Duplex structure of a minimal nucleic acid

Article abstract of DOI:10.1021/ja802788g

The crystal structure of an 8-mer (S)-GNA duplex is presented. As a tool for phasing, the anomalous diffraction of two copper(II) ions within two artificial metallo-base pairs was employed. The duplex structure confirms a canonical Watson-Crick base pairing scheme of GNA with antiparallel strands. The duplex secondary structure is distinct from canonical A- and B-form nucleic acids and can be described as a right-handed helical ribbon wrapped around the helix axis, resulting in a large hollow core. Most intriguingly, neighboring base pairs slide strongly against each other, resulting in extensive interstrand base-base hydrophobic interactions along with unusual hydrophobic intrastrand interactions of nucleobases with their backbone. These results reveal how a minimal nucleic acid backbone can support highly stable Watson-Crick-like duplex formation. Copyright

Full text of DOI:10.1021/ja802788g

Published on Web 06/05/2008
Duplex Structure of a Minimal Nucleic Acid
Mark K. Schlegel, Lars-Oliver Essen,* and Eric Meggers*
Fachbereich Chemie, Philipps-UniVersita¨t Marburg, Hans-Meerwein Strasse, D-35043 Marburg, Germany
Received April 16, 2008; E-mail: essen@chemie.uni-marburg.de; meggers@chemie.uni-marburg.de
All forms of life use nucleic acids containing ribose or deox-
yribose sugars as their genetic material. In order to understand the
necessity for these structurally complicated pentose repeating units,
researchers have investigated nucleic acids with sugar alternatives.1–3
We recently reported a glycol nucleic acid (GNA) with an acyclic
three-carbon propylene glycol phosphodiester backbone (Figure
1a).4 GNA is structurally the most simplified solution for a
phosphodiester-containing nucleic acid backbone and thus consti-
tutes a promising candidate for initial genetic molecules of life.5,6
In addition, due to its unique combination of high duplex stability,
base pairing fidelity, and easy synthetic access of its nucleotides,
GNA comprises an interesting scaffold for future nucleic acid
nanotechnology.
We have now determined the crystal structure of an (S)-GNA
duplex from the self-complementary strand 3′-CGHATHCG-2′ by
X-ray crystallography. The GNA nucleotide H contains the artificial
Figure 1. (a) Constitution of DNA and (S)-GNA oligonucleotides. (b)
Comparison of Watson-Crick base pairs with the hydroxypyridone-Cu
base pair used in this study.
hydroxypyridone nucleobase which forms highly stable homobase
pairs in the presence of copper(II) ions (see Figure 1b).7,8 This
metallo-base pairing scheme was utilized as a convenient handle
to site-selectively introduce two heavy atoms per duplex for phasing
the crystallograhic data.9 In addition, we speculated that the high
stability of duplexes containing such a metallo-base pairing scheme
might be advantageous for growing high quality crystals. In fact,
in the presence of 2 equiv of copper(II) ions, the self-complementary
strand 3′-CGHATHCG-2′ forms a duplex with the astonishing high
melting temperature of 78 °C (4 µM duplex, 10 mM phosphate
buffer, 25 mM NaCl, pH 7.0). For comparison, the related DNA
and GNA sequences 5′(3′)-CGAATTCG-3′(2′) display melting
points of only 26 and 40 °C, respectively, under the same conditions.
Crystals which diffract to 1.3 Å were obtained from a chro-
matographically purified duplex using the sitting drop vapor
diffusion method and 2-methyl-2,4-pentanediol as a precipitant. The
asymmetric unit of the refined structure contains one single strand,
one copper ion, and 86 water molecules (see Supporting Information
for crystallographic data). Individual duplexes are coaxially stacked,
thereby forming a continuous helix within the crystal (Figure 2).
The right-handed (S)-GNA double helix differs significantly from
Figure 2. Overall GNA duplex structure. (a) Continuous packing of octamer
duplexes along the crystallographic z-axis. (b) View along the z-axis with
the canonical A- and B-form nucleic acid helices possessing a very
large helical pitch of 60 Å with 16 residues per turn and a large
helical rise (for a more detailed comparison with B- and A-DNA,
see Table 1).10–12 The base pairs are displaced from the helix axis
(x-displacement) by 5.1 to 8.6 Å, resulting in a very large elliptic
hollow core (Figure 2b). The GNA helix possesses only one large
groove, corresponding to the canonical minor groove, whereas it
