Recognition of mixed-sequence DNA duplexes: Design guidelines for invaders based on 2′-O-(pyren-1-yl)methyl-RNA monomers

Article abstract of DOI:10.1021/jo402085v

The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-O-(pyren-1-yl)methyl- RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2′-O-(pyren-1-yl)methyl- RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2′-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology.

Full text of DOI:10.1021/jo402085v

Article
pubs.acs.org/joc
Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines
for Invaders Based on 2′‑O‑(Pyren-1-yl)methyl-RNA Monomers
Saswata Karmakar, Dale C. Guenther, and Patrick J. Hrdlicka*
Department of Chemistry, University of Idaho, 875 Perimeter Drive, MS 2343, Moscow, Idaho 83844-2343, United States
S
* Supporting Information
ABSTRACT: The development of agents that recognize
mixed-sequence double-stranded DNA (dsDNA) is desirable
because of their potential as tools for detection, regulation, and
modification of genes. Despite progress with triplex-forming
oligonucleotides, peptide nucleic acids, polyamides, and other
approaches, recognition of mixed-sequence dsDNA targets
remains challenging. Our laboratory studies Invaders as an
alternative approach toward this end. These double-stranded
oligonucleotide probes are activated for recognition of mixed-
sequence dsDNA through modification with +1 interstrand
zippers of intercalator-functionalized nucleotides such as 2′-O-
(pyren-1-yl)methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal
DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2′-O-(pyren-1-yl)methyl-
RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and
recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2′-O-(pyren-1-
yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for
recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient
recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular
biology, nucleic acid diagnostics, and biotechnology.
the “nearest-neighbor exclusion principle”,32 resulting in
INTRODUCTION
■
concomitant localized unwinding and destabilization of the
The development of compounds that recognize double-
duplex (i.e., the formation of an “energetic hotspot”; Figure 1).
stranded DNA (dsDNA) in a sequence-specific manner is a
The two strands that constitute an Invader duplex, on the other
research area of considerable interest that is motivated by the
hand, display very strong affinity toward complementary single-
prospect for molecular tools that can detect, regulate, and
stranded DNA (ssDNA), as duplex formation results in
modify genes.1−3 Significant advances have been made with
strongly stabilizing interactions between the pyrene and
triplex-forming oligonucleotides,4−6 peptide nucleic acids
flanking nucleobases (Figure 1).27−31 We have recently
(PNAs),7,8 polyamides,9,10 pseudocomplementary PNA,11−13
demonstrated that the stability difference between Invader
γ-PNA,14,15 engineered proteins,16,17 and other ap-
probes and probe−target complexes can be used to realize
proaches.18−26 However, the development of alternative
mixed-sequence recognition of (i) linear dsDNA,27,28,30 (ii)
DNA hairpins,29,31 and (iii) chromosomal DNA.29
strategies for specific mixed-sequence recognition of dsDNA
under physiological conditions remains a very desirable goal
because of the limitations of current probe technologies, which
include target sequence restrictions, insufficient binding affinity,
need for low-salinity conditions, poor cellular uptake, and/or
challenging synthesis.
Our laboratory is exploring so-called Invaders as an
alternative strategy for mixed-sequence recognition of
dsDNA.27−31 These double-stranded oligonucleotide probes
are activated for dsDNA recognition through modification with
one or more +1 interstrand zipper arrangements of intercalator-
modified nucleotide monomers such as 2′-O-(pyren-1-yl)-
methyl-RNA monomers (Figure 1; for a formal definition of
the zipper terminology, see the Experimental Section).
Presumably, the intercalating pyrene moieties are forced into
the same region of the duplex core, which causes a violation of
In the present work, we systematically studied the influence
that the nucleobase moieties of the 2′-O-(pyren-1-yl)methyl-
RNA monomers, used to construct hotspots, have on the
dsDNA recognition efficiency of Invader duplexes. It is
important to gain this insight, as it will guide the future design
of Invader duplexes for applications in molecular biology,
nucleic acid diagnostics, and biotechnology.
RESULTS AND DISCUSSION
■
Synthesis of 2′-O-(Pyren-1-yl)methyl-RNA Phosphor-
amidites. The corresponding phosphoramidites of monomers
Received: September 20, 2013
Published: November 6, 2013
© 2013 American Chemical Society
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Figure 1. Illustration of the Invader concept for recognition of mixed-sequence dsDNA (droplets denote intercalating pyrene moieties) and
structures of the monomers used in this study.
a
Scheme 1. Synthesis of 2′-O-(Pyren-1-yl)methyl-RNA-GiBu Phosphoramidite 5
a
Py = pyren-1-yl; TMSCl = trimethylsilyl chloride; DMTrCl = 4,4′-dimethoxytrityl chloride; PCl = 2-cyanoethyl-N,N-diisopropylchlorophosphor-
amidite.
A,33 C,33 and U34 were synthesized as previously described.
The novel N2-isobutyryl-protected 2′-O-(pyren-1-yl)-
methylguanosine phosphoramidite 5 was obtained from
guanosine (1) following the same general strategy that was
used by others for the synthesis of the corresponding ABz and
CBz phosphoramidites (Scheme 1).33 Thus, unprotected
guanosine was treated with 1-chloromethylpyrene in the
presence of sodium hydride, which afforded O2′-alkylated
nucleoside 2 in a low but consistent yield (24%). The low yield
is due to concomitant formation of O3′/O5′/N3′-alkylated
products (results not shown). The following N2-isobutyryl
protection of the nucleobase to give 3 was accomplished in 74%
yield using a transient protection protocol.35 Subsequent O5′-
dimethoxytritylation afforded nucleoside 4 in 88% yield, which
upon treatment with 2-cyanoethyl-N,N-diisopropylchlorophos-
oligodeoxyribonucleotides (ONs) using machine-assisted
solid-phase DNA synthesis (4,5-dicyanoimidazole as the
activator, 15 min) in ∼98% stepwise coupling yield.
Design of the Study. In order to systematically study the
influence that hotspot composition has on the dsDNA
recognition efficiency of Invader duplexes, we designed 10
model probes that vary only in the nature of the central hotspot
(see Table 1). The inherent symmetry of the energetic hotspots
along with the utilized sequence context (i.e., hotspots flanked
by A:T pairs on either side) allowed us to study the 16 possible
hotspot designs using 10 probes.
