Reversible metal-dependent destabilization and stabilization of a stem-chelate-loop probe binding to an unmodified DNA target

Article abstract of DOI:10.1021/bc3003293

Herein, we report the discovery of a novel DNA probe with a stem-chelate-loop structure, wherein the stability of the probe-target duplex can be modulated lower or higher using a narrow concentration range of dilute transition metal ions (0.1-10 μM). Oligonucleotide probes containing two terpyridine (TPY) ligands separated by 15 bases of single-stranded DNA, with or without a flanking 5 base self-complementary DNA stem, were tested in thermal transition studies with linear target DNA and varying amounts of ZnCl ₂. Without the stem, addition of Zn²⁺ resulted only in reversible destabilization of the probe-target duplex, consistent with assembly (up to 1 equiv Zn²⁺) and disassembly (excess Zn²⁺) of the intramolecular Zn²⁺-(TPY)₂ chelate. Surprisingly, probes including both the intramolecular chelate and the stem gave a probe-target duplex that was reversibly destabilized and stabilized upon addition of Zn ²⁺ by ±5-7 °C, a phenomenon consistent with assembly and then disassembly of the entire stem-Zn²⁺-(TPY)₂ motif, including the DNA stem. Stem-chelate-loop probes containing dipicolylamine (DPA) ligands exhibited no metal-dependent stabilization or destabilization. The stem-Zn²⁺-(TPY)₂ motif is readily introduced with automated synthesis, and may have broad utility in applications where it is desirable to have both upward and downward, reversible metal-dependent control over probe-target stability involving an unmodified DNA target.

Full text of DOI:10.1021/bc3003293

Communication
pubs.acs.org/bc
Reversible Metal-Dependent Destabilization and Stabilization of a
Stem-Chelate-Loop Probe Binding to an Unmodified DNA Target
Joel R. Morgan,* David V. X. Nguyen, Angela R. Frohman, Sara R. Rybka, and John A. Zebala
Syntrix Biosystems, Inc., 215 Clay Street NW, Suite B-5, Auburn, Washington 98001, United States
S
* Supporting Information
ABSTRACT: Herein, we report the discovery of a novel
DNA probe with a stem-chelate-loop structure, wherein the
stability of the probe−target duplex can be modulated lower or
higher using a narrow concentration range of dilute transition
metal ions (0.1−10 μM). Oligonucleotide probes containing
two terpyridine (TPY) ligands separated by 15 bases of single-
stranded DNA, with or without a flanking 5 base self-
complementary DNA stem, were tested in thermal transition
studies with linear target DNA and varying amounts of ZnCl2.
Without the stem, addition of Zn2+ resulted only in reversible
destabilization of the probe−target duplex, consistent with assembly (up to 1 equiv Zn2+) and disassembly (excess Zn2+) of the
intramolecular Zn2+-(TPY)2 chelate. Surprisingly, probes including both the intramolecular chelate and the stem gave a probe−
target duplex that was reversibly destabilized and stabilized upon addition of Zn2+ by 5−7 °C, a phenomenon consistent with
assembly and then disassembly of the entire stem-Zn2+-(TPY)2 motif, including the DNA stem. Stem-chelate-loop probes
containing dipicolylamine (DPA) ligands exhibited no metal-dependent stabilization or destabilization. The stem-Zn2+-(TPY)2
motif is readily introduced with automated synthesis, and may have broad utility in applications where it is desirable to have both
upward and downward, reversible metal-dependent control over probe-target stability involving an unmodified DNA target.
ovel nucleic acid chemistries have potential use across
many new and existing applications in nanotechnol-
N
ogy,1−3 genomics,4,5 medicine,6−8 and chemistry.9,10 Probe
association/dissociation with a nucleic acid target according to
Watson−Crick base-pairing rules is the pivotal property
exploited in these applications, and is characterized by the
midtransition, or melting temperature (Tm).11 It would be
desirable to be able to tailor the thermodynamics of probe
association and dissociation to a native DNA target in a facile
manner for a particular application. Adding salt (e.g., NaCl,
MgCl2) is well-known to increase DNA duplex stability, but is a
nonspecific intervention that results in large changes in ionic
strength that can have unintended effects in a given application
(e.g., if enzymes are present that are sensitive to changes in
ionic strength).12 Previous attempts to orthogonally increase
duplex stability required installations of metal-chelate moieties
in both probe and target that preclude modulating binding
stability to native DNA.13,14 Herein, we report a new approach
for modulating Tm lower or higher as a function of dilute
transition metal-ion concentrations using a novel DNA probe
with a stem-chelate-loop structure (Figure 1).