lacks a major groove which is instead a convex surface. Therefore,
the GNA helix structure might be best described as a helical ribbon
loosely wrapped around the helix axis. With this, the GNA duplex
resembles more the recently disclosed hexose containing nucleic
acids HNA and homo-DNA rather than the canonical B- and A-form
nucleic acids.13,14
approximate distances d1 ) 7.0 Å and d2 ) 4.5 Å (defined from the helix
axis to the center of the closest atom). (c) Packing of duplexes within the
crystal. The structure has been deposited in the Protein Data Bank under
PDB code 2JJA.
dinate to a central copper(II) ion in a square planar fashion with a
slight propeller twist of 15°. The metallo-base pair appears to fit
well into the overall helix structure without any major distortions
even though the C1′-C1′ distance of 12.7 Å is expanded by 2.0 Å
compared to the standard Watson-Crick base pairs (Figure 3).
Within the crystallized 8-mer duplex, the propylene glycol
nucleotides adopt two different conformations with respect to the
torsional angles between C2′-O and C3′-O (Figure 4b). Whereas
nucleotides of Watson-Crick base pairs maintain a gauche
conformation with an average torsional angle γ of 70°, the glycol
at the hydroxypyridone nucleotides assume an anti conformation
All natural base pairs are engaged in standard Watson-Crick
hydrogen bonding, whereas the two hydroxypyridone bases coor-
9
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10.1021/ja802788g CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Comparison of Average Helical Parameters for GNA,
Maybe the most interesting feature of this GNA duplex structure
is the large average slide between neighboring base pairs of 3.4 Å
(Figure 4). This is a consequence of the large backbone-base
inclination, ranging for this duplex from 42 to 50° as compared to
0° for B-DNA but similar to the unnatural hexose nucleic acids.13,14
This inclination results in an almost complete absence of intrastrand
base-base stacking, which is the predominant stacking interaction
in A- and B-form nucleic acids, but extensive interstrand base-base
stackings. In order to compensate for the solvent-exposed base
resulting from the large base pair slide, the CH2 group of the
propylene glycol backbone is participating in packing against
nucleobases of the same strand. Thus, in this simplified GNA double
helix, the backbone is directly involved in hydrophobic interactions
with the π-system, which might contribute to the high duplex
stability of GNA.
B-DNA, and A-DNAa
(S)-GNA
B-DNA
A-DNA
helical sense
residues per turn
helical pitch (Å)
helical rise (Å)
x-displacement (Å)
tilt (°)
right
16
60
3.8
-7.0
0
-2.8
22.9
-3.4
5.4
right
10
34
3.4
0.1
-0.1
0.6
36
right
12
34
2.9
-4.2
0.1
8.0
31
-1.5
5.9
roll (°)b
twist (°)b
slide (Å)b
0.2
7.0
P-P distance (Å)c
a Data for GNA were calculated using the program Curves (ref 12).
Data for B- and A-DNA were taken from refs 10 and 11. b Local base
pair step parameters. c Average intrastrand P-P distances.
In conclusion, the here presented GNA duplex structure reveals
how a minimal nucleic acid backbone can support antiparallel
duplex formation in a Watson-Crick fashion. With its helical ribbon
structure, the GNA double helix differs significantly from the
canonical A- and B-form nucleic acids. Particularly intriguing are
the extensive interstrand base-base stacking interactions and the
participation of the GNA backbone in hydrophobic packing against
nucleobases. Efforts to learn from this structure about the intrinsic
reasons for the high duplex stability are in progress.
Acknowledgment. Financial support from LRSM-MRSEC
(University of Pennsylvania) and the Philipps-University Marburg
are gratefully acknowledged. We thank Prof. Martin Egli (Vander-
bilt University) for helpful discussions.
Supporting Information Available: Experimental details, spectro-
scopic data, and crystallographic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 3. Electron density of the H-Cu-H base pair (a) and the terminal
G-C base pair (b). The C1′-C1′ distances are indicated.
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Figure 4. Details of the GNA duplex structure. (a) A single GNA octamer
duplex. (b) The backbone conformation with gauche and anti referring to
the torsional angle between C2′-O and C3′-O. (c) Interstrand stacking of
two adjacent base pairs.
(γ ) 165 and 172°) (Figure 1a for annotations, Figure 4). As
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