DNA Hybridization Characteristics of ONs Modified
with 2′-O-(Pyren-1-yl)methyl-RNA Monomers. Virtually
all of the individual Invader strands display strongly increased
affinity towardand increased duplex stability withcomple-
mentary single-stranded DNA compared with unmodified ONs
(ΔTm up to +13.0 °C, as shown in the first two ΔTm columns
in Table 1; ΔΔG293 values down to −15 kJ/mol, as shown in
the first two ΔG293 two columns in Table 2). Interestingly, the
G monomer has a considerably smaller stabilizing effect than
the other 2′-O-(pyren-1-yl)methyl-RNA monomers and is even
destabilizing in some cases [ΔTm = 3−9 °C and ΔΔG293 = 3−
phoramidite and Hunig’s base provided the target phosphor-
̈
1
amidite 5 in 78% yield. The disappearance of H NMR signals
from exchangeable protons upon D2O addition, along with
results from other one- and two-dimensional NMR experiments
and high-resolution MALDI-MS, confirmed the proposed
structures of nucleosides 2−5. The modified phosphoramidites
were used to incorporate monomers A/C/G/U into
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Table 1. Thermal Denaturation Temperatures (Tm) and Deviation of Additivity (DA) Values of 9-Mer DNA Duplexes Modified
a
with 2′-O-(Pyren-1-yl)methyl-RNA Monomers
Tm [ΔTm] (°C)
ON
sequence
upper strand vs ssDNA
42.0 [+6.5]
lower strand vs ssDNA
Invader duplex
dsDNA
35.5
DA (°C)
−17.0
ON1
ON20
5′-GTGA AC TGC
3′-CACT TG ACG
34.5 [−1.0]
24.0 [−11.5]
ON2
5′-GTGA AG TGC
3′-CACT TC ACG
46.0 [+8.5]
40.0 [+8.5]
48.5 [+13.0]
46.0 [+6.5]
54.5 [+11.0]
42.0 [+6.5]
40.0 [−3.5]
42.5 [+13.0]
37.5 [+6.0]
42.5 [+5.0]
38.5 [+7.0]
45.5 [+10.0]
45.0 [+5.5]
53.5 [+10.0]
39.0 [+3.5]
38.0 [−5.5]
42.5 [+13.0]
42.5 [+11.0]
32.5 [−5.0]
25.0 [−6.5]
35.5 [ 0.0]
33.0 [−6.5]
41.5 [−2.0]
26.0 [−9.5]
26.0 [−17.5]
26.5 [−3.0]
26.5 [−5.0]
37.5
31.5
35.5
39.5
43.5
35.5
43.5
29.5
31.5
−18.5
−22.0
−23.0
−18.5
−23.0
−20.0
−8.5
ON19
ON3
ON18
5′-GTGA AT TGC
3′-CACT TA ACG
ON4
ON17
5′-GTGA CA TGC
3′-CACT GU ACG
ON5
ON16
5′-GTGA CC TGC
3′-CACT GG ACG
ON6
ON15
5′-GTGA CG TGC
3′-CACT GC ACG
ON7
ON14
5′-GTGA GA TGC
3′-CACT CU ACG
ON8
ON13
5′-GTGA GC TGC
3′-CACT CG ACG
ON9
ON12
5′-GTGA UA TGC
3′-CACT AU ACG
−29.0
−22.0
ON10
ON11
5′-GTGA UT TGC
3′-CACT AA ACG
a
ΔTm = change in Tm value relative to the corresponding unmodified reference duplex. Tm values were determined from the first derivative maxima
of the thermal denaturation curves (A260 vs T) recorded in medium salt buffer ([Na+] = 110 mM, [Cl−] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4))
using a 1.0 μM concentration of each strand. DA = ΔTm(Invader duplex) − ΔTm(upper strand vs ssDNA) − ΔTm(lower strand vs ssDNA).
Example of DA calculation: DAON10:ON11 = ΔTm(ON10:ON11) − ΔTm(ON10:ssDNA) − ΔTm(ssDNA:ON11) = −5.0 °C − 6.0 °C − 11.0 °C =
−22.0 °C. A/C/G/T = adenin-9-yl/cytosin-1-yl/guanin-9-yl/thymin-1-yl DNA monomers. For the structures of A/C/G/U, see Figure 1.
Table 2. Changes in Gibbs Free Energy upon Duplex Formation (ΔG293) and Binding Energies (ΔG2re9c3) of 9-Mer Invaders at
a
293 K
ΔG293 [ΔΔG293] (kJ/mol)
293
ON
sequence
upper strand vs ssDNA lower strand vs ssDNA
Invader duplex
dsDNA
ΔG (kJ/mol)
rec
ON1
5′-GTGA AC TGC
3′-CACT TG ACG
−51 1 [−5]
−62 1 [−15]
−48 0 [−4]
−58 0 [−12]
−56 1 [−5]
−63 1 [−8]
−50 0 [−4]
−50 1 [+4]
−55 1 [−14]
−46 1 [−3]
−46 0 [ 0]
−50 1 [−3]
−48 0 [−4]
−56 0 [−10]
−54 0 [−3]
−64 1 [−9]
−49 1 [−3]
−48 0 [+6]
−53 1 [−12]
−54 0 [−11]
−37 0 [+9]
−46
−47
−44
−46
−51
−55
−46
−54
−41
−43
0
−14
−23
−13
−26
−18
−24
−14
−5
ON20
ON2
ON19
5′-GTGA AG TGC
3′-CACT TC ACG
−42 0 [+5]
−39 0 [+5]
−42 0 [+4]
−41 0 [+10]
−48 1 [+7]
−39 0 [+7]
−39 1 [+15]
−38 1 [+3]
−38 1 [+5]
0
0
0
1
0
0
0
1
1
ON3
ON18
5′-GTGA AT TGC
3′-CACT TA ACG
ON4
ON17
5′-GTGA CA TGC
3′-CACT GU ACG
ON5
ON16
5′-GTGA CC TGC
3′-CACT GG ACG
ON6
ON15
5′-GTGA CG TGC
3′-CACT GC ACG
ON7
ON14
5′-GTGA GA TGC
3′-CACT CU ACG
ON8
ON13
5′-GTGA GC TGC
3′-CACT CG ACG
ON9
ON12
5′-GTGA UA TGC
3′-CACT AU ACG
−29
−19
ON10
ON11
5′-GTGA UT TGC
3′-CACT AA ACG
a
ΔΔG293 is measured relative to ΔG293 for unmodified DNA duplexes (dsDNA). ΔG293 values were determined via baseline fitting of thermal
293
denaturation curves. ΔG = ΔG293(upper strand vs ssDNA) + ΔG293(lower strand vs ssDNA) − ΔG293(Invader duplex) − ΔG293(dsDNA). “ ”
denotes standard deviation.
