Optimized DNA targeting using N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA

Article abstract of DOI:10.1039/b417101b

Incorporation of N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA (bipyridyl-functionalized 2′-amino locked nucleic acid) monomers into DNA strands enables high-affinity targeting of complementary DNA with excellent Watson-Crick selectivity in the presence

Full text of DOI:10.1039/b417101b

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Optimized DNA targeting using N,N-bis(2-pyridylmethyl)-b-alanyl
29-amino-LNA{
B. Ravindra Babu, Patrick J. Hrdlicka, Christine J. McKenzie and Jesper Wengel*
Received (in Cambridge, UK) 11th November 2004, Accepted 20th December 2004
First published as an Advance Article on the web 3rd February 2005
DOI: 10.1039/b417101b
shown in the caption below Table 1; the stepwise coupling yield of
amidite 3 was y98% using 1H-tetrazole as catalyst and 10 min
coupling time).
Incorporation of N,N-bis(2-pyridylmethyl)-b-alanyl 29-amino-
LNA (bipyridyl-functionalized 29-amino locked nucleic acid)
monomers into DNA strands enables high-affinity targeting of
complementary DNA with excellent Watson–Crick selectivity
in the presence of divalent metal ions. Positioning of bipyridyl-
functionalized 29-amino-LNA monomers in two complemen-
tary DNA strands in a ‘‘39-end zipper’’ constitution allows
modulation of duplex stability, i.e., a strong stabilizing effect
with one equivalent of divalent metal ion per bipyridyl pair, or a
strong destabilizing effect with an excess of divalent metal ions.
The effect of the bipyridyl-functionalized 29-amino-LNA
monomer X on duplex stability was studied by UV thermal
denaturation experiments (Tables 1 and 2). Single denaturation
transitions with hyperchromicities .5% were observed for all
duplexes involving modified ONs.{ A single incorporation into a
9-mer mixed base sequence (Table 1, ON1–ON3) induced a strong
increase in duplex stability towards the DNA complement
(DTm 5 +4 to +6 uC relative to the unmodified DNA:DNA
reference duplex). These experiments were conducted in the
presence of EDTA to remove traces of free divalent metal ions,
and similar Tm values have been reported for other N-acylated
29-amino-LNAs.12 Identical denaturation experiments conducted
in the presence of one equivalent or excess (10 equivalents) of Ni2+,
Cu2+ or Zn2+ ions induced further duplex stabilization against
complementary DNA with metal ion induced additional increases
in Tm values of +6 uC, +2 uC and +4/+5 uC, respectively. Addition
of divalent metals ions had no effect on the stability of the
reference DNA:DNA duplex (Table 1), duplexes formed between
ON1, ON2 or ON3 and their RNA complements (data not
shown), nor DNA:DNA duplexes containing a single N-benzoyl-
or N-(1-adamantylmethyl)carbonyl-29-amino-LNA monomer
(data not shown). An additive effect on DNA binding was
observed upon incorporation of two monomers X (ON4 and ON5;
overall increases in Tm values of approximately +23 uC, +13 uC and
+20 uC in the presence of Ni2+, Cu2+ or Zn2+, respectively). The
affinity-increase upon incorporation of monomer X occurs with
excellent Watson–Crick base pairing selectivity (Table 2). To the
Chemical modification of oligonucleotides (ONs) that structurally
mimic RNA has enabled high-affinity targeting of RNA for
diagnostic and therapeutic applications.1,2 The development of ON
analogues capable of high-affinity targeting of DNA has proven
significantly more difficult. Fully or partially modified LNA
(locked nucleic acid)3–5 and pyrene-functionalized ONs6–10 offer
solutions to this challenge although their general applicability is
hampered by, e.g., the tendency to self-complementarity5 or lack of
sequence generality and selectivity of binding.6,9,10 Continued
efforts towards general and sequence selective high-affinity DNA
targeting are therefore needed to improve the fidelity of DNA
diagnostics and to enable in vivo targeting of DNA. We herein
present an answer to this challenge employing ONs containing
bipyridyl-functionalized 29-amino-LNA monomers (X, Scheme 1).