Figure 1. Representative bimolecular fluorescence-quenching assay
design for detecting probe hybridization with target DNA. Target
hybridization results in stem and chelate dissociation, probe-target
duplex formation and fluorescence quenching. Key: Thick black line,
probe DNA; blue line, target DNA; green circle, metal−ion; gray circle
quencher; large red star, unquenched fluorophore; and small red star,
quenched fluorophore.
piperidine linked TPY,15,16 were added to (S)-dimethoxytrityl-
glycidol,17−19 and the resulting secondary alcohol was
phosphitylated (experimental details are provided as Support-
ing Information). The cyanoethyl phosphoramidites 1 and 2
were used together with the four canonical base phosphor-
amidities, to synthesize 6-carboxyfluorescein (FAM), Black-
Hole Quencher-120 (BHQ1), and chelate ligand (TPY and
DPA) modified oligonucleotides 3−10, by automated DNA
synthesis (Table 1). The fluorescent target oligonucleotide 10
was complementary to each probe’s 15-base nonstem DNA
sequence. A bimolecular fluorescence-quenching assay was
Our findings address the challenge of orthogonally
controlling probe-target duplex stability without modifying
the target nucleic acid or significantly altering solution ionic
strength, capabilities that together will find broad utility in
enzymatic reactions and other applications of probe associa-
tion/dissociation with native DNA.
Received: June 20, 2012
Revised: August 8, 2012
Published: September 18, 2012
Terpyridyl and dipicolyl phosphoramidites, 1 and 2, were
synthesized as in Scheme 1. The secondary amines, DPA or
© 2012 American Chemical Society
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Scheme 1. Synthesis of TPY and DPA Cyanoethyl
Phosphoramidites
Figure 2. Probe-target stability ('Tm) of stemless (4:10) and stemmed
(6:10) TPY-containing probe-target duplexes in the absence of free
transition metal-ions, i.e., 5 μM EDTA (E), or in the presence of the
indicated equivalents of ZnCl2; [probe] = 600 nM, [target 10] = 100
(a) HNR2, K2CO3, CH3CN, 80 °C; (b) 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite, 1H-tetrazole, CH3CN.
Table 1. Oligonucleotides: No-Chelate Control (3, 5),
Chelate-Ligand Modified (4, 6, 7, 8, 9), and Target (10)
nM, 10 mM MOPS, 1 mM MgCl2, pH = 7.5. Normalized melting
2+
̀
temperature ('Tm) at each E or Zn : 4:10 Tm = 4:10 Tm − 3:10 Tm
̀
and 6:10 Tm = 6:10 Tm − 5:10 Tm. Error bars are the SD for three
repetitions (Supporting Information).
a
#
modified oligonucleotide
CCA-(AAA)3-ACC-BHQ1
3
4
TPY- CCA-(AAA)3-ACC-TPY-BHQ1
ZnCl2 reversed the probe−target destabilization, returning the
'Tm to the value of the metal-free control (Figure 2, orange
segment). Maximum destabilization of 4:10 at 1 equiv of Zn2+
is consistent with assembly of all Zn2+-(TPY)2 chelates into
4(Zn2+) complexes (Figure 3B). Energy provided by the Zn2+-
5
CGCTC-CCA-(AAA)3-ACC-GAGCG-BHQ1
CGCTC-TPY-CCA-(AAA)3-ACC-TPY-GAGCG -BHQ1
CGCTC-TPY-CCA-(AAA)3-ACC-GAGCG -BHQ1
CGCTC−CCA-(AAA)3-ACC-TPY-GAGCG -BHQ1
CGCTC-DPA-CCA-(AAA)3-ACC-DPA-GAGCG -BHQ1
FAM-GGT-(TTT)3-TGG
6
7
8
9
10
a
Underlined base sequences indicate the self-complementary stem.
selected so probe-target hybridization could be monitored
independent of intramolecular events such as TPY intercala-
tion, stem-formation, or fluorophore quenching by TPY
(Figure 1).
Thermal transitions were measured by fluorescence spec-
troscopy for the complementary probe-target duplexes 3:10,
4:10, 5:10, 6:10, and 7:10 with varying concentrations of
ZnCl2 from substoichiometric (1/16 equiv) to excess (32
equiv) (see Supporting Information). The Tm of a probe/target
duplex is the sum of contributions from chelate (c), nonchelate
probe elements (nc), and nonspecific metal-ion effects on
duplex stability that vary with each metal-ion concentration
(ns). The normalized melting temperature ('Tm) for each
probe−target duplex was computed at each Zn2+ concentration
by subtracting from the duplex Tm (c+nc+ns), the Tm of the
corresponding no-chelate control duplex (nc+ns). The 'Tm is
therefore a measure of changes in probe-target stability
contributed solely by the chelate (see Supporting Information
for raw data).
Figure 3. Model of probe 4 and target 10 hybridization with (A)
EDTA, (B) 1 equiv of Zn2+, or (C) excess Zn2+. The color coding in
each panel designates predominant probe and target species in the
corresponding colored segments in Figure 2.
