DNA-selective hybridization and dual strand invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized 4′-C- piperazinomethyl-DNA

Article abstract of DOI:10.1039/b402414a

Incorporation of a novel pyren-1-ylcarbonyl-ftmctionalized 4′-C-piperazinomethyl-DNA monomer into oligodeoxynucleotides leads to increased thermal stability of duplexes with DNA complements but reduced thermal stability of duplexes with RNA complements. T

Full text of DOI:10.1039/b402414a

DNA-selective hybridization and dual strand invasion of short
double-stranded DNA using pyren-1-ylcarbonyl-functionalized
4A-C-piperazinomethyl-DNA†
Torsten Bryld, Torben Højland and Jesper Wengel*
Nucleic Acid Center‡, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark. E-mail: jwe@chem.sdu.dk; Fax: +45 6615 8780; Tel: +45 65502510
Received (in Cambridge, UK) 17th February 2004, Accepted 18th March 2004
First published as an Advance Article on the web 13th April 2004
Incorporation of a novel pyren-1-ylcarbonyl-functionalized 4A-
C-piperazinomethyl-DNA monomer into oligodeoxynucleo-
tides leads to increased thermal stability of duplexes with DNA
complements but reduced thermal stability of duplexes with
RNA complements. This DNA-selective hybridization§ is
explored for recognition of double-stranded DNA by a novel
dual strand invasion approach.
RNA target. These data are similar to those obtained for the
corresponding 4A-C-(N-methyl)piperazinomethyl monomer1 thus
confirming the applicability of the C4A position for conjugation of
ONs.7–11
The tendency towards DNA-selective hybridization§ observed
for ONs containing monomer X, and the fact that RNA monomers
derivatized at the O2A position with pyrene-containing moie-
ties12–14 and pyrene-containing pseudo-nucleotides (intercalating
nucleic acids)15 are among the very rare ON modifications that
induce DNA-selectivity, prompted us to investigate the pyren-
1-ylcarbonyl-functionalized 4A-C-piperazinomethyl-DNA mono-
mer Z. As seen in Table 1, incorporation of a single Z monomer
induces a remarkable stabilization of duplexes formed with DNA
complements (ON3 and ON9, DTm values of +7.0 and +9.0 °C) but
a significant destabilization of the duplexes formed with RNA
complements (DTm values of 28.0 and 24.0 °C). Incorporation of
two Z monomers (ON5, ON10 and ON11) substantiates this trend,
although the increase in thermal stability per modification towards
DNA is significantly lower than that observed for the examples
with incorporation of only one Z monomer. Notably, incorporation
of three Z monomers (ON7) leads to a small increase in the thermal
stability of the duplex with DNA but no hybridization (above 10
°C) with the RNA complement. These results strongly suggest the
ability to engineer the relative thermal stability of short DNA:DNA
and DNA:RNA duplexes by incorporation of one or a few 4A-C-
(pyren-1-ylcarbonyl)piperazinomethyl monomer(s). The generality
of this concept needs to be further studied, but it is encouraging that
it is operational when monomer Z is neighboured both by two
purine nucleotides (ON3) and by two pyrimidine nucleotides
(ON9). For comparison, the effect of one 2A-O-(pyrenylmethyl)-
RNA monomer was reported to be strongly sequence dependent.12
Functionalized oligonucleotides are being studied in our laboratory
for Ångström-scale chemical engineering1–3 towards applications
within nucleic acid nanotechnology.3,4 4A-C-Piperazinomethyl-
DNA monomers (Fig. 1) are interesting building blocks in this
context as the distal nitrogen atom allows attachment of a range of
functionalities facing the minor groove of nucleic acid duplexes. To
