5′-Tethered Stilbene Derivatives as Fidelity- and Affinity-Enhancing Modulators of DNA Duplex Stability

Article abstract of DOI:10.1021/ja0394434

A series of 5′-linked stilbene-DNA conjugates with different substituents in the distal aromatic ring of the stilbene was prepared, and the effect of the modifications on duplex stability was determined via UV-melting curves. A trimethoxystilbene derivative as a 5′-substituent increases duplex melting points by up to 12.2 °C per modification. With this alkoxystilbene substituent, terminal mismatches in DNA duplexes lower the melting point by up to 23.4 °C over the perfectly matched control, whereas terminal mismatches in unmodified DNA cause melting point depressions of no more than 6.1 °C. An aminomethylstilbene substituent linked to an oligopyrrolamide minor groove binder increases the melting point of an all-A/T decamer by up to 32.7 °C, thus shifting the melting point into a range typical for duplexes with statistical G/C-content. An affinity- and selectivity-enhancing effect was also observed when the trimethoxystilbene cap was employed on a small DNA microarray. The phosphoramidite of the trimethoxystilbene can be readily employed in automatic DNA synthesis, facilitating the generation of DNA chips with improved fidelity. Copyright

Full text of DOI:10.1021/ja0394434

Published on Web 03/26/2004
5′-Tethered Stilbene Derivatives as Fidelity- and Affinity-Enhancing
Modulators of DNA Duplex Stability
Zeynep Dogan, Ralph Paulini,† Jan A. Rojas Stu¨tz, Sukunath Narayanan, and Clemens Richert*
Institute for Organic Chemistry, UniVersity of Karlsruhe (TH), D-76131 Karlsruhe, Germany
Received November 5, 2003; E-mail: cr@rrg.uka.de
Table 1. UV-Melting Pointsa (°C) of DNA Duplexes
Oligonucleotides serve as hybridization probes, primers, and
b
b
biomedical agents. DNA chips are arrays of immobilized oligo-
nucleotides for massively parallel hybridization.1 The sequence
selectivity of oligonucleotides themselves is limited, and cross
hybridization between strands with partial complementarity limits
the usefulness of DNA chips.2 The termini of duplexes are hot spots
of low base pairing fidelity due to fraying and few neighboring
base pairs affected. Even for short oligonucleotides, a mismatch at
the terminus lowers the UV-melting point by only a few degrees
and the free energy of binding by <1 kcal/mol, both for DNA3
and for RNA.4 This is less than the variation in binding constants
between strands with different sequence and G/C content.5 Because
in a typical DNA chip experiment,2 over 105 probe sequences
compete for their targets, the low selectivity at the termini is
significant.
Since nature did not evolve DNA to serve as a hybridization
probe, but as a polymeric carrier of information read by fidelity-
enhancing polymerases, chemically modifying oligonucleotides may
improve fidelity. Some polymerase primers employ short sequences
with fidelity-enhancing molecular appendages to achieve improved
selectivity,6 but routine protocols for DNA chips do not employ
fidelity-enhancing elements for the termini of DNA probes. Wider
searches for such elements have been limited to acylamido
substituents7 that have to be introduced through peptide coupling
to aminodeoxynucleotides. Here, we report on fidelity-enhancing
5′-caps that can be introduced via automated phosphoramidite
chemistry.
duplex
150 mM
1M
∆Tm to control (v)c
(1v)2
31.6 ( 1.7
50.6 ( 0.4
39.1 ( 3.9
43.6 ( 1.1
48.9 ( 0.5
51.9 ( 0.4
43.9 ( 0.7
42.9 ( 1.3
29.8 ( 0.5
38.2 ( 0.5
32.7 ( 0.3
44.9 ( 0.8
31.5 ( 0.8
36.0 ( 0.9
30.0 ( 1.0
41.2 ( 0.3
27.2 ( 0.7
36.9 ( 0.7
33.5 ( 0.5
55.3 ( 0.7
n.d.