We have recently reported that N-functionalized 29-amino-LNA
derivatives hybridize with complementary DNA or RNA with
binding affinities as observed for LNA.11–13 We therefore decided
to study 29-amino-LNA functionalized with metal-chelating
moieties aiming at modulating hybridization processes and at
making structurally well-defined metal arrays.§14–22
Phosphoramidite building block 3, suitable for the incorpora-
tion of monomer X into ONs, was synthesized as shown in
Scheme 1. Removal of the N-trifluoroacetyl group of 59-
O-dimethoxytrityl-29-amino-LNA nucleoside 1,11,12 followed by
treatment with N,N-bis(2-pyridylmethyl)-b-alanine23 in the pre-
sence of EDC gave nucleoside 2 as a 1:2 mixture of rotamers.",I
Subsequent phosphitylation of the 39-hydroxy group afforded
phosphoramidite 3" (56% yield from 1) which was used for
standard automated solid-phase synthesis of ON1–ON5 (Table 1;
.80% purity by ion exchange HPLC; MALDI-MS data are
{ Electronic supplementary information (ESI) available: table of synthe-
sized 13-mer ONs and thermal denaturation studies thereof; Sample
thermal denaturation curves; Procedure for molecular modelling;
Modelling structures of ON1:DNA. See http://www.rsc.org/suppdata/cc/
b4/b417101b/
Scheme 1 Reagents and conditions: (i) (a) sat NH3 in MeOH (ref. 12), (b)
N,N-bis(2-pyridylmethyl)-b-alanine, EDC?HCl, CH2Cl2, rt (77%, two
steps); (ii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite,
DIPEA, CH2Cl2, rt (73%); (iii) DNA synthesizer. T 5 thymin-1-yl;
DMT 5 4,49-dimethoxytrityl.
*jwe@chem.sdu.dk
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Table 1 9-mer ONs synthesized and thermal denaturation studies at different concentrations of divalent metal ionsa
Tm (DTm)/uC
Ni2+
Cu2+
Zn2+
+ EDTA
1 equiv.b
Excessc
28
1 equiv.b
Excessc
28
1 equiv.b
Excessc
28
DNA
DNA
59-GTG ATA TGC
39-CAC TAT ACG
28 (ref)
28
28
28
ON1
DNA
59-GTG AXA TGC
39-CAC TAT ACG
34 (+6)
32 (+4)
33 (+5)
38 (+10)
39 (+11)
34 (+6)
39 (+11)
47 (+19)
40 (+12)
38 (+10)
39 (+11)
51 (+23)
51 (+23)
53 (+25)
47 (+19)
48 (+20)
40 (+12)
38 (+10)
40 (+12)
51 (+23)
51 (+23)
24 (24)
50 (+22)
20 (28)
36 (+8)
34 (+6)
nd
36 (+8)
34 (+6)
nd
38 (+10)
37 (+9)
38 (+10)
48 (+20)
48 (+20)
36 (+8)
46 (+18)
50 (+22)
39 (+11)
37 (+9)
DNA
ON2
59-GTG ATA TGC
39-CAC XAT ACG
DNA
ON3
59-GTG ATA TGC
39-CAC TAX ACG
38 (+10)
48 (+20)
48 (+20)
31 (+3)
ON4
DNA
59-GTG AXA XGC
39-CAC TAT ACG
41 (+13)
41 (+13)
40 (+12)
42 (+14)
56 (+28)
41 (+13)
41 (+13)
20 (28)
39 (+11)
,10
DNA
ON5
59-GTG ATA TGC
39-CAC XAX ACG
ON1
ON2
59-GTG AXA TGC
39-CAC XAT ACG
ON1
ON3
59-GTG AXA TGC
39-CAC TAX ACG
47 (+19)
37 (+9)
ON4
ON5
a
59-GTG AXA XGC
39-CAC XAX ACG
Thermal denaturation temperatures [Tm values/uC (DTm 5 change in Tm value calculated relative to the DNA:DNA reference duplex)]
measured as the maximum of the first derivative of the melting curve (A260 vs. temperature) recorded in medium salt buffer ([Na+] 5 110 mM,
[Cl2] 5 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 mM concentrations of the two complementary strands, and a micromolar extinction
coefficient of 14.6 for monomer X; The Tm values are based on at least two independent denaturation experiments giving Tm values varying
within ¡ 0.5 uC. A 5 adenin-9-yl DNA monomer, C 5 cytosin-1-yl DNA monomer, G 5 guanin-9-yl DNA monomer, T 5 thymin-1-yl
b
DNA monomer; see Scheme 1 for structure of monomer X; ‘‘+EDTA’’ y0.1 mM EDTA. 1 equiv. refers to 1 equivalent of metal ion per X
monomer for experiments with DNA complements, and to 0.5 equivalent of metal ion per X monomer for experiments with ON1:ON2,
c
ON1:ON3 or ON4:ON5. [M2+] 5 10 mM; NiSO4?6H2O, CuCl2?2H2O and ZnCl2 were used; ‘‘nd’’ 5 not determined; MALDI-MS m/z
([M 2 H]2; found/calc): ON1, 3031/3033; ON2, 2960/2962; ON3, 2956/2962; ON4, 3309/3313; ON5, 3242/3242.