rec
12 kJ/mol less favorable than for A/C/U monomers, as shown
in Tables 1 and 2; only ONs with identical 3′-flanking
nucleotides, e.g. ON1, ON5, ON14, and ON13, should be
compared (vide infra)]. Absorbance spectra of single-stranded
ON1−ON20 and their duplexes with ssDNA were recorded to
determine whether the low stability of G-modified duplexes is
linked to differences in pyrene binding modes (Figure S2 in the
Supporting Information). Small hybridization-induced bath-
ochromic shifts of pyrene absorption maxima, consistent with
increased interactions between pyrene and nucleobases and
hence pyrene intercalation,36 were generally observed for all of
the ONs (Δλ = 0−4 nm; Table 3). While G-modified ONs
displayed slightly smaller bathochromic shifts, the results do
not rule out pyrene intercalation as a likely binding mode. We
speculate that the destabilizing effect of the G monomer is
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Table 3. Absorption Maxima in the 300−400 nm Region for Single-Stranded ON1−ON20, the Corresponding Duplexes with
a
ssDNA, and Invaders Comprising These ONs
λmax [Δλ] (nm)
ON
sequence
upper strand lower strand upper strand vs ssDNA lower strand vs ssDNA Invader duplex
ON1
5′-GTGA AC TGC
3′-CACT TG ACG
349
349
349
352
349
349
349
349
348
348
351
349
351
352
350
348
349
351
349
349
352 [+3]
352 [+3]
352 [+3]
352 [ 0]
352 [+3]
352 [+3]
351 [+2]
351 [+2]
352 [+4]
351 [+3]
352 [+1]
352 [+3]
352 [+1]
353 [+1]
352 [+2]
352 [+4]
352 [+3]
352 [+1]
352 [+3]
352 [+2]
349
350
349
344
344
345
344
343
345
345
ON20
ON2
ON19
5′-GTGA AG TGC
3′-CACT TC ACG
ON3
ON18
5′-GTGA AT TGC
3′-CACT TA ACG
ON4
ON17
5′-GTGA CA TGC
3′-CACT GU ACG
ON5
ON16
5′-GTGA CC TGC
3′-CACT GG ACG
ON6
ON15
5′-GTGA CG TGC
3′-CACT GC ACG
ON7
ON14
5′-GTGA GA TGC
3′-CACT CU ACG
ON8
ON13
5′-GTGA GC TGC
3′-CACT CG ACG
ON9
ON12
5′-GTGA UA TGC
3′-CACT AU ACG
ON10
ON11
5′-GTGA UT TGC
3′-CACT AA ACG
a
Conditions as described in footnote a of Table 1. T = 5 °C, 1.0 μM concentration of each strand.
instead linked to a weakening of the G:C base pair following
pyrene intercalation.37
each other and destabilize duplexes when placed in +1
interstrand zippers. This is corroborated by the steady-state
fluorescence emission spectra of Invader duplexes, which
generally feature pyrene−pyrene excimer signals at λem ≈ 490
nm (Figure S7 in the Supporting Information), implying a
coplanar arrangement of the pyrene moieties with an
interplanar separation of ∼3.4 Å.20,27,28,31,40−45
As has been previously observed for U-modified ONs,38 the
nucleotide flanking a 2′-O-(pyren-1-yl)methyl-RNA monomer
on the 3′-side has a major influence on the duplex stability.
Thus, the stabilizing effect of 2′-O-(pyren-1-yl)methyl-RNA
monomers is greater when they are flanked by a 3′-purine
(ΔTm = 0−12 °C and ΔΔG293 = 3−11 kJ/mol more stabilizing
than with 3′-flanking pyrimidines; e.g., compare ΔTm and
ΔΔG293 for ON9, ON17, ON10, and ON14 in Tables 1 and
2). This finding is consistent with the 3′-directed intercalating
binding mode that has been proposed for the pyrene moiety on
the basis of NMR structures of U-modified DNA duplexes,39 as
the larger aromatic surface area of a 3′-purine likely facilitates
stronger stacking interactions with the pyrene moiety.
Similar conclusions are reached on the basis of the
thermodynamic data: (i) the formation of Invader duplexes is
3−15 kJ/mol less favorable than the formation of the
corresponding reference DNAs (see the ΔΔG293 values in the
“Invader duplex” column in Table 2) and (ii) the ΔΔG293
values of Invader duplexes are 5−29 kJ/mol less favorable than
would be expected for additive contributions from the two 2′-
293
rec
O-(pyren-1-yl)methyl-RNA monomers (see the ΔG values in
Thermostability and Thermodynamics of Invader
Duplexes. The influence on duplex stability upon incorpo-
ration of a second monomer can be additive, more than
additive, or less than additive relative to a corresponding singly
modified duplex. This is readily quantified in terms of Tm values
by the “deviation from additivity” (DA), which is defined as
DAON :ON ≡ ΔTm(ONX:ONY) − ΔTm(ONX:ssDNA) −
Table 2). Invader duplex destabilization is dominated by
unfavorable enthalpy (ΔΔH = 20−80 kJ/mol, results not
shown), presumably as +1 interstrand monomer arrangements
perturb stacking and/or nearby base pairing as a consequence
of violating the “nearest-neighbor exclusion principle”.32
Arrangement of monomers in +1 interstrand zippers results
in a localized region with one intercalator per base pair (Figure
1). Local perturbation of the duplex is further corroborated by
the broad melting profiles (Figure S1 in the Supporting
Information) and the blue-shifted pyrene absorption maxima
observed for Invader duplexes (Δλ = 1−9 nm relative to
probe−target duplexes; Table 3), which are indicative of weaker
pyrene−nucleobase interactions. Previous modeling and NMR
studies of Invader duplexes based on the closely related 2′-N-
(pyren-1-yl)methyl-2′-amino-α-L-LNA monomers also strongly
suggested localized duplex perturbation.31
X
Y
ΔTm(ssDNA:ONY) or expressed in terms of ΔG293 by the
term ΔGr2e9c 3(ONX:ONY) = ΔG293(ONX:ssDNA) +
ΔG293(ssDNA:ONY) − ΔG293(ONX:ONY) − ΔG293(dsDNA),
where ONX:ONY is an Invader duplex with a +1 interstrand
zipper arrangement of 2′-O-(pyren-1-yl)methyl-RNA mono-
293
rec
mers. ΔG also serves as an estimate for the thermodynamic
potential of Invader duplexes for recognition of isosequential
dsDNA targets (vide infra).