-/-
19.0/21.8
7.5/-
(1a)2
(1b)2
(1c)2
47.1 ( 0.5
54.1 ( 0.3
55.8 ( 0.2
47.7 ( 0.3
46.1 ( 0.5
36.1 ( 0.3
45.5 ( 0.6
38.8 ( 0.9
50.3 ( 0.9
35.9 ( 0.6
42.9 ( 0.5
38.5 ( 3.2
46.9 ( 0.4
35.2 ( 0.5
45.4 ( 1.7
19.0 ( 0.5
25.2 ( 0.7
33.0 ( 0.4
43.5 ( 0.9
27.4 ( 0.9
51.7 ( 1.4
24.6 + 1.3
12.0/13.6
17.3/20.6
20.3/22.3
12.3/14.2
11.3/12.6
-/-
(1d)2
(1e)2
(1f)2
(1g)2
2Tv:7
2Te:7
2Cv:9
2Ce:9
2Av:10
2Ae:10
2Gv:8
2Ge:8
2Tv:11
2Te:11
3v:12
3h:12
3i:12
8.4/9.4
-/-
12.2/11.5
-/-
4.5/7.0
-/-
11.2/8.4
-/-
9.7/10.2
-/-
<15
17.3 ( 0.8
22.6 ( 0.7
34.8 ( 1.0
19.1 ( 0.9
41.9 ( 0.8
n.d.
-/6.2
-/14.0
-/24.5
-/8.4
3j:12
3k:12
3l:12
-/32.7
-/5.6
3m:12
a Mean of three Tm’s ( SD at pH 7, 3.0 ( 0.8 µM strand concentration
(entries 1-8; 19-25), or 1.5 ( 0.2 µM strand concentration (entries 9-18).
b [salt]; entries 1-18, NH4OAc; entries 19-25, NaCl in 10 mM phosphate
buffer. c At 150 mM/1 M salt.
Table 2. Melting Points for Duplexes with Terminal Mismatches
The present caps are stilbenes, a class of compounds known to
bind to DNA.8,9 Stilbene-DNA conjugates form very stable bridged
hairpins10 and are well known from elegant work on electron
transfer.11 To our knowledge, the effect of singly appended stilbenes
on base pairing fidelity has not been studied. However, Letsinger,
Lewis, Egli, and co-workers have identified diethers12 and stilbene-
carboxamides13 as optimized bridges for hairpins. Singly linked
stilbenecarboxamide a (Figure 1) was the lead for our study. To
exclude possible (but unlikely) intercalation of the stilbene ring
system, tricyclic b was prepared, which cannot intercalate. Six
different carboxylic acids were coupled on-support to protected
DNA with a 5′-aminopropanol residue (Supporting Information),
followed by deprotection, producing 1a-g.
Tm (°C)a
∆Tm
∆Tm for control duplexb
b
duplex
mismatch
2Te:8
2Te:9
2Te:10
2Ae:7
2Ce:7
2Ge:7
3l:13
T:C
T:G
T:T
A:A
C:A
G:A
T:C
T:G
T:T
35.8 ( 0.9
39.0 ( 0.9
36.5 ( 0.8
29.5 ( 1.5
26.9 ( 0.7
30.5 ( 0.9
34.9 ( 1.5
37.7 ( 2.4
37.4 + 3.7
-9.7
-6.5
-9.0
-2.1
-1.4
-1.9
-2.2
-6.1
-4.2
-13.4
-23.4
-16.4
-7.0
c
-
c
3l:14
3l:15
-4.2
-4.5
-
c
-
a Mean of three Tm’s ( SD; 1.5 µM strands and 1 M NH4OAc (entries
1-6), or 3 µM strands, 150 mM NaCl, 10 mM phosphate (entries 7-9).
b To perfectly matched duplex. c Control too low for accurate determination.