(TPY)2 chelate in 4(Zn2+) must be overcome in order for the
probe to hybridize to the target, resulting in destabilization of
the probe−target duplex at equilibrium.22,23 Excess Zn2+ (>1
equiv) populates both TPY ligands of 4, resulting in
disassembly of the Zn2+-(TPY)2 chelate and formation of
unconstrained probe 4(Zn2+)2 (Figure 3C) having probe−
target duplex stability equal to that of unconstrained probe 4 in
the metal-free control (Figure 3A).
Similar to the duplex 4:10, the 'Tm of the duplex 6:10 also
decreased as a function of Zn2+, reaching a minimum at 1 equiv
of metal ion (Figure 2, yellow segment). Surprisingly, however,
excess ZnCl2 beyond 1 equiv not only reversed the
destabilization of the probe−target duplex, but also resulted
in a net 5 °C stabilization of the probe−target duplex relative to
the metal-free control; a significant increase in 'Tm of 12 °C
overall (Figure 2, orange segment). Maximum destabilization of
the duplex 6:10 at 1 equiv of Zn2+ is consistent with assembly
In control transitions absent free metal ions (i.e., excess
EDTA), TPY-containing probe−target duplexes 4:10 and 6:10
were both stabilized by ∼2 °C relative to their TPY-free
counterparts (Figure 2, gray segment). TPY-base-pair stacking
interactions provide a duplex stabilizing force,21 particularly
with 3′ adjacent purine nucleobases.13
In the presence of Zn2+, the 'Tm of stemless probe−target
duplex 4:10 decreased as a function of Zn2+ (i.e., the probe−
target duplex was destabilized), reaching a minimum at 1 equiv
(600 nM) of metal ion (Figure 2, yellow segment). Excess
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Communication
of all stem-Zn2+-(TPY)2 motif chelates into 6(Zn2+) complexes
analogous to 4(Zn2+) complexes (Figure 4B). In distinction to
metal, possibly because the TPY is neighbored with a 3′ purine
and thus is alternatively engaged in more favorable stem base-
stacking interactions. Therefore, the stem disruptor function is
embodied in the 5′ TPY lacking an adjacent purine, as in 7:10.
Neither duplex 7:10 nor 8:10 exhibited a duplex destabilizing
effect at 1 equiv of Zn2+, indicating both TPY motifs are
required for stem-stabilization. The structural basis for the
metal-mediated disruption of the DNA stem by the 5′ TPY is
unknown, but is believed to involve the formation of a putative
TPY-Zn2+-nucleobase complex that precludes Watson−Crick
base-pairing in the stem (Figure 4C). The complex returns the
probe not to a bimetal stem-constrained probe, but instead to
the unconstrained configuration 6(Zn2+)2, which exhibits a net
5 °C stabilization of the probe-target duplex relative to the
stem-constrained metal-free control 6 (Figure 4A).
Except for replacement of the two TPY ligands with DPA
ligands, the DPA -containing probe−target duplex 9:10 is
identical to duplex 6:10 (Table 1). DPA and TPY have
equivalent ability to chelate Zn2+, and yet, the duplex 9:10
exhibited no metal-dependent change in 'Tm and no metal-free
stabilization of its Tm relative to the DPA-free counterpart 5:10
(Supporting Information). The basis for these striking
differences in metal-dependent and metal-independent behav-
ior between duplexes 9:10 and 6:10 are unclear. However, the
lack of metal-free stabilization in 9:10 suggests that, compared
to TPY, DPA has limited ability to stack with adjacent base-
pairs in the probe−target duplex. Additionally, terpyridyl and
dipicolyl phosphoramidites 1 and 2 install TPY and DPA
ligands with slightly different interstem spacing, respectively
(Scheme 1). The metal-dependent and metal-independent
phenomena seen with TPY-containing probes 4 and 6, may
therefore mechanistically depend on base-stacking and/or
precise placement of the TPY ligands.
Figure 4. Model of stemmed probe 6 and target 10 hybridization with
(A) EDTA, (B) 1 equiv of Zn2+, or (C) excess Zn2+. The color coding
in each panel designates predominant probe and target species in the
corresponding colored segments in Figure 2.
4(Zn2+), the energy provided by both the Zn2+-(TPY)2 chelate,
and the duplex stem must be overcome in order for 6(Zn2+) to
hybridize to target. The stem alone contributes a 5 °C
reduction in probe−target Tm compared to the stemless
analogue (see Supporting Information). If excess Zn2+ (>1
equiv) populated the two TPY ligands of 6 and resulted in only
the disassembly of the Zn2+-(TPY)2 chelate, the probe−target
duplex stability would be predicted to equal that of 6 in the
metal-free control in analogous fashion to the stemless probe 4.