In agreement with our preliminary results,29−31 Invader
duplexes are strongly destabilized relative to singly modified
duplexes, irrespective of the monomers used to construct the
energetic hotspot (DA ≪ 0 °C; Table 1). In fact, all of the
studied Invader duplexes display lower Tm values than the
corresponding unmodified dsDNAs (ΔTm between −17.5 and
0.0 °C; see the “Invader duplex” column in Table 1). Evidently,
the two 2′-O-(pyren-1-yl)methyl-RNA monomers influence
The specific nature of the energetic hotspot influences the
relative magnitude of Invader duplex destabilization. Thus,
Invaders with hotspots composed of G monomers are more
destabilized relative to the corresponding unmodified dsDNA
than Invader duplexes composed of other 2′-O-(pyren-1-
yl)methyl-RNA monomers (see the ΔTm and ΔΔG293 values of
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Figure 2. Invader-mediated recognition of DNA hairpins. (a) Illustration of the recognition process. (b) Sequences and Tm values of DNA hairpins
with isosequential stems (conditions of thermal denaturation experiments; see Table 1). (c−f) Gel electropherograms illustrating dose−response
experiments between (c) DH1 and ON9:ON12, (d) DH2 and ON6:ON15, (e) DH3 and ON10:ON11, and (f) DH4 and ON8:ON13. (g) Dose−
response curves. (h−j) Gel electropherograms illustrating incubation of DH1−DH3 with a 500-fold excess of ON9:ON12, ON6:ON15, or
ON10:ON11, respectively. Experimental conditions: separately preannealed probes and targets (34.4 nM) were incubated for 15 h at room
temperature in 1× HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 10% sucrose, 1.4 mM spermine tetrahydrochloride, pH 7.2) and
then run on 12 or 16% nondenaturing PAGE (performed at 70 V, 2 h, ∼4 °C) using 0.5× TBE as a running buffer (45 mM Tris, 45 mM boric acid, 1
mM EDTA). DIG = digoxigenin.
ON1:ON20, ON5:ON16, ON7:ON14, and ON8:ON13 in the
“Invader duplex” columns in Tables 1 and 2).
Binding Energy for Invader-Mediated dsDNA Recog-
nition. As mentioned above, the binding energy for
recognition of isosequential dsDNA targets using Invader
selected for their wide range of ΔG2re9c3 and absolute Tm values
were evaluated using the electrophoretic mobility shift assay
that we recently introduced and validated (Figure 2a).29,31
Briefly described, digoxigenin (DIG)-labeled DNA hairpins
(DHs) comprising 9-mer double-stranded stems linked via T10
loops serve as model dsDNA targets. The unimolecular nature
of the hairpins confers extra stability to the stem region, which
renders it a more challenging target than isosequential dsDNA
targets [compare the Tm values of hairpins (Figure 2b) and
isosequential dsDNA targets (Table 1)]. Invader-mediated
recognition of DNA hairpins is expected to yield recognition
complexes with lower electrophoretic mobilities on non-
denaturing PAGE gels than DNA hairpins alone (Figure 2a).
Indeed, room-temperature incubation of Invader duplexes
with their respective DNA hairpin targets results in dose-
duplexes (i.e., the process depicted in Figure 1) is described by
293
ΔG2re9c3; highly negative ΔG
values signify that the two
rec
resulting probe−target duplexes are much more stable than the
Invader and target duplexes. Interestingly, all of the Invader
duplexes studied herein display favorable binding energies for
293
rec
recognition of isosequential dsDNA targets (ΔG between
−29 and −5 kJ/mol; Table 2). Invader duplexes with energetic
hotspots comprising exclusively C and/or U monomers display
293
rec
the most favorable binding energies (ΔG between −29 and
−24 kJ/mol; Table 2), which largely reflects the greater
stabilization of probe−target duplexes when 2′-O-(pyren-1-
yl)methyl-RNA monomers are flanked by 3′-purines. Con-
comitantly, Invader duplexes with hotspots exclusively compris-
dependent formation of recognition complexes (Figure 2c−g).
293
rec
The results indicate that the ΔG value and the dsDNA
recognition efficiency are correlated. Thus, the Invader duplex
293
ing A and/or G monomers display the least favorable binding
with the greatest recognition potential (ON9:ON12, ΔG
=
rec
293
rec
energies (ΔG between −14 and −5 kJ/mol; Table 2). The
−29 kJ/mol; Table 2) results in ∼80% recognition of DH1
when used in 100-fold excess (Figure 2g), while ON8:ON13,
which exhibits the lowest recognition potential (ΔG = −5
kJ/mol; Table 2), hardly results in any recognition of DH4
binding energies of Invaders with other hotspots fall between
293
rec
293
rec
these two extremes (ΔG between −23 and −14 kJ/mol;
Table 2). Importantly, these results indicate that incorporation
of +1 interstrand zippers of 2′-O-(pyren-1-yl)methyl-RNA
monomers is a general strategy to activate probes for dsDNA
recognition.
(Figure 2g). ON6:ON15 and ON10:ON11, which were
293
rec
predicted to have intermediate recognition potentials (ΔG
= −24 and −19 kJ/mol, respectively; Table 2), display
intermediate dsDNA recognition efficiencies (Figure 2g). It is
interesting to note that dsDNA recognition is slightly more
Invader-Mediated Recognition of DNA Hairpins. The
dsDNA recognition characteristics of four Invader duplexes
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Table 4. Thermal Denaturation Temperatures (Tm) and Deviation of Additivity (DA) Values of 14-Mer DNA Duplexes
a
Modified with 2′-O-(Pyren-1-yl)methyl-RNA Monomers
Tm [ΔTm] (°C)
ON
Invader
upper strand vs ssDNA
71.0 [+20.0]
lower strand vs ssDNA
Invader duplex
54.0 [+3.0]
dsDNA target
51.0
DA (°C)
−34.0
ON21
ON22
5′-TUA CTC ACG AUG CT
3′-AAU GAG TGC TAC GA
68.0 [+17.0]
ON23
ON24
5′-TUA CTC ACG ATG CT
3′-AAU GAG UGC TAC GA
67.5 [+16.5]
63.5 [+12.5]
56.0 [+5.0]
63.5 [+12.5]
61.0 [+10.0]
54.0 [+3.0]
40.5 [−10.5]
47.5 [−3.5]
38.0 [−13.0]
51.0
51.0
51.0
−39.5
−26.0
−21.0
ON25
ON26
5′-TTA CTC ACG ATG CT
3′-AAT GAG TGC TAC GA
ON27
ON28
5′-TTA CTC ACG ATG CT
3′-AAT GAG TGC TAC GA
a
See Table 1 for experimental conditions and definitions.
