A novel silver(I)-mediated DNA base pair

Article abstract of DOI:10.1021/ja0279951

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base p

Full text of DOI:10.1021/ja0279951

Published on Web 10/25/2002
A Novel Silver(I)-Mediated DNA Base Pair
Nicole Zimmermann, Eric Meggers, and Peter G. Schultz*
The Scripps Research Institute, Department of Chemistry, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received August 2, 2002
Scheme 1. Synthesis of the SPy Phosphoramidite 1a
Selective Watson-Crick hydrogen bonding between the natural
base pairs dA:dT and dC:dG is the key element for information
storage and retrieval in DNA. Over the years efforts to find
additional base pairs have focused on bases that pair through altered
hydrogen-bonding patterns1 or hydrophobic interactions.2,3 We and
others recently reported a third strategy which involves metal-
dependent pairing of two nucleobases.4,5 Our first-generation
metallo-base pair consisted of a planar tridentate pyridine-2,6-
dicarboxylate (Dipic) nucleobase and a monodentate pyridine (Py)
nucleobase.4 When these bases are introduced into the middle of a
15-nucleotide duplex, the duplex displays thermal stability in the
presence of equimolar Cu2+ comparable to that of a duplex with a
dA:dT base pair at the same site. However, this metallo-base pair
has only modest pairing selectivity. To further explore the scope
of metallo-base pairing in DNA and to improve the properties of
our first-generation metallo-base pair, we are investigating additional
metal-ligand complexes. According to the principle of hard and
soft acids and bases (HSAB-principle), replacement of the O-atoms
of the Dipic-ligand with softer donor atoms such as S or N should
improve the stability of complexes with softer, late transition
metals.6 We therefore decided to substitute the Dipic ligand with
the S,N,S-ligand 2,6-bis(ethylthiomethyl)pyridine (SPy) which is
known to form stable complexes with a variety of different transition
metals (Figure 1).7
a Conditions: (a) SOCl2, THF (86%). (b) NaSCH3, EtOH/CH2Cl2
(10:1) (97%). (c) TFA, CH2Cl2 (83%). (d) DMTr-Cl, NEt3, py (67%). (e)
(iPr)2NP(Cl)CH2CH2CN, (iPr)2NEt, CH2Cl2 (76%).
Table 1. Sequence of the 15-mer DNA Duplex 7 and
Denaturation Temperatures (Tm) for Various Combinations of
X and Ya
5′-CACATTAXTGTTGTA-3′
3′-GTGTAATYACAACAT-5′
7
X )
Y )
SPy
Py
A
T
C
G
SPy
42.5
(23.4)b
35.0
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
Figure 1. Replacement of the Dipic nucleobase with a 2,6-bis(ethylthio-
methyl)pyridine (SPy) nucleobase.
Py
25.4
25.0
30.6
26.1
d
(23.5)b
30.5
(24.6)b
n.d.c
A
T
26.5
39.4
38.6
31.4
26.6
34.2
27.5
30.5
The corresponding SPy phosphoramidite 1 was synthesized from
diol 2, an intermediate in the synthesis of the Dipic nucleobase
(Scheme 1).4 After conversion to the dichloride 3, treatment with
sodium thiomethoxide afforded the 2,6-bis(ethylthiomethyl)pyridine
nucleoside 4. Tritylation followed by phosphitylation provided
phosphoramidite 1. Incorporation of 1 into the 15-nucleotide 7 was
achieved by standard protocols for automated DNA synthesis, and
identities of all oligonucleotides were confirmed by MALDI-TOF
MS. The thermal stability of the duplexes 7 (X, Y ) dSPy, dPy,
dA, dT, dC, and dG) in the presence of different transition metals
(Ni2+, Pd2+, Pt2+, Cu2+, Ag+, Au3+) was evaluated by determining
their melting temperatures (Tm) using UV spectroscopy.8 Of these
metals only Ag(I) shows a metal-dependent base pairing, and the
corresponding Tm-values are reported in Table 1.
29.4
n.d.c
(39.1)b
25.3
C
G
29.6
30.1
n.d.c
n.d.c
32.5
30.1
25.5
41.1
39.1
d
28.1
(39.6)b
a Melting temperature experiments were carried out on a Cary 300-Bio
UV/vis spectrophotometer. The heating rates were 0.5 °C min-1. Conditions:
5 mM sodium phosphate, 50 mM sodium perchlorate, pH 7 with 1 µM
silver(I) nitrate and 1 µM DNA duplex. b Melting temperature in the absence
of silver(I) nitrate. c Not determined. d No sigmoidal melting curve was
observed in the temperature range between 15 and 65 °C.
The dSPy:dSPy base-pair shows a very high pairing stability in
the sequence context of duplex 7. In the presence of one equivalent
of Ag(I) this self-pair is even more stable than the natural dC:dG
and dA:dT base pairs (Tm ) 42.5, 41.1, and 39.4 °C for dSPy:dSPy,
dC:dG, and dA:dT, respectively, Table 1) (Figure 2). The mixed
* To whom correspondence should be addressed. E-mail: schultz@scripps.edu.
9
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J. AM. CHEM. SOC. 2002, 124, 13684-13685