explore this opportunity we have synthesized oligodeoxynucleo-
tides (ONs) containing the 4A-C-(N-pyren-1-ylcarbonyl)piperazino-
methyl thymidine monomer Z and evaluated its effect on nucleic
acid hybridization in a direct comparison with the corresponding 4A-
C-piperazinomethyl monomer X (Fig. 1, T = thymin-1-yl).
The phosphoramidite building blocks 1 and 2 (for description of
synthesis of 1 and 2, see ESI) were used to synthesize ON2–ON7
and ON9–ON11 (Table 1) on an automated DNA synthesizer. The
coupling yields of 1 and 2 were 98% and 90% (10 min coupling
time, 1H-tetrazole as activator), respectively, and ~ 99% for
unmodified DNA phosphoramidites (2 min coupling time, 1H-
tetrazole as activator). Phosphoramidite 2 was used for synthesis of
ON7 and ON9–ON11 and phosphoramidite 1 for synthesis of ON2
and ON4. In addition, 1 was also used for synthesis of ON3 and
ON5 employing an on-column conjugation approach involving
selective removal of the Fmoc groups and subsequent reaction with
1-pyrenecarboxylic acid and HBTU in DMF following essentially
a procedure published for synthesis of 5A-end conjugated ONs.5,6
The composition of the synthesized ONs was verified by MALDI-
MS¶ and the purity ( > 80%) by capillary gel electrophoresis.
The hybridization properties of the modified ONs were deter-
mined in a medium salt buffer and compared with those of the
reference DNA strands ON1 and ON8 (Table 1). Incorporation of
one, two or three 4A-C-piperazinomethyl monomer(s) X (ON2,
ON4 and ON6) induced a small increase in the thermal denatura-
tion temperature (Tm value) with DTm values (change in Tm value
per modification) of approximately +2 °C towards the DNA
complement and virtually no change in the Tm value towards the
Table 1 ONs synthesized and thermal denaturation studiesa
DNA target
MM-DNA RNA target
Tm (DTm)/°C Tm/°C
Tm (DTm)/°C
ON1 5A-GTGATATGC 29 (ref)
13/18/18
14/23/18
19/29/23
28 (ref)
28 (±0)
ON2 5A-GTGAXATGC 31 (+2.0)
ON3 5A-GTGAZATGC 36 (+7.0)
ON4 5A-GCAXAXCAC 34 (+2.5)
ON5 5A-GCAZAZCAC 37 (+4.0)
ON6 5A-GXGAXAXGC 34 (+1.7)
ON7 5A-GZGAZAZGC 31 (+0.7)
20 (28.0)
26 (21.0)
20 (24.0)
26 (20.7)
< 10 ( < 26.0)
ON8 5A-GTGTTTTGC
32 (ref)
32 (ref)
ON9 5A-GTGTZTTGC 41 (+9.0)
ON10 5A-GTGZTZTGC 34 (+1.0)
ON11 5A-GZGTTTZGC 34 (+1.0)
19/25/24
28 (24.0)
21 (25.5)
20 (26.0)
a Thermal denaturation temperatures [Tm values (DTm = change in Tm
value per modification calculated relative to the Tm value of reference ON1
or ON8)] measured as the maximum of the first derivative of the melting
curve (A260 vs. temperature; 10 °C to 80 °C with an increase of 1 °C/min)
recorded in medium salt buffer (10 mM sodium phosphate, 100 mM sodium
chloride, pH 7.0) using ca. 1 mM concentrations of the two complementary
strands. A, C, G and T are standard DNA monomers. In the column “MM-
DNA” are listed Tm values recorded for mis-matched DNA target strands
containing a single mis-matched nucleotide; the values are listed in the
following order (indicating the mis-matched nucleotide in the central
position of the target DNA): C/G/T.
Fig. 1 Structures of monomers X and Z and phosphoramidites 1 and 2.
† Electronic supplementary information (ESI) available: short description
of synthesis of the phosphoramidites 1 and 2 and procedure used to record
fluorescence emission spectra. See http://www.rsc.org/suppdata/cc/b4/
b402414a/
‡ A research center funded by the Danish National Research Foundation for
studies on nucleic acid chemical biology.
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C h e m . C o m m u n . , 2 0 0 4 , 1 0 6 4 – 1 0 6 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