Table 5. Changes in Gibbs Free Energy upon Duplex Formation (ΔG293) and Thermodynamic dsDNA-Targeting Potentials
a
(ΔG2re9c3) of 14-Mer Invaders at 293 K
ΔG293[ΔΔG293] (kJ/mol)
293
ON
Invader
upper strand vs ssDNA lower strand vs ssDNA
Invader duplex
dsDNA target ΔG (kJ/mol)
rec
ON21
ON22
5′-TUA CTC ACG AUG CT
3′-AAU GAG TGC TAC GA
−74 1 [−8]
−83 1 [−17]
−78 1 [−12]
−68 1 [−2]
−91 0 [−25]
−73 1 [−7]
−74 1 [−8]
−75 1 [−9]
−58 1 [+8]
−66
−66
−66
−66
1
1
1
1
−41
−45
−38
−30
ON23
ON24
5′-TUA CTC ACG ATG CT
3′-AAU GAG UGC TAC GA
−45 1 [+21]
−48 1 [+18]
−47 0 [+19]
ON25
ON26
5′-TTA CTC ACG ATG CT
3′-AAT GAG TGC TAC GA
ON27
ON28
5′-TTA CTC ACG ATG CT
3′-AAT GAG TGC TAC GA
a
See Table 2 for definitions and conditions.
Figure 3. Invader-mediated recognition of DNA hairpins with 14-mer isosequential stem regions. (a−d) Gel electropherograms illustrating dose−
response experiments between DH5 and (a) ON21:ON22, (b) ON23:ON24, (c) ON25:ON26, and (d) ON27:ON28. (e) Sequence and Tm of
DNA hairpin DH5 (for conditions of thermal denaturation experiments, see Table 1). (f) Dose−response curves (see Figure 2 for experimental
conditions).
efficient with the more strongly activated ON6:ON15
compared with ON10:ON11, in spite of the significantly
greater Invader (Tm = 41.5 vs 26.5 °C, respectively; Table 1)
and duplex target (69.5 vs 59.0 °C, respectively; Figure 2b)
thermostabilities. This indicates that Invader-mediated dsDNA
recognition can occur at experimental temperatures that are
considerably lower than the Tm values of the Invader and/or
target duplexes, which is likely to prove important for biological
applications where the experimental temperature is not easily
adjustable.
efficient recognition of dsDNA. Moreover, incubation of
Invader duplexes ON9:ON12, ON6:ON15, and
ON10:ON11 with DNA hairpins that feature fully base-paired
but non-isosequential stem regions (i.e., one or two base pair
deviations relative to the Invader probes) fails to produce
significant amounts of recognition complexes even when the
Invaders are used in 500-fold excess, which demonstrates that
the recognition process proceeds with excellent fidelity (Figure
2h−j).
Properties of 14-Mer Invader Duplexes. Next, we set
out to examine whether the design principles inferred from the
9-mer study can be applied to longer and more densely
modified Invader duplexes. Toward this end, we designed four
14-mer Invaders, each modified with three energetic hotspots
Less than 40% recognition of DH1 is observed when single-
stranded ON9 or ON12 is used at 500-fold molar excess
(Figure S8 in the Supporting Information), which demonstrates
that both strands of Invader duplexes are needed to ensure
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293
that were dispersed in a similar manner and selected to afford
differentially activated probes (see Table 4). For example, the
three hotspots used in Invader duplex ON21:ON22 comprise
the relative contributions of probe−target duplexes to ΔG
rec
relative to Invader duplexes; Table 2). Invaders with hotspots
constructed using one or two G monomers (or two A
monomers) are the least activated constructs for dsDNA
recognition. However, a beneficial probe architecture can
compensate for a suboptimal choice of energetic hotspots for
construction of Invader duplexes (Figure 3). The insight gained
from this study will guide the design of efficient dsDNA-
targeting Invaders to enable applications in molecular biology,
nucleic acid diagnostics, and biotechnology.
C and/or U monomers and were expected to strongly promote
293
rec
dsDNA recognition on the basis of the observed ΔG values
in the 9-mer model study (Table 2). Conversely, the three
hotspots used in Invader duplex ON27:ON28 comprise A and/
or G monomers and therefore were expected to result in a less
efficient probe. Invaders ON23:ON24 and ON25:ON26 were
expected to fall between these two extremes.
The thermal denaturation data (Table 4) and thermody-
namic data (Table 5) exhibit the expected trends with a couple
of exceptions. ON21 and ON22 display the highest affinity
toward ssDNA targets in this series, while ON27 and ON28
display the lowest affinity toward ssDNA targets (first two Tm
columns in Table 4). The observed changes in Gibbs free
energy upon duplex formation between individual probe
strands and ssDNA corroborate these findings (first two
ΔG293 columns in Table 5), except that ON21:ssDNA is less
stable than expected. The Invader duplexes are destabilized
relative to the reference duplexes. It should be noted that
ON21:ON22 is the most thermostable of the evaluated
Invaders (see the ΔTm and ΔΔG293 values in the “Invader
duplex” columns in Tables 4 and 5, respectively). As a result,
the dsDNA recognition potential decreases in the order
EXPERIMENTAL SECTION
■
2′-O-(Pyren-1-yl)methylguanosine (2). 1-Chloromethylpyrene
(1.86 g, 7.41 mmol) was added to a suspension of guanosine (1) (3.00
g, 10.6 mmol) and NaH (60% suspension in mineral oil, 0.64 g, 26.5
mmol) in anhydrous DMSO. The reaction mixture was stirred for ∼14
h at room temperature, at which point water (100 mL) was added.