However, relative to the metal-free control, excess Zn2+ resulted
in stabilization of the 6:10 probe−target duplex by an amount
equal to that provided by the duplex stem (i.e., ∼5 °C), a
phenomenon consistent with the disassembly of the entire
stem-Zn2+-(TPY)2 motif, including the DNA stem.
To characterize the mechanism underlying this stem
disassembly further, two control probes (7 and 8) were made
with a sequence identical to 6, except bearing only a single TPY
each (Table 1). The duplex 7:10 was increasingly stabilized as a
function of metal (Figure 5). The duplex 8:10 exhibited no 2
°C TPY-duplex stabilization or change in 'Tm as a function of
Other metallo-chelate-nucleobase polymer assemblies have
been described in the literature. We previously reported the
development of PNA-based probes that exhibited enhanced
binding specificity.23 In an analogous arrangement, Kramer24
̈
and Moreau21 introduced oligonucleotides with antipodal TPY
groups that underwent macrocyclization in the presence of a
coordinating transition metal. Chelate-modified oligonucleo-
tides consisting of unnatural metallo-base pairs (e.g., T-Hg+-T)
are known that exhibit exceptional duplex stability.25−34
However, to our knowledge this is the first report of a novel
stem-chelate-loop nucleobase polymer assembly that permits
metal-dependent stabilization and destabilization of probe-
target binding to unmodified DNA. We demonstrated here that
a DNA probe with the stem-chelate-loop structure consisting of
two TPY ligands is maximally stabilized with 1 equiv of Zn2+ via
an intramolecular chelate (i.e., TPY-Zn2+-TPY). However, with
increasing amounts of supra-stoichiometric Zn2+, the probe−
target duplex exhibits increased stability consistent with
disassembly of both the intramolecular chelate and the adjacent
Watson−Crick base-paired stem. The 'Tm of the probe−target
duplex was modulated by a total of 12 °C over a narrow
concentration range of Zn2+ ions (0.1−10 μM). This
concentration range is 4 and 5 orders of magnitude more
dilute than the range of MgCl2 and NaCl that nonspecifically
affects a similar change in duplex Tm, respectively.12
The stem-Zn2+-(TPY)2 motif is introduced readily with
automated oligonucleotide synthesis, and the range of 'Tm
values accessible could be expanded at the synthesis stage by
adjusting stem stability, by altering the stem length or G/C
content, or by employing alternate stem types.22,25,35−39 The
Figure 5. Probe-target duplex stability ('Tm) of monoterpyridine
controls (7:10) and (8:10) in the absence of free transition metal ions,
i.e., 5 μM EDTA (E), or in the presence of the indicated equivalents of
ZnCl2.
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(13) Kalek, M., Madsen, A. S., and Wengel, J. (2007) Effective
modulation of DNA duplex stability by reversible transition metal
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stem-chelate-loop structure may thus have broad utility in life
science applications, as well as in the development of DNA-
based nanodevices and electronic components, all applications
where it may be useful to have reversible upward and
downward, metal-dependent control over duplex stability.1,40
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an unlocked nucleic acid terpyridine monomer and binding of divalent
metal ion in nucleic acid duplexes. J. Org. Chem. 74, 8838−8841.
ASSOCIATED CONTENT
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■
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2,2′:6′,2″-terpyridines for applications in macromolecular chemistry
and nanoscience. Eur. J. Org. Chem., 3769−3776.
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* Supporting Information
Experimental procedures and spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(16) Johansson (2006) Pd-catalyzed amination of chloro-terpyridine
for the preparation of amine-containing ruthenium (II) complexes.
Synthesis, 2585−2589.
AUTHOR INFORMATION
(17) Schlegel, M. K., Zhang, L., Pagano, N., and Meggers, E. (2009)
Metal-mediated base pairing within the simplified nucleic acid gna.
Org. Biomol. Chem. 7, 476−482.
■
Corresponding Author
*E-mail: jmorgan@syntrixbio.com, tel 253.833.8009, fax
253.833.8127.
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nucleic acid. J. Am. Chem. Soc. 127, 4174−4175.
Notes
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propane-2,3-diol combinatorial monomers. Tetrahedron Lett. 37,
3911−3934.
The authors declare the following competing financial
interest(s): All of the authors are current (J.R.M., D.V.X.N.,
J.A.Z.) or former (A.R.F., S.R.R.) employees of Syntrix
Biosystems (Auburn, WA). Syntrix Biosystems manufactures
dipicolylamine cyanoethyl phosphoramidite, for commercial
sale through Glen Research (Sterling, VA).
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acceptor energy transfer, U.S. Patent 7,109,312, 2006.
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ACKNOWLEDGMENTS
■
We thank the National Cancer Institute (Grant No.
R44CA094612 and R44CA094612-S2) for supporting this
research.
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