UV-melting points of self-complementary 1a-g showed strong
interactions between stilbenes and DNA, as evidenced by melting
point (Tm) increases (Table 1). For (1e)2, the highest melting points
were detected, followed by 1a, and electron deficient pentafluoride
1d. Difluoride 1c, aminomethylstilbene 1f, and 1g each gave Tm
increases in the range of 6-7 °C per modification, whereas tricyclic
1b showed only half that effect. For the most duplex-stabilizing
cap e, phosphoramidite 5 was synthesized, which can be used
directly on DNA synthesizers. With 5, modified sequences 2Ae-
Te were prepared. Mismatch discrimination at the terminus was
improved in the presence of e, both for the terminal and for the
penultimate base pair of sequence 2T (Table 2 and Table S1,
Supporting Information). The affinity- and fidelity-enhancing effect
was also found for A:T, C:G, and G:C as terminal base pairs (Tables
1 and 2), with ∆Tm’s of up to -23.4 °C (∆∆G° of 6.3 kcal/mol)
for a terminal C:A mismatch. An overhang in the target strand
(sequence 11), a situation typical for hybridization to longer DNA,
did not reduce the duplex-stabilizing effect. In fact, the +10.2 °C
∆Tm over control for 2Te:11 at 1 M salt is one of the higher ∆Tm’s
found for cap e and octamer 2. It is greater than that for 2Te:7.
† Current address: Organic Chemistry Laboratory, ETH Hoenggerberg, HCI,
CH-8093 Zurich, Switzerland.
9
4762
J. AM. CHEM. SOC. 2004, 126, 4762-4763
10.1021/ja0394434 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Figure 1.
rich sequences. More elaborate minor groove binders17 should bind
other sequences. Modified hybridization probes may thus alleviate
both poor base pairing fidelity at the termini and the low duplex
stability of A/T-rich sequences, two issues central to increasing
the fidelity of DNA chips. High fidelity DNA chip data are
needed,18 and they may be obtained, if better biostatistics are
combined with improved molecular recognition on the chip.
Acknowledgment. Technical assistance by A. Hochgesand and
support by DFG (RI 1063/2; FOR 434) are acknowledged.
Supporting Information Available: Experimental protocols, NMR
or MS spectra for all new compounds (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 2. Hybridization of 4Te/4Ce/4Tv/4Cv with Cy3-labeled 16; (left)
integration of fluorescence of spots, (right) fluorescence scan.
This encouraged us to attach the trimethoxystilbene to hybridiza-
tion probes immobilized on glass surfaces as part of a small DNA
microarray.14 Incubation with target 16 gave a more than 2-fold
stronger signal for 4Te over 4Tv (Figure 2). Even without
optimizing hybridization conditions and without picking a well
discriminating base pair, there was a clearly detectable discrimina-
tion against a terminal mismatch (4Ce), which was almost absent
for the unmodified controls (4Tv/4Cv).
Next, it was tested whether stilbene caps can bind synergistically
with oligopyrrolamides, when incorporated in DNA-minor groove
binder conjugates.15 When netropsin-derived pyrrolamides16 were
linked to all-A/T sequence 3 via aminomethyl stilbene g, introduced
through phosphoramidite 6, the resulting caps were strongly duplex
stabilizing (Table 1, entries 19-25). On the level of dimers, two
â-alanine linkers were better than one (caps i and j). A terminal
nitro group (cap k), which interferes with proper hydrogen bonding
in the minor groove by displaying an N-O moiety where an N-H
hydrogen bond donor is required, gave lower duplex stability.
Tetramer l, when linked through 2 â-alanines, gave a melting point
increase of 32.7 °C, shifting the Tm of the all-A/T duplex 3 into a
range typical for duplexes with higher G/C content. Control tetramer
m again had almost no effect on the Tm. Also, the base pairing
fidelity at the terminal base pair capped by the stilbene portion of
the DNA decamer is satisfactory (Table 2, entries 7-9).
Our data suggest that trimethoxystilbene caps selectively stabilize
duplexes with a matched terminal base pair. As binding elements
they may be called “orthogonal”, since they increase the affinity
for target strands without themselves offering binding motives for
other DNA sequences (as an elongation of the sequences would).
Molecular modeling shows that the methyl group of the 4′-methoxy
substituent protrudes out of the plane of the stilbene ring system
and may thus “gauge” the size of base pairs. The aminomethyl-
stilbene combined with minor groove binders produces “embracing
caps” that induce a very substantial melting point increase for A/T-
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