The resulting solids were filtered off, washed with water (3 × 20 mL),
and dried. The filtrate was extracted with EtOAc (3 × 50 mL), and the
organic layer was dried (Na2SO4). The aqueous layer was partially
evaporated (∼100 mL remaining) and left overnight at rt, which
resulted in an additional crop of solids. The solids and organic layer
were combined and purified by silica gel column chromatography (4−
8% v/v MeOH in CH2Cl2) to afford an off-white residue, which was
precipitated from acetone to afford nucleoside 2 (0.90 g, 24%) as a
white solid. Rf = 0.3 (10% MeOH in CH2Cl2, v/v). MALDI-HRMS:
m/z 520.1598 ([M + Na]+, C27H23N5O5Na+, calcd 520.1591). 1H
NMR (DMSO-d6): δ 10.58 (s, 1H, ex, NH), 8.27−8.32 (m, 3H, Py),
8.01−8.21 (m, 6H, Py), 7.97 (s, 1H, H8), 6.38 (s, 2H, 2ex, NH2), 5.97
(d, 1H, J = 6.7 Hz, H1′), 5.40−5.43 (m, 2H, 1 ex, 3′-OH, CH2Py),
5.20−5.23 (d, 1H, J = 11.4 Hz, CH2Py), 5.09 (t, 1H, ex, J = 5.2 Hz, 5′-
OH), 4.61−4.64 (m, 1H, H2′), 4.43−4.47 (m, 1H, H3′), 4.00−4.03
(m, 1H, H4′), 3.63−3.68 (m, 1H, H5′), 3.57−3.61 (m, 1H, H5′). 13C
NMR (DMSO-d6): δ 156.6, 153.6, 151.2, 135.3 (C8), 131.3, 130.65,
130.63, 130.2, 128.7, 127.30 (Py), 127.26 (Py), 127.1 (Py), 126.2
(Py), 125.23 (Py), 125.18 (Py), 124.4 (Py), 123.9, 123.7, 123.5 (Py),
116.7, 86.1 (C4′), 84.6 (C1′), 81.3 (C2′), 69.9 (CH2Py), 69.0 (C3′),
61.3 (C5′).
ON23:ON24
ON27:ON28 (see the ΔG values in Table 5).
>
ON21:ON22
>
ON25:ON26
>
293
rec
Incubation of these Invader duplexes with their DNA hairpin
target DH5 results in dose-dependent recognition of the mixed-
sequence dsDNA stem region (GC content ∼43%) in all four
cases, with ON21:ON22 as the most efficient probe (50%
recognition at ∼125-fold excess; Figure 3). It is noteworthy that
even Invader ON27:ON28 featuring less desirable energetic
hotspots recognizes DH5, albeit with the lowest efficiency in
this probe series. Importantly, this suggests that beneficial
probe architectures can compensate for the effect of suboptimal
energetic hotspots.
2-N-Isobutyryl-2′-O-(pyren-1-yl)methylguanosine (3). Nu-
cleoside 2 (0.26 g, 0.52 mmol) was dried through repeated
coevaporation with anhydrous pyridine and redissolved in anhydrous
pyridine (6 mL). Trimethylsilyl chloride (0.5 mL, 3.92 mmol) was
added, and the reaction mixture was stirred for 2 h at rt. After the
mixture was cooled to 0 °C, isobutyryl chloride (0.15 mL, 1.57 mmol)
was added over 10 min with continued cooling. This mixture was then
stirred for 3 h at rt and then cooled to 0 °C. Water (2 mL) was added,
and the mixture was stirred at 0 °C for 10 min and then for another 5
min at rt, at which point aq. NH3 (28%, 5 mL) was added. The
resulting solution was stirred at rt for 15 min and evaporated to near
dryness. The residual material was taken up in CH2Cl2 (100 mL) and
washed with water (2 × 60 mL). The organic phase was dried
(Na2SO4) and evaporated to dryness, and the resulting crude material
was purified by silica gel column chromatography (1−3% v/v MeOH
in CH2Cl2) to afford nucleoside 3 (220 mg, 74%) as a pale-yellow
solid. Rf = 0.6 (10% v/v MeOH in CH2Cl2). MALDI-HRMS: m/z
In summary, the design principles inferred from the 9-mer
study can be confidently applied to longer and more densely
modified Invader duplexes, but a systematic examination of the
interplay between probe architecture (i.e., number and position
of energetic hotspots) and dsDNA recognition efficiency is
necessary to gain a more complete picture of Invader probe
design. Studies along these lines are ongoing and will be
presented in due course. Nonetheless, the results presented
herein will already guide the design of efficient Invaders for
recognition of mixed-sequence dsDNA.
CONCLUSION
■
The present study demonstrates that incorporation of +1
interstrand zippers of 2′-O-(pyren-1-yl)methyl-RNA monomers
is a general approach toward activation of double-stranded
oligodeoxyribonucleotide probes for recognition of mixed-
sequence dsDNA targets. The recognition efficiency of the
probes is, however, influenced by the composition of the
energetic hotspots. Thus, Invader duplexes with hotspots
comprising exclusively C and/or U monomers display very
favorable dsDNA-binding affinities since (i) the resulting
probe−target duplexes are most strongly stabilized when 2′-
O-(pyren-1-yl)methyl-RNA monomers are flanked by 3′-
purines (Tables 1 and 2) and (ii) probe−target stabilization
is more important than Invader duplex destabilization in driving
the favorable thermodynamics of dsDNA recognition (compare
590.2008 ([M + Na]+, C31H29N5O6 Na+, calcd 590.2010). H NMR
1
(DMSO-d6): δ 11.85 (s, 1H, ex, NH), 11.30 (s, 1H, ex, NH), 8.21−
8.28 (m, 3H, Py), 8.19 (s, 1H, H8), 8.01−8.14 (m, 5H, Py), 7.94−7.97
(d, 1H, J = 7.8 Hz, Py), 5.94 (d, 1H, J = 6.7 Hz, H1′), 5.43−5.46 (m,
2H, 1 ex, 3′-OH, CH2Py), 5.14−5.16 (d, 1H, J = 11.4 Hz, CH2Py),
5.08 (t, 1H, ex, J = 5.2 Hz, 5′-OH), 4.65−4.68 (m, 1H, H2′), 4.46−
4.48 (m, 1H, H3′), 4.01−4.03 (m, 1H, H4′), 3.57−3.67 (m, 2H, H5′),
2.56−2.61 (septet, 1H, J = 6.7 Hz, CHMe2), 1.04 (d, 3H, J = 6.7 Hz,
CH3), 1.00 (d, 3H, J = 6.7 Hz, CH3). 13C NMR (DMSO-d6): δ 179.8,
154.5, 148.5, 147.8, 137.2 (C8), 131.2, 130.62, 130.58, 130.1, 128.7,
127.3 (Py), 127.2 (Py), 127.07 (Py), 127.06 (Py), 126.1 (Py), 125.2
(Py), 125.1 (Py), 124.3 (Py), 123.8, 123.6, 123.5 (Py), 119.9, 86.5
(C4′), 84.7 (C1′), 81.6 (C2′), 70.2 (CH2Py), 69.2 (C3′), 61.3 (C5′),
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34.5 (CHMe2), 18.7 (CH3), 18.6 (CH3). A trace impurity of CH2Cl2
was identified in the 13C NMR spectrum of 3.