The ability to discriminate mis-matched complements as efficiently
as the reference ON1 was confirmed for the singly-modified ON2,
ON3 and ON9 (Table 1).
invasion. However, addition of a mixture of ON7 and RNA to the
preformed duplex ON1:DNA induced efficient duplex invasion as
shown by the absence of an excimer band in the spectrum recorded
of this mixture.∑ The importance of addition also of the RNA strand
strongly indicates this strand to partake in duplex formation with
the ON1 strand of the original DNA duplex during strand invasion,
and the process to involve dual strand invasion (Fig. 2).
The above example is the first of dual invasion of a mixed-
sequence DNA duplex not employing the concept of pseudo-
complementary nucleobases.21–23 The key to this novel approach is
the DNA-selectivity induced by incorporation of the 4A-C-(pyren-
1-ylcarbonyl)piperazinomethyl monomer Z. That medium salt
conditions and a preformed dsDNA target were applied are
encouraging towards fulfilling the goal of developing a general
method for sequence-specific molecular recognition of mixed-
sequence dsDNA. However, studies involving longer and more
biologically relevant double-stranded DNA target segments are
needed to establish the scope and limitations of this novel dual
strand invasion strategy.
Steady-state fluorescence emission spectra of ON3 and ON5
hybridized to DNA and RNA revealed typical pyrene monomer
fluorescence emission bands between 380 and 410 nm (data not
shown). A significant difference in pyrene monomer fluorescence
when hybridized with DNA or RNA was not observed. This
suggests that the DNA-selective hybridization induced by mono-
mer Z is due to favorable interaction of the pyrene units with the
DNA:DNA duplex within the minor groove rather than inter-
calation of the pyrene units, a reasoning which is similar to that
proposed for the 2A-O-(pyrenylmethyl)-RNA monomer.12 Molec-
ular modelling and NMR experiments are ongoing to investigate
the molecular basis for the hybridization characteristics of 4A-C-
piperazinomethyl-DNA.
We decided to explore the DNA-selectivity induced by monomer
Z for targeting double-stranded DNA (dsDNA). Currently, se-
quence-specific recognition of dsDNA by oligonucleotide ana-
logues is hampered by target sequence limitations and the
requirement of unnatural salt concentrations, although progress has
been accomplished using triplex-forming oligonucleotides,16
strand invading PNA,17,18 LNA19,20 and pseudo-complementary
DNA21,22 or PNA.23 In the latter approach, nucleobase analogues
are used that sterically prevent cross-hybridization while allowing
recognition of natural DNA. The absence of cross-hybridization
between ON7, containing three Z monomers, and its RNA
complement, suggests the use of ON7+RNA as a reagent mixture
for recognition of the two complementary segments of a dsDNA
duplex (Fig. 2).
We used the duplex ON1:DNA as a dsDNA target model and the
changes in fluorescence emission of ON7 upon hybridization to
monitor the processes in solution (Fig. 2; ON7 was present in ca.
2/3 molar ratio to the target). An excimer band at 430–520 nm is
seen in the fluorescence emission spectrum of single-stranded
ON7, which can be explained by the flexibility of the single
stranded ON7 allowing the pyrene units to form pyrene–pyrene
pairs. No excimer band is observed for the mixture of ON7 and
DNA indicating formation of a rigid duplex structure. However, for
the mixture of ON7 and RNA, an excimer band is evident and the
fluorescence emission spectrum resembles the spectrum of ON7
alone. This corroborates the inability of ON7 and RNA to form a
duplex above 10 °C at the applied conditions. The strand invasion
experiments were performed at 10 °C under medium salt conditions
(110 mM Na+). First ON7 was added to the preformed target duplex
ON1:DNA. The strong excimer band observed throughout the
experiment (24 h) revealed the absence of significant duplex
We thank the Danish National Research Foundation for financial
support, Ms Britta M. Dahl for ON synthesis and Dr Michael
Meldgaard, Exiqon A/S, for MALDI-MS analysis.
Notes and references
§ By “DNA-selective hybridization” is understood the formation of
duplexes with DNA complements that are significantly more thermally
stable than duplexes formed with RNA complements.
¶ MALDI-MZ: m/z ([M 2 H]2 found/calcd.) 2852/2853 (ON2), 3122/3125
(ON3), 2879/2878 (ON4), 3336/3332 (ON5), 3048/3048 (ON6),
3730/3731 (ON7), 3061/3059 (ON9), 3387/3388 (ON10), 3387/3387
(ON11).
∑ In Fig. 2 is shown the spectrum recorded 60 min after addition of ON7 and
RNA; this spectrum was identical to that recorded 24 h after the addition,
whereas gradually decreasing excimer band intensity was observed in the
spectra recorded during the first minutes after the addition.
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Fig. 2 Dual strand invasion of dsDNA (ON1:DNA duplex). Fluorescence
emission spectra of ON7,DNA (15 min), ON7,RNA (15 min), ON7 alone
(15 min), ON1,DNA after addition of ON7 (24 h), and ON1,DNA after
addition of ON7,RNA (60 min, see schematic drawing); in parentheses is
shown for each spectrum the elapsed time after mixing at which the
spectrum was recorded. ON7 was used in ca. 0.15 mM concentration ( ~ 2/3
molar ratio to the target); see Table 1 for buffer used.
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