10.1021/ja0279951 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Fifteen-nucleotide duplexes containing multiple metallo-base
pairs (S ) SPy, P ) Py).
In summary we have generated a novel silver(I)-mediated
metallo-base pair consisting of two 2,6-bis(ethylthiomethyl)-pyridine
SPy nucleobases. This self-pair has a remarkably high pairing
stability and selectivity which rivals that of the natural base pairs
dA:dT and dC:dG. Efforts are underway to determine the exact
coordination geometry of this new base pair by X-ray analysis. In
addition, we plan to investigate potentially novel electronic proper-
ties of DNA duplexes containing multiple metallo-base pairs.
Figure 2. Ag(I)-mediated base pairs between dSPy:dPy and dSPy:dSPy.
dSPy:dPy base-pair is stabilized by 11.5 °C in the presence of Ag(I)
and is only 4.4 °C less stable than a dA:dT base pair (Tm ) 35.0
and 39.4 °C for dSPy:dPy and dA:dT, respectively). It is noteworthy
that the dPy-dPy self-pair is only stabilized by 0.8 °C in the
presence of Ag(I) in this sequence context. For comparison, Tm-
values of the natural base pairs dA:dT and dC:dG increase by
0.3 and 1.5 °C, respectively, in the presence of one equivalent
Ag(I). Shionoya and co-workers recently reported a much higher
increase in Tm of 6.8 °C for exactly the same dPy-dPy base pair in
a 21-nucleotide dT10PyT10/dA10PyA10 duplex, indicating that the
stabilization is context-dependent.5b,9 The relationship between
thermal stability and increasing number of S-donor atoms in the
series dPy:dPy, dSPy:dPy, and dSPy:dSPy clearly shows the
stabilizing effect of additional thioether substituents on the stability
of the metallo-base pair. Ag(I) generally has a high affinity for
sulfur ligands but normally prefers a linear, trigonal, or tetrahedral
metal coordination.10 The coordination geometry of the dSPy:dSPy
self-pair with six potential donor atoms is therefore not obvious,
and efforts are underway to obtain a structure of a DNA duplex
containing this metallo-base pair.
To store genetic information, an unnatural base pair should not
only be highly stable but also highly selective against mispairing
with the natural nucleobases. Mispairs with the dSPy nucleobase
are destabilized by 12.0-13.1 °C relative to dSPy:dSPy, and by
4.5-5.6 °C relative to dSPy:dPy (Table 1). For comparison,
mispairs between the natural nucleobases are 6.9-15.6 °C less
stable than natural base pairs in this sequence context. Mispairs
with the Py nucleobase are destabilized by 4.4-10.0 °C relative to
dSPy:dPy. These data suggest that the dSPy:dSPy self-pair exhibits
pairing stability and selectivity rivalling that of the natural base
pairs, whereas the dSPy:dPy shows a moderate pairing stability
and selectivity.
Acknowledgment. We thank the A. v. Humboldt Foundation
for a Feodor Lynen Fellowship for N.Z. We are also grateful to
the German Research Foundation for a fellowship for E.M. This
work was supported by the National Institutes of Health (GM
64403).
Supporting Information Available: Experimental procedures and
characterization data (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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Automated DNA synthesis offers a convenient method for
constructing DNA duplexes containing metal ions in defined
locations. Encouraged by the high pairing stabilities of both the
dSPy:dSPy and the dSPy:dPy metallo-base pair, oligonucleotides
containing multiple metallo-base pairs were synthesized (Figure 3).
UV-melting experiments revealed that the dSPy:dSPy self-pair can
replace natural base pairs with these duplexes at multiple sites and
still form stable duplexes (Tm ) 39.1 and 44.6 °C for 8 and 9,
respectively).11 The stability of the mixed dSPy:dPy base pair is
apparently too low to allow the substitution of natural base pairs
at multiple sites (Tm ) 37.0 and 25.6 °C for 10 and 11, respectively).
(8) Tm-data for all other metal ions except Ag+ is not shown.
(9) In contrast to ref 5b there is no further significant increase in Tm for the
dPy:dPy self-pair using more than one equivalent of Ag(I) within the
sequence context of duplex 7.
(10) Cotton, F. A.; Wilkinson, G.; Murillo, C. A.; Bochmann, M. In AdVanced
Inorganic Chemistry, 6th ed.; John Wiley & Sons: New York, 1999; pp
1084-1092.
(11) Conditions: 5 mM sodium phosphate, 50 mM sodium perchlorate, pH 7,
1 µM DNA duplex and 2 µM (8/10)/3 µM (9/11) silver(I) nitrate.
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