(>90%) was verified by ion-pair reversed-phase HPLC running in
analytical mode.
Thermal Denaturation Experiments. ON concentrations were
estimated using the following extinction coefficients (OD/μmol) at
260 nm: dA (15.2), dC (7.05), dG (12.0), T (8.4), and pyrene (22.4).
Quartz optical cells with a path length of 1.0 cm were used. Strands
were mixed, denatured through heating to ∼70 °C, and cooled to the
starting temperature of the experiment. Thermal denaturation curves
(1.0 μM final concentration of each strand) were recorded on a
Peltier-controlled UV/vis spectrophotometer using a medium salt
buffer (100 mM NaCl, 0.1 mM EDTA, and pH 7.0 adjusted with 10
mM Na2HPO4 and 5 mM Na2HPO4). A temperature ramp of 0.5 °C/
min was used in all of the experiments. Thermal denaturation
temperatures (Tm) were determined as the maxima of the first
derivatives of the denaturation curves. The experimental temperatures
ranged from at least 15 °C below Tm (although not below 3 °C) to at
least 20 °C above Tm. Reported Tm values are averages of at least two
experiments within 1.0 °C, unless otherwise mentioned.
5′-O-(4,4′-Dimethoxytrityl)-2-N-isobutyryl-2′-O-(pyren-1-yl)-
methylguanosine (4). Nucleoside 3 (200 mg, 3.56 mmol) was
coevaporated with anhydrous pyridine (3 × 1.5 mL) and redissolved in
anhydrous pyridine (3 mL). To this was added 4,4′-dimethoxytrityl
chloride (DMTrCl) (300 mg, 0.53 mmol) and N,N-dimethyl-4-
aminopyridine (DMAP) (∼10 mg), and the reaction mixture was
stirred at rt for ∼14 h. The reaction mixture was diluted with CH2Cl2
(20 mL), and the organic phase sequentially washed with water (2 ×
10 mL) and sat. aq. NaHCO3 (2 × 10 mL). The organic phase was
evaporated to near dryness, and the resulting crude material was
coevaporated with abs. EtOH and toluene (2:1 v/v, 3 × 2 mL) and
purified by silica gel column chromatography (0−5% v/v MeOH in
CH2Cl2) to afford nucleoside 4 (270 mg, 88%) as a pale-yellow foam.
Rf = 0.8 (7% v/v MeOH in CH2Cl2). MALDI-HRMS: m/z 892.3325
1
([M + Na]+, C52H47N5O8.Na+, calcd 892.3317). H NMR (DMSO-
d6): δ 11.90 (s, 1H, ex, NH), 11.34 (s, 1H, ex, NH), 8.25−8.32 (m,
3H, Py), 8.04−8.15 (m, 5H, Py), 8.03 (s, 1H, H8), 7.99−8.01 (d, 1H, J
= 7.8 Hz, Py), 7.32−7.34 (m, 2H, DMTr), 7.19−7.25 (m, 7H, DMTr),
6.79−6.82 (m, 4H, DMTr), 5.97 (d, 1H, J = 5.5 Hz, H1′), 5.49−5.51
(d, 1H, J = 11.7 Hz, CH2Py), 5.47 (d, 1H, ex, J = 5.4 Hz, 3′-OH),
5.22−5.25 (d, 1H, J = 11.7 Hz, CH2Py), 4.72 (ap t, 1H, J = 5.5 Hz,
H2′), 4.45−4.48 (m, 1H, H3′), 4.11−4.14 (m, 1H, H4′), 3.710 (s, 3H,
CH3O), 3.706 (s, 3H, CH3O), 3.28−3.31 (m, 1H, H5′, overlap with
H2O signal), 3.18−3.22 (m, 1H, H5′), 2.63 (septet, 1H, J = 6.7 Hz,
CHMe2), 1.04−1.07 (2d, 6H, both J = 6.7 Hz, CH3). 13C NMR
(DMSO-d6): δ 179.8, 158.0, 154.5, 148.5, 147.9, 144.7, 137.1 (C8),
135.4, 135.3, 131.1, 130.7, 130.6, 130.1, 129.6 (DMTr), 128.7, 127.7
(DMTr), 127.6 (DMTr), 127.31 (Py), 127.29 (Py), 127.2 (Py), 127.0
(Py), 126.6 (DMTr), 126.2 (Py), 125.3 (Py), 125.2 (Py), 124.4 (Py),
123.9, 123.7, 123.5 (Py), 120.1, 113.1 (DMTr), 85.6, 85.1 (C1′), 84.2
(C4′), 80.8 (C2′), 70.3 (CH2Py), 69.4 (C3′), 63.9 (C5′), 54.9
(CH3O), 34.6 (CHMe2), 18.8 (CH3), 18.7 (CH3).
3′-O-(2-Cyanoethoxy(diisopropylamino)phosphinyl)-5′-O-
(4,4′-dimethoxytrityl)-2-N-isobutyryl-2′-O-(pyren-1-yl)-
methylguanosine (5). Nucleoside 4 (260 mg, 0.29 mmol) was
coevaporated with anhydrous 1,2-dichloroethane (2 × 2 mL) and
redissolved in anhydrous CH2Cl2 (7 mL). To this was added
anhydrous N,N-diisopropylethylamine (DIPEA) (213 μL, 1.19 mmol)
and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (PCl re-
agent) (133 μL, 0.59 mmol), and the reaction mixture was stirred at
rt for ∼3 h, whereupon abs. EtOH (1 mL) and CH2Cl2 (20 mL) were
sequentially added. The organic phase was washed with sat. aq.
