion technique using the Nucleic Acid Miniscreen
(Hampton Research, Aliso Viejo, CA).21 Droplets (2 µL) containing
oligonucleotide (0.6 mM), sodium cacodylate (20 mM, pH 6.0),
potassium chloride (40 mM), magnesium chloride (10 mM),
spermine tetrahydrochloride (6 mM), and 2-methyl-2,4-pentanediol
(MPD; 5% (v/v)) were equilibrated against a reservoir of MPD (1
mL, 35%). Crystals were mounted in nylon loops without further
cryo-protection and frozen in liquid nitrogen. Diffraction data were
collected on the undulator beamline X25 at the National Synchro-
tron Light Source (NSLS). Diffraction data were processed with
the program HKL2000.22 Selected crystal data and refinement
parameters are listed in Table 2.
Structure Refinement. The structure was determined by the
Molecular Replacement (MR) technique with the program MOL-
REP,23 using a single strand of an A-form DNA (PDB ID 411D)
as the search model. Initially the single strand was refined as an
all-DNA model and the refinement was carried out with the program
Refmac,24 randomly setting aside 8% of the reflections for
calculating the R-free.25 Water molecules were added into regions
of superimposed (2Fo - Fc) sum and (Fo - Fc) difference Fourier
electron density. The refinement was carried out in both space
groups P41 and P41212. However, refinement in the former space
group resulted in higher values for R-free and R-work (typically
around 3-4% difference), thereby indicating the higher symmetry
is correct. At a later stage refinement was continued and the
phosphorus and the two nonbridging oxygen atoms between the
T4 and A5 residues replaced by a carbon. The refinement of the
structure clearly reveals all the atoms of the formacetal linkage.
Data Deposition. Final coordinates and structure factors have
been deposited in the Protein Data Bank (http://www.rcsb.org): PDB
ID code 3HR3.
Synthesis of Formacetal Modified Oligonucleotides. Formac-
etal containing RNAs and DNAs were synthesized by following
and modifying the reported procedures.4,10 For details, see the
Supporting Information.
UV Thermal Melting and Osmotic Stress. Melting of each
oligonucleotide (2 µM) was done in 10 mM sodium cacodylate,
0.1 mM EDTA, and 300 mM NaCl in presence of 0, 5, 10, 15, and
20% weight/volume of each of the three osmolytes in Table 1.
Oligonucleotide concentration were calculated using extinction
coefficients obtained using the nearest-neighbor approximation.19
Absorbance vs temperature profiles were measured at 260 nm on
a Varian Bio 100 spectrometer equipped with a six-position Peltier
temperature controller. The temperature was increased at 0.5 °C
per minute. Five samples were measured concurrently in the double-
beam mode. At temperatures below 15 °C the sample compartment
was flushed with dry nitrogen gas.
The melting temperatures and thermodynamic parameters were
obtained using Varian Cary software (Version 02.00). The experi-
mental absorbance vs temperature curves were converted into a
fraction of strands remaining hybridized (R) vs temperature curves
by fitting the melting profile to a two-state transition model, with
linearly sloping lower and upper base lines. The melting temper-
atures (Tm) were obtained directly from the temperature at R )
0.5. The final Tm was an approximation of at least five to eight
measurements. The thermodynamic parameters were determined
using two different methods20 as described below:
(i) from the width at the half-height of differentiated melting
curve (Table 1, column 4). The fraction of strands remaining
hybridized (R) vs temperature curves were converted into dif-
ferentiated melting curves (δR/δ(Tm-1) vs Tm) using Varian Cary
software (Version 02.00). The width of the of the differentiated
melting curve at the half-height is inversely proportional to the van’t
Hoff transition enthalpy; for a bimolecular transition ∆H ) 10.14/
(T1-1-T2-1) where T1 is the lower temperature at one-half of (δR/
δ(Tm-1)) and T2 is the upper temperature at one-half of (δR/
δ(Tm-1)).20 The final -∆H is the average of at least 10 measurements.
Full experimental data are given in Supporting Information Tables
S2-S4 and in ref13 for OL1 and OL3.
(ii) from the van’t Hoff plot of ln K vs 1/Tm (Table 1, columns
6-7). For a bimolecular transition of self-complementary strands
the equilibrium constant K ) R/[2C(1 - R)2] where C is the total
strand concentration (C ) 2 × 10-6 M). The van’t Hoff plot (ln K
vs 1/Tm) is linear with -∆H/R as the slope and ∆S/R as the intercept
(R is the universal gas constant 1.986 cal/mol/K). All fitting and
calculation operations were done using Varian Cary software
(Version 02.00) using settings for a bimolecular transition of self-
complementary strands. The final -∆H and -∆S is the average of
at least 10 measurements. Full experimental data are given in
Supporting Information Tables S2-S4 and in ref13 for OL1 and
OL3.
Acknowledgment. We thank Binghamton University, NIH (R01
GM071461 to E.R. and R01 GM055237 to M.E.), and NSF-NATO
(fellowship DGE-0410935 to A.K.) for support of this research.
We also thank Dr. Annie He´roux for the X-ray data collection at
beamline X25 of the National Synchrotron Light Source (NSLS),
Brookhaven National Laboratory, New York. Financial support for
the beamline comes principally from the Offices of Biological and
Environmental Research and of Basic Energy Sciences of the US
Department of Energy, and from the National Center for Research
Resources of the National Institutes of Health.
Supporting Information Available: Experimental procedures,
details of thermal melting and osmotic stress experiments, and
copies of 1H and 13C NMR data. This material is available free
of charge via the Internet at http://pubs.acs.org.
The changes in the number of water molecules associated with
the melting process ∆nW were determined as described previously
by Spink and Chaires14 and by us:13 ∆nW ) -∆H/R)[d(Tm-1)/
d(lnaw)], where -∆H is the enthalpy determined from the width at
the half-height of differentiated melting curve in pure buffer and R
is the universal gas constant (1.986 cal/mol/K). The experimentally
determined values of water activity (lnaw) at given cosolute
JA904926E
(21) Berger, I.; Kang, C. H.; Sinha, N.; Wolters, M.; Rich, A. Acta Cryst.
D 1996, 52, 465–468.
(22) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307–326.
(23) Vagin, A. A.; Teplyakov, A. J. Appl. Crystallogr. 1997, 30, 1022–
1025. Collaborative Computational Project, Number 4, Acta Cryst. D
1994, 50, 760-763.
(24) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Cryst. D 1997,
53, 240–255.
(25) Bru¨nger, A. T. Nature 1992, 355, 472–475.
(19) Puglisi, J. D.; Tinoco, I., Jr. Methods Enzymol. 1989, 180, 304–324.
(20) Breslauer, K. J. Methods Enzymol. 1995, 259, 221–242.
9
J. AM. CHEM. SOC. VOL. 131, NO. 41, 2009 14937