Journal of the American Chemical Society
COMMUNICATION
Table 1. Thermodynamic Stabilities of Selected DNA
Duplexes
duplex
sequence at 5 μM duplex
TM (°C)
ΔG (kcal/mol)
1
2
3
4
5
6
7
d(CCGG AAAA CGCC)
d(CCGG AAAHA CGCC)
63.4
61.0
58.3
60.9
51.1
44.4
54.7
-19.1 ( 0.6
-16.4 ( 0.4
-14.8 ( 0.3
-16.4 ( 2.3
-13.9 ( 0.2
-10.8 ( 0.4
-15.2 ( 0.7
d(CCGG AAACH A CGCC)
3
d(CCGG AAACH OHA CGCC)
2
d(CCGG (AH)4 CGCC)
d(CCGG (ACH )4 CGCC)
3
Figure 2. Structural water mimic interacting with the carbonyl of the
cross-strand dT one base pair removed.
d(CCGG (ACH OH)4 CGCC)
2
of hydration but additionally introduces further steric effects and
additional destabilization of the duplex. The sequence containing
four hydroxymethyl substituents in principle has the greatest
steric effects in the minor groove, but the hydroxyl groups appear
to provide complementary stabilizing interactions through a
modified water structure guided by favorable interactions from
the -CH2OH groups. A possible interaction mimicking the
corresponding water molecule is illustrated in Figure 2.
CD spectra of the various modified helices (Figure 3) also
offer some insight into the effects of various analogues. All of the
spectra suggest the presence of B-form helices. The CD spectra
for the native sequence 1 and sequence 5 containing four dc3A
residues are the most alike of the four spectra, suggesting similar
chromophoric stacking (dA vs dc3A) within the helix. The
interactions of the purine heterocycles are dramatically different
for duplex 6, in which four methyl groups have been added to the
central core of base pairs. This spectrum suggests a quite different
stacking of the dmc3A chromophores, leading to dramatic shifts
in the positive and negative displacements in the observed CD
spectrum. The final spectrum, for duplex 7, is intermediate in
character relative to those of duplexes 5 and 6. It seems likely that
the minor-groove interactions present with the structural water
mimic drive the conformation back toward a more nativelike
B-form, but much like the temperature data, the final native B-like
structure is not fully attained.
in dimethyl sulfoxide followed by treatment with fluoride ion.
Confirmation of the complete deprotection of the oligonucleo-
tides was obtained by mass spectral analysis.
We prepared three types of oligonucleotides: (i) those con-
taining 3-deazaadenine, (ii) those containing 3-deaza-3-methyl-
adenine, and (iii) those containing 3-deaza-3-hydroxymethyla-
denine. The design parameters were as follows: The first modified
oligos did not contain the N3 nitrogen, a site that is used in the
formation of the spine of hydration but otherwise introduces no
unfavorable steric effects. In the methyl-containing sequences, the
N3 nitrogen was also removed, and additional unfavorable steric
effects were introduced into the minor groove through the pre-
sence of one or more methyl groups. In the final sequences, hy-
droxyls were introduced onto the unfunctionalized methyls to
generate a covalently bound water mimic capable of interacting
by hydrogen bonding in the minor groove.
Thermodynamic parameters were obtained for the formation
of all duplexes (Table 1). Even the simple elimination of a single N3
nitrogen from a centrally located dA residue (duplex 2) resulted in a
2.4 °C decrease in the melting temperature (TM), consistent with
previous reports.11 In that same study, the introduction of three dA
residues lacking the N3 nitrogen resulted in a 12 °C change in TM,
which also compares favorably with the 12.3 °C reduction in TM
observed here with four deazaadenines present (duplex 5). We then
examined the methyl-substituted sequences (duplexes 3 and 6). The
3-deaza-3-methyl analogues both lack the N3 nitrogen but also add
steric bulk within the minor groove. Introduction of a single methyl
group (duplex 3) reduced the TM value by 2.7 °C relative to a
sequence containing a single 3-deaza analogue (duplex 2) and by
5.1 °C relative to the unmodified standard. The presence of four
analogues containing methyls (duplex 6) reduced the TM by a
dramatic 19.0 °C, attesting to the duplex instability resulting from
increased steric effects in the minor groove. The hydroxymethyl
group is sterically even larger than a methyl group and could have an
even more dramatic destabilizing effect should the hydroxy groups be
unable to take part in advantageous interactions in the minor groove.
Introduction of one -CH2OH group did result in helix destabiliza-
tion relative to the control sequence (compare the TM for duplex 4
with that for duplex 1). However, relative to the single-methyl
sequence (duplex 3), the TM for the hydroxymethyl sequence was
some 2.6 °C higher. This observation is further supported by the
sequence with four -CH2OH groups (duplex 7), which exhibited a
TM 10.3 °C higher than that for the sequence containing four
methyl groups. No significant additional cooperativity appeared to
be present for any analogue duplex.
The data presented here suggest that simple methyl groups
located at the C3 position of dA residues are sufficient for dra-
matic structural perturbation but that the introduction of a -OH
group as a structural water mimic assists in partial stabilization of
the analogue structure, presumably through interactions within
the minor groove.
The simplest interpretation of these results is that the absence
of hydrogen-bonding acceptors in the minor groove destabilizes
the duplex as a result of disruption of the ordered spine of hydra-
tion. The presence of four methyl groups also disrupts the spine
Figure 3. CD spectra (ellipticity vs wavelength at 3 μM duplex concen-
tration) of duplexes 1 (blue), 5 (red), 6 (green), and 7 (purple) obtained
in 20 mM sodium phosphate (pH 7) and 1 M NaCl.
1767
dx.doi.org/10.1021/ja1103684 |J. Am. Chem. Soc. 2011, 133, 1766–1768