Table 2 Melting temperatures (Tm values) towards matched and
singly mismatched complementary DNAa
ON4 and ON5 with excess of Cu2+ ions. Conversely, upon
incorporation of two X monomers in a ‘‘1 + 1 59-end zipper’’
constitution (ON1:ON3), stabilization corresponding to an
additive effect of X monomers was induced with one equivalent
as well as with an excess of divalent metal ions. A very similar
affinity-enhancing effect on DNA binding and directional
preference was observed upon incorporation of monomer X in a
mixed 13-mer sequence.{ A similar hybridization pattern for ONs
modified with alkyl-linked terpyridine ligands at the 59-end (or
39-end) of duplexes in the presence of transition metals has been
reported, but high-affinity targeting of DNA was not realized.17
Encouraged by the effect of monomer X on DNA binding we
attempted to rationalize these results by simplified molecular
modelling studies.{ MMFF force field24 calculations of 9-mer
DNA-duplexes containing a single monomer X (ON1:DNA) or
two monomers X arranged in a ‘‘1 + 1 59-end zipper’’ constitution
(ON1:ON3) and one Zn2+-ion** indicate that the metal ion
induced increased binding affinity towards DNA may arise from
complexation of a divalent metal ion with the N,N-bis(2-
pyridylmethyl)-b-alanyl ligand, the phosphate backbone of either
of the two strands near the brim of the minor groove and solvent
molecules/counter ions [Fig. 1 (left) and S1,{ ON1:ON3 and
ON1:DNA, respectively].{{ The lowest energy model structure
of the 9-mer duplex where two monomers X are positioned in a
‘‘1 + 1 39-end zipper’’ constitution [ON1:ON2; Fig. 1 (right)]
containing a single Zn2+-ion suggests formation of an interstrand
DNA target (39-CAC TYT ACG)
+ EDTA
Ni2+ (10 mM)
Y:
A
T
G
C
A
T
G
C
59-GTG AXA TGC (ON1)
59-GTG AXA XGC (ON4)
34 17 19 14
38 24 24 21
40 24 19 20
51 34 32 31
a
Tm values for the matched sequences are shown in boldface type.
See below Table 1 for details.
best of our knowledge, the approach introduced herein constitutes
the most efficient way yet described of enhancing DNA binding
affinity with preserved Watson–Crick selectivity.
Hybridization studies involving two complementary 9-mers each
containing one or two X monomers revealed several interesting
features. When positioned in a ‘‘1 + 1 39-end zipper’’ constitution
(duplex ON1:ON2), addition of one equivalent of Ni2+ ion leads to
a remarkable increase in Tm value (+19 uC relative to ON1:ON2
measured with EDTA; +25 uC relative to the reference
DNA:DNA duplex). However, with an excess of Ni2+ ions,
significant destabilization was observed, even relative to the
reference DNA:DNA duplex. Similar experimental observations
were made with a ‘‘2 + 2 39-end zipper’’ constitution (ON4:ON5)
and, to a variable degree, also with Cu2+ and Zn2+ ions (Scheme 1).
Notably, no duplex formation above 10 uC was observed between
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Notes and references
§ Conjugates of metal chelators have been studied for, e.g., metal-mediated
base pairing,14–16 metal-mediated joining of the ends of complementary
ONs,17 modulation of DNA hairpin stability,18 metal- and template-
mediated ON assembly,19 association of metal ions at internal positions of
duplexes,20,21 and RNA cleavage by synthetic metallonucleases.22
"
13C NMR data for compound 2: d (CDCl3, major rotamer) 172.0, 164.3,
158.7, 158.6, 158.3, 150.3, 148.9, 144.6, 137.0, 136.8, 135.7, 135.5, 134.6,
130.2, 130.1, 128.2, 128.0, 127.1, 124.0, 123.4, 122.4, 122.3, 113.3, 113.2,
110.3, 89.1, 87.6, 86.6, 70.1, 64.2, 60.1, 59.8, 55.3, 51.8, 51.6, 32.7, 12.7. 31P
NMR data for compound 3: d (CDCl3) 150.6, 150.2, 149.0.
I The ratio between rotamers when bipyridyl-functionalized nucleotides
are present in an ON is unknown. The denaturation curves display smooth
single transitions (see ESI{).