NaHCO3 (10 mL) and evaporated to near dryness, and the resulting
residue was purified by silica gel column chromatography (40−70% v/
v EtOAc in petroleum ether) to afford the desired phosphoramidite 5
(250 mg, 78%) as a white foam. Rf = 0.8 (5% v/v MeOH in CH2Cl2).
MALDI-HRMS: m/z 1092.4389 ([M + Na]+, C61H64N7O9P Na+,
calcd 1092.4395). 31P NMR (CDCl3): δ 150.39, 150.35.
Synthesis and Purification of Modified ONs. The correspond-
ing phosphoramidites of monomers A, C, and U (ABz, CBz, U) were
obtained as previously described.33,34 The phosphoramidite of
monomer G was obtained as described above. Modified ONs were
synthesized on an automated DNA synthesizer (0.2 μmol scale;
succinyl-linked LCAA-CPG support) using a ∼50-fold molar excess of
2′-O-(pyren-1-yl)methyl-RNA phosphoramidites in anhydrous aceto-
nitrile (at 0.02 M) and extended coupling (4,5-dicyanoimidazole as the
activator, 15 min, ∼98% coupling yield) and oxidation (45 s). Standard
protocols were used with DNA phosphoramidites. Cleavage from the
solid support and removal of protecting groups was accomplished
using 32% aq. ammonia (55 °C, 12 h). ONs were purified via ion-pair
reversed-phase HPLC using a triethylammonium acetate buffer−
water/acetonitrile (v/v) gradient, detritylated (80% aq. AcOH), and
precipitated from acetone (−18 °C for 12−16 h). The identities of all
modified ONs were verified through MALDI-MS/MS analysis
recorded in positive ion mode on a quadrupole time-of-flight tandem
mass spectrometer equipped with a MALDI source using anthranilic
acid as a matrix (Table S1 in the Supporting Information). Purity
Determination of Thermodynamic Parameters. Thermody-
namic parameters for duplex formation were determined through
baseline fitting of denaturation curves using the software provided with
the UV/vis spectrometer. Bimolecular reactions, two-state melting
behavior, and a heat capacity change of ΔCp = 0 upon hybridization
were assumed.46 A minimum of two experimental denaturation curves
were each analyzed at least three times to minimize errors arising from
baseline choice. Averages and standard deviations are listed.
Absorption Spectra. UV−vis absorption spectra (range: 300−400
nm) were recorded at 5 °C using the same samples and
instrumentation as in the thermal denaturation experiments.
Steady-State Fluorescence Emission Spectra. Steady-state
fluorescence emission spectra were recorded on a Peltier-controlled
fluorimeter using the same samples as in the thermal denaturation
experiments (i.e., in nondeoxygenated Tm buffer). Spectra were
obtained as averages of five scans using an excitation wavelength of λex
= 350 nm, excitation slit = 5.0 nm, emission slit = 2.5 nm, and a scan
speed of 600 nm/min. Spectra were recorded at 5 °C to ascertain
maximal duplex formation.
Recognition of DNA Hairpins in Cell-Free Assay. This assay,
which was chosen in lieu of footprinting experiments to avoid the use
of 32P-labeled targets, was performed in a similar manner as previously
described.29,31 Unmodified DH1−DH5 were obtained from commer-
cial sources and used without further purification. DH1−DH5 were 3′-
DIG-labeled using the second-generation DIG Gel Shift Kit (Roche
Applied Bioscience) as recommended. The DIG-labeled ONs were
diluted and used in the recognition experiments without further
purification. Preannealed Invader duplexes (85 °C for 2 min, cooled to
room temperature over 15 min) and DIG-labeled dsDNA targets (34.4
nM) were mixed and incubated in HEPES buffer (50 mM HEPES, 100
mM NaCl, 5 mM MgCl2, 10% sucrose, 1.44 mM spermine
tetrahydrochloride, pH 7.2) for 15 h at room temperature. The
reaction mixtures were diluted with 6× DNA loading dye (Fermentas)
and loaded onto a 12 or 16% nondenaturing polyacrylamide gel.
Electrophoresis was performed using a constant voltage of 70 V for 2 h
at ∼4 °C using 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mM
EDTA) as a running buffer. Gels were blotted onto a positively
charged nylon membrane (Roche Applied Bioscience) using a
constant voltage of 100 V at ∼4 °C. The membranes were exposed
to anti-digoxigenin-AP Fab fragments as recommend by the
manufacturer of the DIG Gel Shift Kit, transferred to a hybridization
jacket, and incubated with the substrate (CSPD) in detection buffer
for 10 min at 37 °C. The chemiluminescence of the formed product
was captured on X-ray film, which was developed using an X-ray film
developer. The resulting bands were quantified using a multi-imager
equipped with appropriate software. Recognition efficiency was
determined as the intensity ratio between the recognition complex
band and the total lane. Averages of three independent experiments
are reported along with standard deviations. Nonlinear regression was
used to fit data points from dose−response experiments.
Definition of “Interstrand Zipper Arrangement”. The
following nomenclature describes the relative arrangement between
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two monomers positioned on opposing strands in a duplex. The
number n describes the distance measured in number of base pairs and
has a positive value if a monomer is shifted toward the 5′-side of its
own strand relative to a second reference monomer on the other
strand. Conversely, n has a negative value if a monomer is shifted
toward the 3′-side of its own strand relative to a second reference
monomer on the other strand.
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ASSOCIATED CONTENT
* Supporting Information
General experimental section; MS data for modified ONs;
representative thermal denaturation curves and absorption
spectra; steady-state fluorescence emission spectra; absorption
maxima for 14-mer probes and duplexes; and NMR spectra for
nucleosides 2−5. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
*E-mail: hrdlicka@uidaho.edu. Phone: 208-885-0108.
Notes
The authors declare the following competing financial
interest(s): Dr. Hrdlicka is the inventor of intellectual property
involving the Invader technology.
■
ACKNOWLEDGMENTS
■
This study was supported by Award R01 GM088697 from the
National Institute of General Medical Sciences, National
Institutes of Health; Awards IF13-001 and IF14-012 from the
Higher Education Research Council, Idaho State Board of
Education; and Minitube of America. We thank Dr. Lee
Deobald (EBI Murdock Mass Spectrometry Center, University
of Idaho) for assistance with mass spectrometric analysis and
Dr. Carolyn Bohach (Food Science, University of Idaho) for
access to gel documentation stations.
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