Fig. 1 Lowest energy structure of ON1:ON3 (‘‘1 + 1 59-end zipper’’)
including one Zn2+-ion (to the left), and lowest energy structure of
ON1:ON2 (‘‘1 + 1 39-end zipper’’) including one Zn2+-ion (to the right).
For clarity hydrogens, sodium ions and bond orders have been omitted.
Colouring scheme: nucleobases, yellow; sugar–phosphate backbone, red;
N,N-bis(2-pyridylmethyl)-b-alanyl ligand, blue; Zn2+-ion, black.
** Zn2+ ions were used as a model divalent metal ion due to the availability
of their parameters in the MMFF force field.24 The Cu2+ and Ni2+ ions will
show greater ligand field preferences in their coordination geometries
compared with a Zn2+ ion. The consequent effect on binding modes and
topology is a likely origin of the observed differences in the thermal
denaturation temperatures.
complex between a divalent metal ion and the N,N-bis(2-
pyridylmethyl)-b-alanyl ligands of the two monomers X.{{ A
similar interstrand complexation in the ‘‘1 + 1 59-end zipper’’
constitution (ON1:ON3; see Fig. 1) seems precluded due to the
spatial separation of the N,N-bis(2-pyridylmethyl)-b-alanyl
ligands. Interestingly, the lowest energy structure of ON1:ON2
including two Zn2+-ions (results not shown) does not indicate
formation of an interstrand complex but rather points towards
complexation of divalent metal ions in a similar manner as
described for ON1:DNA and ON1:ON3. Although molecular
modelling does not provide compelling evidence for this, we
speculate that the observed destabilization of duplexes containing
monomers X in a ‘‘1 + 1 39-end zipper’’ constitution (ON1:ON2)
upon addition of excess divalent metal ions results from steric or
electrostatic repulsion of two spatially close metal complexes.
Additional biophysical studies have been initiated to further
elucidate the structural basis of the observed effects.
{{ The lowest energy structure of ON1:DNA (Fig. S2{) suggests that
complexation of a divalent metal ion buried in the minor groove with the
carbonyl functionality of the N,N-bis(2-pyridylmethyl)-b-alanyl ligand,
nearby nucleobases or solvent molecules is an alternative explanation for
duplex stabilization.
{{ The model structure does not yield a well-defined coordination complex
but shows the two metal chelators and backbone phosphate oxygen atoms
2+
˚
in proximity (approx. 3 A) of the Zn ion. It is clear that further
refinement of the model necessitates that the double helix is not restrained,
as was the case for our calculations. Also because of computing limitations,
the possible role of chloride ions in the metal coordination spheres was not
explored.
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A strategy for optimized high-affinity DNA targeting, using
bipyridyl-functionalized 29-amino-LNA in the presence of divalent
metal ions, has been introduced. The observed metal-induced
increases in duplex stability without compromising Watson–Crick
base-pairing selectivity suggest bipyridyl-functionalized 29-amino-
LNA9s as candidates for optimal probes for DNA targeting.
Furthermore, since positioning of bipyridyl-functionalized
29-amino-LNA monomers in two complementary DNA strands
allows engineering of duplex stability, probes with reduced (or no)
tendency for self-complementarity can be designed.
11 S. K. Singh, R. Kumar and J. Wengel, J. Org. Chem., 1998, 63, 10035.
A 29-amino-LNA is in this article defined as an ON containing at least
one 29-amino-29-deoxy-29-N,49-C-methylene-b-D-ribofuranosyl nucleo-
tide monomer.
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We thank The Danish National Research Foundation and
EU-FP6 (CIDNA; proposal no. 505669-1) for funding, Ms B. M.
Dahl, University of Copenhagen and Ms. Kirsten Østergaard,
University of Southern Denmark, for oligonucleotide synthesis,
and Dr M. Meldgaard, Exiqon A/S, and Dr Kenneth B. Jensen,
University of Southern Denmark, for MALDI-MS analysis.
B. Ravindra Babu, Patrick J. Hrdlicka, Christine J. McKenzie and
Jesper Wengel*
17 L. Zapata, K. Bathany, J.-M. Schmitter and S. Moreau, Eur. J. Org.
Chem., 2003, 1022.
Nucleic Acid Center,{ I Department of Chemistry, University of
Southern Denmark, DK-5230, Odense M, Denmark.
E-mail: jwe@chem.sdu.dk; Fax: +45 6615 8780; Tel: +45 6550 2510
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{ A research center funded by the Danish National Research Foundation
for studies on nucleic acid chemical biology.
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This journal is ß The Royal Society of Chemistry 2005
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